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Figure S2. Secretome analysis of PSC-conditioned medium. (A) Flow-chart describing procedure for conditioned medium collection, processing and proteome analysis of the PSC secretome. (B) Conditioned medium collected from ten different PSC cultures were subjected to proteomic analysis by LC-MS/MS. Graph indicates the number of proteins identified with high confidence from each sample. (C) The proteins detected by LC-MS/MS were interrogated in terms of functional annotation by DAVID Bioinformatics Resource tool. The representative GO terms cluster groups with top 10 enrichment score are presented. The horizontal axis represents the significance (p value) for each term, while the vertical axis represents the GO categories for biological processes. GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes; PSC, pancreatic stellate cell; PSC-CM, PSC-conditioned medium; SFM, serum-free DMEM; STRING, search tool for the retrieval of interacting genes/proteins. (PPTX 3662 kb)

Figure S1. Experimental set-up for co-culture experiments. Monolayer of PCCs (A), PSCs (B), direct co-culture of PCCs with PSCs (C) and indirect co-culture of PSCs with PCCs (D). PCC, pancreatic cancer cell; PSC, pancreatic stellate cell. (PPTX 61 kb)

Figure S5. Both FN-inhibitor (RGDS) and ERK-inhibitor (PD98059) block PSC-CM induced chemoresistance to gemcitabine. PCCs seeded on 96-well plates were incubated with SFM or PSC-CM for 24 h and/or RGDS (20 μM) or PD98059 (20 μM) for 4 h prior to incubation with gemcitabine (10 μM) for 48 h. Cell viability was determined using the MTT assay. Data are the mean ± SEM of triplicate determinations. *p

Table S1. Clinicopathological features of pancreatic adenocarcinoma (PADC) tumors. (DOCX 14 kb)

Background & Aims Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide. Currently, no specific pharmacologic therapy is available for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies in genetic mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine protein kinase (STK)25 as a critical regulator of hepatic lipid partitioning and NAFLD/NASH. Here, we studied the metabolic benefit of liver-specific STK25 inhibitors on NAFLD development and progression in a mouse model of diet-induced obesity. Methods We developed a hepatocyte-specific triantennary N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotide (ASO) targeting Stk25 and evaluated its effect on NAFLD features in mice after chronic exposure to dietary lipids. Results We found that systemic administration of hepatocyte-targeting GalNAc-Stk25 ASO in obese mice effectively ameliorated steatosis, inflammatory infiltration, hepatic stellate cell activation, nutritional fibrosis, and hepatocellular damage in the liver compared with mice treated with GalNAc-conjugated nontargeting ASO, without any systemic toxicity or local tolerability concerns. We also observed protection against high-fat-diet–induced hepatic oxidative stress and improved mitochondrial function with Stk25 ASO treatment in mice. Moreover, GalNAc-Stk25 ASO suppressed lipogenic gene expression and acetyl-CoA carboxylase protein abundance in the liver, providing insight into the molecular mechanisms underlying repression of hepatic steatosis. Conclusions This study provides in vivo nonclinical proof-of-principle for the metabolic benefit of liver-specific inhibition of STK25 in the context of obesity and warrants future investigations to address the therapeutic potential of GalNAc-Stk25 ASO in the prevention and treatment of NAFLD., Graphical abstract

In past decade, type 2 diabetes (T2D) and associated metabolic complications have become a major global threat for human health with epidemic increase in incidence. Currently available treatments for T2D have several limitations and side effects making it difficult safely reach adequate metabolic control. Therefore, it is important to identify novel therapeutic targets for metabolic regulation, which could complement current treatments for T2D. T2D is closely associated with ectopic lipid deposition. Recent evidence suggests that hepatic lipid deposition is not merely a consequence of the metabolic syndrome but rather that non-alcoholic fatty liver disease (NAFLD), and progression to non-alcoholic steatohepatitis (NASH), exacerbate hepatic and systemic insulin resistance and actively contribute to the pathogenesis of the metabolic syndrome. However, no pharmacological treatment is approved for NAFLD/NASH, to date. Serine/threonine protein kinase 25 (STK25) is broadly expressed in mouse, rat and human tissues. Previous studies by our research group in the rat myoblast cell line L6 by small interfering RNA (siRNA) have shown that STK25 is involved in regulation of glucose uptake and lipid oxidation. Studies presented in this thesis work demonstrate that the transgenic mice overexpressing STK25 challenged with a high-fat diet display a shift in the metabolic balance in peripheral tissues from lipid oxidation to lipid storage, resulting in a systemic insulin resistance. In contrast, Stk25 knockout mice show better-preserved systemic insulin sensitivity via an opposite shift in the metabolic balance in peripheral tissues from lipid storage to lipid utilization. We found that STK25 coats lipid droplets in mouse liver and human hepatocytes. Our studies performed in mouse liver, and in hepatocytes from both mouse and human, suggest that STK25 regulates hepatic lipid partitioning by controlling β-oxidation and very low-density lipoprotein (VLDL)-triacylglycerol (TAG) secretion. Increased activity of STK25 reduces liver lipolytic capacity, which in turn results in lower availability of lipids for hepatic β-oxidation and VLDL-TAG secretion, leading to enhanced lipid storage. Upon inactivation of STK25 reciprocal increase in liver lipolytic capacity, β-oxidation and VLDL-TAG secretion was seen, which likely account for the reduced hepatic lipid storage. We also found a statistically significant positive correlation between STK25 mRNA expression in human liver biopsies and hepatic fat content. Taken together, our studies suggest that inhibition of STK25 enables the reduction of ectopic lipid deposition and improves insulin sensitivity and glucose utilization in peripheral tissues. Our studies highlight STK25 as a potential drug target for prevention and/or treatment of T2D, NAFLD and NASH.