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Background: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. Methods: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). Results: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). Conclusions: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior. [ABSTRACT FROM AUTHOR]

Background: The chloride intracellular channels (CLICs) family includes six ion channels (CLIC1-CLIC6) expressed on the cellular level and secreted into interstitial fluid and blood. They are involved in the physiological functioning of multiple systems as well as the pathogenetic processes of cancer. CLICs play essential roles in the tumor microenvironment. The current systematic review aimed at identifying and summarizing the research of CLICs in oncology on clinical material to assess CLICs' potential as novel biomarkers and personalized therapy targets. Materials and methods: The authors systematically searched the PubMed database for original articles concerning CLIC research on clinical material of all types of cancer -- fluids and tissues. Results: Fifty-three articles investigating in summary 3944 clinical samples were qualified for the current review. Studied material included 3438 tumor samples (87%), 437 blood samples (11%), and 69 interstitial fluid samples (2%). Studies investigated 21 cancer types, mostly hepatocellular carcinoma, colorectal, ovarian, and gastric cancer. Importantly, CLIC1, CLIC2, CLIC3, CLIC4, and CLIC5 were differently expressed in cancerous tissues and patients' blood compared to healthy controls. Moreover, CLICs were found to be involved in several cancer-associated signaling pathways, such as PI3K/AKT, MAPK/ERK, and MAPK/p38. Conclusion: CLIC family members may be candidates for potential novel cancer biomarkers due to the contrast in their expression between cancerous and healthy tissues and secretion to the interstitial fluid and blood. CLICs are investigated as potential therapeutic targets because of their involvement in cancer pathogenesis and tumor microenvironment. [ABSTRACT FROM AUTHOR]

Mouse models of breast cancer have revealed that tumour‐bearing hosts must express the oxidoreductase CLIC4 to develop lung metastases. In the absence of host CLIC4, primary tumours grow but the lung premetastatic niche is defective for metastatic seeding. Primary breast cancer cells release EVs that incorporate CLIC4 as cargo and circulate in plasma of wildtype tumour‐bearing hosts. CLIC4‐deficient breast cancer cells also form tumours in wildtype hosts and release EVs in plasma, but these EVs lack CLIC4, suggesting that the tumour is the source of the plasma‐derived EVs that carry CLIC4 as cargo. Paradoxically, circulating EVs are also devoid of CLIC4 when CLIC4‐expressing primary tumours are grown in CLIC4 knockout hosts. Thus, the incorporation of CLIC4 (and perhaps other factors) as EV cargo released from tumours involve specific signals from the surrounding stroma determined by its genetic composition. Since CLIC4 is also detected in circulating EVs from human breast cancer patients, future studies will address its association with disease. [ABSTRACT FROM AUTHOR]

Abstract Mouse models of breast cancer have revealed that tumour‐bearing hosts must express the oxidoreductase CLIC4 to develop lung metastases. In the absence of host CLIC4, primary tumours grow but the lung premetastatic niche is defective for metastatic seeding. Primary breast cancer cells release EVs that incorporate CLIC4 as cargo and circulate in plasma of wildtype tumour‐bearing hosts. CLIC4‐deficient breast cancer cells also form tumours in wildtype hosts and release EVs in plasma, but these EVs lack CLIC4, suggesting that the tumour is the source of the plasma‐derived EVs that carry CLIC4 as cargo. Paradoxically, circulating EVs are also devoid of CLIC4 when CLIC4‐expressing primary tumours are grown in CLIC4 knockout hosts. Thus, the incorporation of CLIC4 (and perhaps other factors) as EV cargo released from tumours involve specific signals from the surrounding stroma determined by its genetic composition. Since CLIC4 is also detected in circulating EVs from human breast cancer patients, future studies will address its association with disease.

Chloride intracellular channel proteins (CLICs) display ubiquitous expression, with each member exhibiting specific subcellular localisation. While all CLICs, except CLIC3, exhibit a highly conserved putative nuclear localisation sequence (NLS), only CLIC1, CLIC3 and CLIC4 exist within the nucleus. The CLIC4 NLS, 199‐KVVAKKYR‐206, appears crucial for nuclear entry and interacts with mouse nuclear import mediator Impα isoform 1, omitting the IBB domain (mImpα1ΔIBB). The essential nature of the basic residues in the CLIC4 NLS has been established by the fact that mutating out these residues inhibits nuclear import, which in turn is linked to cutaneous squamous cell cancer. Given the conservation of the CLIC NLS, CLIC1 likely follows a similar import pathway to CLIC4. Peptides of the CLIC1 (Pep1; Pep1_S C/S mutant) and CLIC4 (Pep4) NLSs were designed to examine binding to human Impα isoform 1, omitting the IBB domain (hImpα1ΔIBB). Molecular docking indicated that the core CLIC NLS region (KKYR) forms a similar binding pattern to both mImpα1ΔIBB and hImpα1ΔIBB. Fluorescence quenching demonstrated that Pep1_S (Kd ≈ 237 μM) and Pep4 (Kd ≈ 317 μM) bind hImpα1ΔIBB weakly. Isothermal titration calorimetry confirmed the weak binding interaction between Pep4 and hImpα1ΔIBB (Kd ≈ 130 μM) and the presence of a proton‐linked effect. This weak interaction may be due to regions distal from the CLIC NLS needed to stabilise and strengthen hImpα1ΔIBB binding. Additionally, this NLS may preferentially bind another hImpα isoform with different flexibility properties. [ABSTRACT FROM AUTHOR]

Numerous academic literature suggests that amyloid-β (Aβ) deposition, tau protein phosphorylation, and irreversible neuronal death are the three major causes of AD. The chloride intracellular channel (CLIC) protein family not only regulates the polarisation of neurons, but also has important implications for neuronal survival. Chloride intracellular channel 4 (CLIC4) can be pathologically activated by cyclin-dependent kinase 5 (Cdk5), which causes a significant increase in the expression of CLIC4 and mediates neuronal apoptosis. CLIC4 knockdown inhibits H2O2-induced neuronal apoptosis; however, the relationship between CLIC4 and AD remains unknown. In the present study, we showed that CLIC4 expression was elevated in the hippocampus of AD mice; knockdown of hippocampal CLIC4 alleviated Aβ25–35-induced cognitive impairment in mice; overexpression of hippocampal CLIC4 accelerated Aβ deposition and tau protein hyperphosphorylation in young AD mice (APP/PS1 mice at three months of age). CLIC4 overexpressing mice had a longer escape latency compared to controls in behavioural testing (Morris water maze and T-maze tests). By Co-immunoprecipitation/mass spectrometry (Co-IP/MS) of HT22 cells to identify proteins that specifically bind to CLIC4, we found interactions with CCAAT enhancer binding protein (C/EBPβ); a critical pathway involved in the development of various neurodegenerative diseases. In addition, the knockdown of hippocampal CLIC4 alleviated AD-like pathology by inhibiting the C/EBPβ/AEP signaling pathway. These data suggest an essential role for high CLIC4 expression in the pathophysiology of AD and reveal that inhibition of CLIC4 expression may provide an opportunity for treatment. [ABSTRACT FROM AUTHOR]

The airway epithelium is located at the interactional boundary between the external and internal environments of the organism and is often exposed to harmful environmental stimuli. Inflammatory response that occurs after airway epithelial stress is the basis of many lung and systemic diseases. Chloride intracellular channel 4 (CLIC4) is abundantly expressed in epithelial cells. The purpose of this study was to investigate whether CLIC4 is involved in the regulation of lipopolysaccharide (LPS)-induced inflammatory response in airway epithelial cells and to clarify its potential mechanism. Our results showed that LPS induced inflammatory response and decreased CLIC4 levels in vivo and in vitro. CLIC4 silencing aggravated the inflammatory response in epithelial cells, while overexpression of CLIC4 combined with LPS exposure significantly decreased the inflammatory response compared with cells exposed to LPS without CLIC4 overexpression. By labeling intracellular chloride ions with chloride fluorescent probe MQAE, we showed that CLIC4 mediated intracellular chloride ion-regulated LPS-induced cellular inflammatory response. [Display omitted] • LPS induces inflammatory response and decreases CLIC4 levels in vivo and in vitro. • CLIC4 inhibits LPS-induced inflammatory response in HBECs. • CLIC4 mediates intracellular chloride ion-regulated LPS-induced cellular inflammatory response. [ABSTRACT FROM AUTHOR]

Simple Summary: Although chloride intracellular channel proteins (CLICs) have been identified as ion channel proteins, their true functions are still elusive. Recent in silico analyses show that CLICs may be prognostic markers in cancer. This review focuses on CLIC2 that plays preventive roles in malignant cell invasion and metastasis. CLIC2 is secreted extracellularly and binds to matrix metalloproteinase 14 (MMP14), while inhibiting its activity. As a result, CLIC2 may contribute to the development/maintenance of junctions between blood vessel endothelial cells and the inhibition of invasion and metastasis of tumor cells. CLIC2 may be a novel therapeutic target for malignancies. CLICs are the dimorphic protein present in both soluble and membrane fractions. As an integral membrane protein, CLICs potentially possess ion channel activity. However, it is not fully clarified what kinds of roles CLICs play in physiological and pathological conditions. In vertebrates, CLICs are classified into six classes: CLIC1, 2, 3, 4, 5, and 6. Recently, in silico analyses have revealed that the expression level of CLICs may have prognostic significance in cancer. In this review, we focus on CLIC2, which has received less attention than other CLICs, and discuss its role in the metastasis and invasion of malignant tumor cells. CLIC2 is expressed at higher levels in benign tumors than in malignant ones, most likely preventing tumor cell invasion into surrounding tissues. CLIC2 is also expressed in the vascular endothelial cells of normal tissues and maintains their intercellular adhesive junctions, presumably suppressing the hematogenous metastasis of malignant tumor cells. Surprisingly, CLIC2 is localized in secretory granules and secreted into the extracellular milieu. Secreted CLIC2 binds to MMP14 and inhibits its activity, leading to suppressed MMP2 activity. CLIC4, on the other hand, promotes MMP14 activity. These findings challenge the assumption that CLICs are ion channels, implying that they could be potential new targets for the treatment of malignant tumors. [ABSTRACT FROM AUTHOR]

Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveal CLICs' contribution to RGC death. • Expression of Clic1 and Clic4 are upregulated in RGCs after axonal injury. • Optimization of AAV2-retro for RGC labeling and gene manipulation. • Integrated use of the AAV2-retro-shRNA and longitudinal retinal fundus imagining. • Concomitant knockdown of Clic1 and Clic4 promotes RGC survival. [ABSTRACT FROM AUTHOR]

Introduction: Proper placentation requires well controlled extravillous trophoblast cell (EVT) migration and invasion. Transforming growth factor β (TGFβ) signaling has been well characterized as negatively regulating EVT migration and invasion. CLIC4 is an enhancer of TGFβ signaling, however CLIC4's function in placentation and its association to placental TGFβ signaling is unknown. Here we aimed to investigate the role of CLIC4 on trophoblast cell function and its relationship to TGFβ signaling.Methods: CLIC4 was immunolocalized in human placenta throughout gestation and the first trimester decidua. siRNA was used to knockdown CLIC4 in a human trophoblast cell line (HTR8/SVneo) to reveal functional consequences of CLIC4 loss on cell adhesion, proliferation, migration and invasion via xCELLigence. qPCR was used to identify downstream targets of CLIC4 in HTR8/SVNeo cells.Results: CLIC4 was widely expressed in the syncytiotrophoblast, cytotrophoblast and decidual cells across all trimesters of pregnancy with no significant difference in staining intensity in the different cellular compartments both across gestation and between compartments. Using immunofluorescent co-localization of CLIC4 and EVT marker HLA-G, we confirmed that CLIC4 localized to the cytoplasm of cell column EVTs in the first trimester decidua and nuclei of some EVTs that invaded in the decidua. Knockdown of CLIC4 in HTR8/SVneo cells significantly elevated cell adhesion, migration and invasion. Analysis of TGFβ signaling downstream targets identified that CDH2 and BAMBI expression were significantly increased after CLIC4 knockdown in HTR8/SVneo cells.Discussion: Our data support an inhibitory role for CLIC4 in regulating trophoblast migration and invasion, likely acting in part via BAMBI and CDH2. [ABSTRACT FROM AUTHOR]

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (Hs CLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (Pg DHAR). In comparison, Hs CLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs. • Stress adapted pearl millet DHA reductase (DHAR) possess higher turnover rate. • Detailed kinetic analysis presented for a plant DHAR and human CLIC1, 3 and 4. • Crystal structures reveal different oxidation states of catalytic cysteine. • Possible implications in crop improvement and anti-cancer drug-discovery. [ABSTRACT FROM AUTHOR]

Background: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. Methods: We used qRT‐PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. Results: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. Conclusions: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes. [ABSTRACT FROM AUTHOR]

Non-coding RNAs (ncRNAs) have shown to act as crucial mediators in atherosclerosis (AS) development. The purpose of our study was to explore the role of Astragaloside IV (ASV) and circular RNA_0000231 (circ_0000231) in AS using AS cell model. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to analyze cell viability and apoptosis. Migration ability was assessed by transwell migration assay and wound healing assay. The inflammatory response was evaluated via enzyme-linked immunosorbent assay (ELISA). Oxidative status was assessed via matching commercial kits. Western blot assay was conducted to detect the expression of monocyte chemoattractant protein 1 (MCP1), intercellular adhesion molecule 1 (ICAM1), and chloride intracellular channel 4 (CLIC4). The levels of circ_0000231, its linear form Rho GTPase activating protein 12 (ARHGAP12), microRNA-135a-5p (miR-135a-5p), and CLIC4 messenger RNA (mRNA) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Circ_0000231–miRNA interactions were established using Starbase and Circbank softwares, while the targets of miR-135a-5p were explored by Starbase software. Dual-luciferase reporter assay and RNA-pull down assay were used to verify these target interactions. ASV suppressed the apoptosis, inflammation, and oxidative stress while recovered the viability and migration ability of HUVECs which were mediated by oxidized low-density lipoprotein (ox-LDL). Circ_0000231 overexpression antagonized the protective role of ASV in ox-LDL-induced HUVECs. MiR-135a-5p was verified as a direct target of circ_0000231, and circ_0000231 contributed to ox-LDL-induced cell injury of HUVECs through down-regulating miR-135a-5p. MiR-135a-5p directly interacted with the 3′ untranslated region (3′-UTR) of CLIC4 mRNA in HUVECs, and miR-135a-5p protected HUVECs against ox-LDL-induced injury through down-regulating CLIC4. ASV protected HUVECs against ox-LDL-induced injury through targeting circ_0000231/miR-135a-5p/CLIC4 axis. Targeting circ_0000231/miR-135a-5p/CLIC4 axis might provide a novel insight to develop effective strategy for AS treatment. [ABSTRACT FROM AUTHOR]

Objective: Necrotizing enterocolitis (NEC) is a gastrointestinal emergency with a severe inflammation storm, intestinal necrosis, and perforation. MicroRNA-146a-5p (miR-146a-5p) has been reported to be a valuable anti-inflammatory factor in various intestinal inflammatory disorders. However, the role of miR-146a-5p in NEC, its effects on nucleotide-binding domain and leucine-rich repeat-containing protein 3 (NLRP3) inflammasome, and its downstream inflammatory factors remain unknown. This study aimed to investigate the role of miR-146a-5p and NLRP3 inflammasome and its downstream inflammatory factors in NEC development.Methods: The expression levels of miR-146a and NLRP3 inflammasome were investigated in intestinal tissues. Next, the mechanism by which miR-146a-5p regulates NLRP3 inflammasome activation was explored in vitro in THP-1 cells. Finally, to identify the effects of miR-146a-5p on NEC in vivo, NEC mice were transinfected with miR-146a-5p overexpression adenovirus before the occurrence of NEC.Results: NLRP3 inflammasome enzymatic protein caspase-1 and its downstream inflammatory factors increased in NEC intestinal samples in both humans and mice, and miR-146a-5p expression level was increased and mainly expressed in the macrophages of the affected intestine. In vitro, only miR-146a-5p mimic inhibited NLRP3 inflammasome downstream inflammatory factors and its upstream protein chloride intracellular channel protein 4 (CLIC4) expression in cellular membrane in the THP-1 cell line, and this only occurred under mild/moderate LPS concentration. MiR-146a-5p overexpression adenovirus transfection reduced CLIC4 cellular membrane expression and inhibited NLRP3 downstream factors increasing in vivo. After the transfection of miR-146a-5p adenovirus, the survival rate of NEC mice was increased, and intestinal injury was ameliorated.Conclusion: MiR-146a-5p inhibited NLRP3 inflammasome downstream inflammatory factors and CLIC4 membrane expression in NEC. Additionally, miR-146a-5p could attenuate inflammation and intestinal injury in the NEC-affected intestine.

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-β signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-β/SMAD3 co-operates with Wnt3a/β-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of β-catenin and GLI expression or activity abolishes TGF-β/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-β, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors

Background and Methods: Acute myeloid leukemia (AML), which starts in the bone marrow, is a group of hematopoietic stem cell disorders. Chloride intracellular channel 4 (CLIC4) is regulated by p53, c-Myc, and TGF-β. It induces the NF-κB-dependent activation of HIF (hypoxia-inducible factor) and participates in tumor growth through its microenvironmental function. However, its prognostic value in AML remains unclear, as well as its co-expression biomarkers. In this study, we evaluated the prognostic significance of CLIC4 expression using two independent large cohorts of cytogenetically normal AML (CN-AML) patients. Multivariable analysis and multi-omics analysis with weighted correlation network analysis (WGCNA) in the CN-AML group were also presented. Based on CLIC4 and its related genes, microRNA–target gene interaction network analysis and downstream gene ontology analysis were performed to unveil the complex functions behind CLIC4.Results: We demonstrated that the overexpression of CLIC4 was notably associated with unfavorable outcome in the two independent cohorts of CN-AML patients [overall survival (OS) and event-free survival (EFS): P < 0.0001, n = 185; OS: P = 0.016, n = 232], as well as in the European LeukemiaNet (ELN) Intermediate-I group (OS: P = 0.015, EFS: P = 0.012, n = 115), the National Comprehensive Cancer Network Intermediate Risk AML group (OS and EFS: P < 0.0001, n = 225), and the non-M3 AML group (OS and EFS: P < 0.0001, n = 435). Multivariable analysis further validated CLIC4 as a high-risk factor in the CN-AML group. Multi-omics analysis presented the overexpression of CLIC4 as associated with the co-expression of the different gene sets in leukemia, up/downregulation of the immune-related pathways, dysregulation of microRNAs, and hypermethylation around the CpG islands, in open sea regions, and in different gene structural fragments including TSS1500, gene body, 5′UTR region, 3′UTR region, and the first exon. By further performing WGCNA on multi-omics data, certain biomarkers that are co-expressed with CLIC4 were also unveiled.Conclusion: We demonstrated that CLIC4 is a novel, potential unfavorable prognosticator and therapeutic target for CN-AML. As having a key role in CN-AML, the interactions between CLIC4 and other genomics and transcriptomics data were confirmed by performing microRNA–target gene interaction network analysis and gene ontology enrichment analysis. The experimental result provides evidence for the clinical strategy selection of CN-AML patients.

The endometrium remodels in each menstrual cycle to become receptive in preparation for embryo implantation which occurs in the mid-secretory phase of the cycle. Failure of blastocyst adhesion and implantation cause infertility. We compared chloride intracellular channel 4 (CLIC4) expression in human endometrium from women with normal fertility and primary unexplained infertility in the mid-secretory/receptive phase of the menstrual cycle. CLIC4 localised to both the epithelial and stromal regions of the endometrium of fertile tissues across the cycle. CLIC4 expression was significantly reduced in the luminal and glandular epithelium and remained unchanged in the stromal region of mid-secretory infertile endometrium compared to fertile endometrium. siRNA knockdown of CLIC4 significantly compromised adhesive capacity of Ishikawa cells (endometrial epithelial cell line). This reduced adhesion and CLIC4 expression was associated with elevated SGK1 , p53 , SIRT1 , BCL2 and MCL1 gene expression in the Ishikawa cells. CLIC4 expression was increased in primary human endometrial stromal cells during decidualization, however, siRNA knockdown of CLIC4 did not affect decidualization. Our data provide evidence that CLIC4 may regulate receptivity and facilitate blastocyst attachment initiating implantation. Reduced CLIC4 levels may be causative of implantation failure in women. • Endometrial receptivity requires is critical for successful embryo implantation. • Chloride channel 4 is abnormally decreased in the endometrium of women with infertility. • Low levels of Chloride channel 4 reduces endometrial cell line adhesion but not decidualization. • Chloride channel 4 alters genes associated with apoptosis and TGFb signalling in endometrial epithelial cells. [ABSTRACT FROM AUTHOR]

Background and Methods: Acute myeloid leukemia (AML), which starts in the bone marrow, is a group of hematopoietic stem cell disorders. Chloride intracellular channel 4 (CLIC4) is regulated by p53, c-Myc, and TGF-β. It induces the NF-κB-dependent activation of HIF (hypoxia-inducible factor) and participates in tumor growth through its microenvironmental function. However, its prognostic value in AML remains unclear, as well as its co-expression biomarkers. In this study, we evaluated the prognostic significance of CLIC4 expression using two independent large cohorts of cytogenetically normal AML (CN-AML) patients. Multivariable analysis and multi-omics analysis with weighted correlation network analysis (WGCNA) in the CN-AML group were also presented. Based on CLIC4 and its related genes, microRNA–target gene interaction network analysis and downstream gene ontology analysis were performed to unveil the complex functions behind CLIC4. Results: We demonstrated that the overexpression of CLIC4 was notably associated with unfavorable outcome in the two independent cohorts of CN-AML patients [overall survival (OS) and event-free survival (EFS): P < 0.0001, n = 185; OS: P = 0.016, n = 232], as well as in the European LeukemiaNet (ELN) Intermediate-I group (OS: P = 0.015, EFS: P = 0.012, n = 115), the National Comprehensive Cancer Network Intermediate Risk AML group (OS and EFS: P < 0.0001, n = 225), and the non-M3 AML group (OS and EFS: P < 0.0001, n = 435). Multivariable analysis further validated CLIC4 as a high-risk factor in the CN-AML group. Multi-omics analysis presented the overexpression of CLIC4 as associated with the co-expression of the different gene sets in leukemia, up/downregulation of the immune-related pathways, dysregulation of microRNAs, and hypermethylation around the CpG islands, in open sea regions, and in different gene structural fragments including TSS1500, gene body, 5′UTR region, 3′UTR region, and the first exon. By further performing WGCNA on multi-omics data, certain biomarkers that are co-expressed with CLIC4 were also unveiled. Conclusion: We demonstrated that CLIC4 is a novel, potential unfavorable prognosticator and therapeutic target for CN-AML. As having a key role in CN-AML, the interactions between CLIC4 and other genomics and transcriptomics data were confirmed by performing microRNA–target gene interaction network analysis and gene ontology enrichment analysis. The experimental result provides evidence for the clinical strategy selection of CN-AML patients. [ABSTRACT FROM AUTHOR]

Chloride intracellular channel 4 (CLIC4) functions in diverse actin‐dependent processes. Upon Rho activation, CLIC4 reversibly translocates from the cytosol to the plasma membrane to regulate cell adhesion and migration. At the plasma membrane, CLIC4 counters the formation of filopodia, which requires actin assembly by the formin mammalian Diaphanous (mDia)2. To this end, mDia2 must be activated through conversion from the closed to the open conformation. Thus, CLIC4 could harness the activation or the open conformation of mDia2 to inhibit filopodium formation. Here, we find that CLIC4 silencing enhances the filopodia induced by two constitutively active mDia2 mutants. Furthermore, we report that CLIC4 binds the actin‐regulatory region of mDia2 in vitro. These results suggest that CLIC4 modulates the activity of the open conformation of mDia2, shedding new light into how cells may control filopodia. [ABSTRACT FROM AUTHOR]

Endothelial cell apoptosis plays an essential role in the pathogenesis of atherosclerosis. MicroRNAs and chloride intracellular channels (CLICs) have been verified to participate in the endothelial cell apoptosis process, however, the underlying molecular mechanisms are still unclear. The main aim of this study was to investigate the biological effects of microRNA-217-5p (miR-217-5p) and CLIC4 on endothelial cell apoptosis in atherosclerosis. An atherosclerotic mouse model (n = 18) was constructed by feeding apolipo protein E knockout ApoE(−/−) mice with high-fat diet for 12 weeks. An atherosclerotic cell model was established by treating human aortic endothelial cells with oxidized low-density lipoprotein (ox-LDL; 50 μg/mL) for 24 h. Quantitative real-time polymerase chain reaction and immunofluorescent staining confirmed the downregulation of miR-217-5p and upregulation of CLIC4 in atherosclerotic endothelial cells. Combined with western blot, flow cytometry assay and Hoechst staining, we demonstrated that miR-217-5p upregulation or CLIC4 knockdown regulated the apoptosis-related genes, ameliorated mitochondrial membrane permeability and therefore inhibited the apoptosis of aortic endothelial cells induced by ox-LDL. We further confirmed that miR-217-5p inhibited apoptosis of endothelial cells through targeting CLIC4 using luciferase report assay and rescue experiments. We revealed for the first time that miR-217-5p inhibited apoptosis of endothelial cells in atherosclerosis and identified CLIC4 as a novel target of miR-217-5p. Our work provides a potential therapeutic approach for the treatment of atherosclerosis. • It is confirmed that miR-217-5p is downregulated and CLIC4 is upregulated in both atherosclerotic mouse model and cell model. • It is verified that upregulation of miR-217-5p or CLIC4 knockdown inhibits apoptosis of atherosclerotic endothelial cells. • It is identified for the first time that CLIC4 is a direct target of miR-217-5p. • We verify for the first time that miR-217-5p inhibits apoptosis of endothelial cells through targeting CLIC4. [ABSTRACT FROM AUTHOR]

The altered expression of chloride intracellular channel 4 (CLIC4) was reported to correlate with tumor progression. Previously, we have shown that the reduced cellular invasion induced by photodynamic therapy (PDT) is associated with suppression of CLIC4 expression in PDT-treated cells. Herein, we attempted to decipher the regulatory mechanisms involved in PDT-mediated CLIC4 suppression in A375 and MDA-MB-231 cells in vitro. We found that PDT can increase the expression and enzymatic activity of DNA methyltransferase 1 (DNMT1). Bisulfite sequencing PCR further revealed that PDT can induce hypermethylation in the CLIC4 promoter region. Silencing DNMT1 rescues the PDT-induced CLIC4 suppression and inhibits hypermethylation in its promoter. Furthermore, we found tumor suppressor p53 involves in the increased DNMT1 expression of PDT-treated cells. Finally, by comparing CLIC4 expression in lung malignant cells and normal lung fibroblasts, the extent of methylation in CLIC4 promoter was found to be inversely proportional to its expression. Taken together, our results indicate that CLIC4 suppression induced by PDT is modulated by DNMT1-mediated hypermethylation and depends on the status of p53, which provides a possible mechanistic basis for regulating CLIC4 expression in tumorigenesis.

Objectives: Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic β-cell apoptosis. Methods: We used βTC-tet cells and islets from β-cell specific Clic4 knockout mice (βClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. Results: We show that cytokines increased Clic4 expression in βTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in βTC-tet cells or its genetic inactivation in islets β-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in βTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets β-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from βTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. Conclusions: Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes β-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.

Background/purpose: 5-Aminolevulinic acid mediated photodynamic therapy (ALA-PDT) has been used for the treatment of precancerous lesions and oral cancers. Compared with traditional treatment such as surgery, ALA-PDT can selectively kill cancer cells and reduce side effects. However, some cells might survive from PDT and may be directly attributable to the limited penetration of light. Therefore, it is necessary to elucidate alterations in cellular behavior and molecular mechanisms in cancer cells that have survived from PDT. Materials and methods: Human tongue squamous carcinoma cells, SCC-4 cells, and mice bearing C26 tumors were used as cancer models in vitro and in vivo. After irradiation with the LD50 (50% lethal dose) light dose, the ability of cellular migration and metastasis related gene expression were assayed. Results: Cells were rounded up and migration ability was shown to have significantly decreased under the influence of irradiation with the LD50 light dose. Gene expression related to metastasis including matrix metalloproteinases (MMPs) and chloride intracellular channel 4 (CLIC4) was also reduced. In addition, in the animal tumor model, mRNA expression of MMP9 and CLIC4 was also noticeably decreased. Conclusion: Our results indicate that PDT could reduce the expression of invasion related genes of surviving cancer cells in vitro as well as in vivo. These could be used as references for clinical PDT of oral cancer in the future.

Background: Premature birth is a significant health care burden. Xenon (Xe) is a general anesthetic with neuroprotective effects. Objectives: Here, we investigate the neuroprotective role of Xe in a lipopolysaccharide (LPS)- and hypoxia-ischemia (HI)-induced white matter damage (WMD) model. Methods: Three-day-old Sprague-Dawley rats were randomly divided into a sham group (group A, n = 24), an LPS + HI group (group B, n = 24), and an LPS + HI + Xe group (group C, n = 72). The onset of Xe inhalation started at 0, 2, and 5 h in subgroups C1, C2, and C3, respectively. Next, we performed TUNEL and hematoxylin and eosin (HE) staining; and examined the expression of CLIC4 and Bcl-2 in brain tissues. Results: HE staining revealed distorted cytoarchitecture, tangled nerve fibers, and pyknosis in group B, while Xe treatment improved these histological alterations in the group C pups. Following LPS and HI insult, the number of apoptotic cells significantly increased in group B at 48 and 72 h (p < 0.05), and Xe significantly alleviated apoptosis (p < 0.001) at 24, 48, and 72 h, respectively. Similarly, CLIC4 mRNA expression was significantly increased in group B (p < 0.05), and Xe produced a marked reduction in CLIC4 mRNA expression in group C subgroups (p < 0.05). Western blotting demonstrated enhanced Bcl-2 expression in group C when compared to group B (p < 0.05). Conclusions: These results demonstrate that LPS and HI successfully induced WMD, and Xe decreased neuronal apoptosis via Bcl-2- and CLIC4-mediated pathways. Moreover, the therapeutic time window of Xe extended for up to 5 h. These findings suggest that Xe can be used as a protective treatment for WMD in premature infants. [ABSTRACT FROM AUTHOR]

Chloride intracellular channel proteins (CLICs) display ubiquitous expression, with each member exhibiting specific subcellular localisation. While all CLICs, except CLIC3, exhibit a highly conserved putative nuclear localisation sequence (NLS), only CLIC1, CLIC3 and CLIC4 exist within the nucleus. The CLIC4 NLS, 199-KVVAKKYR-206, appears crucial for nuclear entry and interacts with mouse nuclear import mediator Impα isoform 1, omitting the IBB domain (mImpα1ΔIBB). The essential nature of the basic residues in the CLIC4 NLS has been established by the fact that mutating out these residues inhibits nuclear import, which in turn is linked to cutaneous squamous cell cancer. Given the conservation of the CLIC NLS, CLIC1 likely follows a similar import pathway to CLIC4. Peptides of the CLIC1 (Pep1; Pep1&#95;S C/S mutant) and CLIC4 (Pep4) NLSs were designed to examine binding to human Impα isoform 1, omitting the IBB domain (hImpα1ΔIBB). Molecular docking indicated that the core CLIC NLS region (KKYR) forms a similar binding pattern to both mImpα1ΔIBB and hImpα1ΔIBB. Fluorescence quenching demonstrated that Pep1&#95;S (K d  ≈ 237 μM) and Pep4 (K d  ≈ 317 μM) bind hImpα1ΔIBB weakly. Isothermal titration calorimetry confirmed the weak binding interaction between Pep4 and hImpα1ΔIBB (K d  ≈ 130 μM) and the presence of a proton-linked effect. This weak interaction may be due to regions distal from the CLIC NLS needed to stabilise and strengthen hImpα1ΔIBB binding. Additionally, this NLS may preferentially bind another hImpα isoform with different flexibility properties., (© 2022 The Authors. Journal of Molecular Recognition published by John Wiley & Sons Ltd.)

In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled “Membrane Channels and Transporters in Cancers.” [ABSTRACT FROM AUTHOR]

The number and diameter of native collaterals in tissues of healthy mice vary widely, resulting in large differences in tissue injury in occlusive diseases. Recent studies suggest similar variation may exist in humans. Collateral variation in mice is determined by genetic background- dependent differences in embryonic collateral formation, by variation in maturation of the nascent collaterals, and by environmental factors such as aging that cause collateral rarefaction in the adult. Recently, formation of the collateral circulation in the brain was found to involve a unique VEGF-A-dependent "arteriolar" angiogenic sprouting-like mechanism. Elsewhere, chloride intracellular protein 4 (CLIC4) was implicated but not investigated directly, prompting the present study. Deletion of Clic4 had no effect on embryonic collaterogenesis. However, during collateral maturation from embryonic day 18.5 to postnatal day 7, reduced mural cell investment was observed and excessive pruning of collaterals occurred. Growth in collateral diameter was reduced. This resulted in 50% fewer collaterals of smaller diameter in the adult and thus larger infarct volume after middle cerebral artery occlusion. During collateral maturation, CLIC4 deficiency resulted in reduced expression of Vegfr2, Vegfr1, Vegfc, and mural cell markers, but not notch-pathway genes. Overexpression of VEGF-A in Clic4-/- mice had no effect on collaterogenesis, but rescued the above defects in collateral maturation by preventing mural cell loss and collateral pruning, thus restoring collateral number and diameter and reducing stroke severity in the adult. CLIC4 is not required for collaterogenesis but is essential for perinatal maturation of nascent collaterals through a mechanism that supports VEGF signaling. [ABSTRACT FROM AUTHOR]

Objectives Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic β-cell apoptosis. Methods We used βTC-tet cells and islets from β-cell specific Clic4 knockout mice (βClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. Results We show that cytokines increased Clic4 expression in βTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in βTC-tet cells or its genetic inactivation in islets β-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in βTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets β-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from βTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. Conclusions Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes β-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad. [ABSTRACT FROM AUTHOR]

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic -- endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum-or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA- dependent integrin trafficking. [ABSTRACT FROM AUTHOR]

Background Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members. Results We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells. Conclusions In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously. [ABSTRACT FROM AUTHOR]

Background CLIC4, a member of the CLIC family of proteins, was recently demonstrated to translocate to the nucleus in differentiating keratinocytes where it potentiates TGFβ-driven gene regulation. Since TGFβ signaling is known to play important roles in the fibrotic response to acute kidney injury, and since CLIC4 is abundantly expressed in kidney, we hypothesized that CLIC4 may play a role in the response to acute kidney injury. Methods Previously described Clic4 null mice were analyzed for the effect of absence of CLIC4 on growth, development and response to kidney injury. Kidney size, glomerular counts and density of peritubular capillaries of matched WT and Clic4 null mice were determined. Cohorts of WT and Clic4 null mice were subjected to the folic acid model of acute kidney injury. Extent of acute injury and long term functional recovery were assessed by plasma blood urea nitrogen (BUN); long term fibrosis/scarring was determined by histochemical assessment of kidney sections and by residual renal mass. Activation of the TGFβ signaling pathway was assessed by semi-quantitative western blots of phosphorylated SMADs 2 and 3. Results CLIC4 is abundantly expressed in the apical pole of renal proximal tubule cells, and in endothelial cells of glomerular and peritubular capillaries. CLIC4 null mice are small, have smaller kidneys with fewer glomeruli and less dense peritubular capillary networks, and have increased proteinuria. The Clic4 null mice show increased susceptibility to folic acid-induced acute kidney injury but no difference in recovery from acute injury, no nuclear redistribution of CLIC4 following injury, and no significant difference in activation of the TGFβ-signaling pathway as reflected in the level of phosphorylation of SMADs 2 and 3. Conclusions Absence of CLIC4 results in morphologic changes consistent with its known role in angiogenesis. These changes may be at least partially responsible for the increased susceptibility to acute kidney injury. However, the absence of CLIC4 has no significant impact on the extent of functional recovery or fibrosis following acute injury, indicating that CLIC4 does not play a major non-redundant role in the TGFβ signaling involved in response to acute kidney injury. [ABSTRACT FROM AUTHOR]

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H 2 O 2 as a ROS-inducing agent. In intact cells, H 2 O 2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H 2 O 2 -induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H 2 O 2 -treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells., Competing Interests: Conflict of interest Authors declare that there are no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)