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Table S2. p65BTK mRNA but not p77BTK mRNA is expressed in NSCLC cell lines. mRNA expression was evaluated by RT-PCR using primers specific for each of the two isoforms [18]. (PDF 90 kb)

Figure S3. p65BTK expression in T cells. FACS-purified CD3 cell lysate was tested for p65BTK expression by BN49 antibody and for p77BTK expression using the anti-BTK (#611117, from Becton Dickinson. 100 pg of purified p77BTK (#B4312, Sigma-Aldrich) were also loaded as a positive control. (PDF 469 kb)

Figure S5. Cell death triggered by the combination of BTK inhibitors and target therapy or SOC chemotherapy is apoptosis. a Caspase-3/7 activation after 24 hs treatment of SK-Lu-1 and NCI-H2228 cells with the indicated drugs, as assessed by luminometric assay. Error bars represent mean ± SEM. n = 3. b Cell viability of SK-Lu-1 and NCI-H2228 cell lines in response to combinations of BTK inhibitor RN486 and EGFR inhibitor Gefitinib or Cisplatin. (T0 = time 0; NT = untreated; RN10 = RN486 10 μM; QVD 10 = Q-VD-OPh 10 μM; GEF20 = Gefitinib 20 μM; CIS10 = Cisplatin 10 μM). X-axis crosses in correspondence of T0 values (before starting the treatment); 72 h values are then expressed as the percentage variation relative to the initial cell number. Data are presented as mean ± SEM. n ≥ 3 independent experiments. (PDF 301 kb)

Figure S1. BN30 antibody characterization. a Western blot analysis of lysates from HCT116p53KO cells harvested 48hs after transfection with control (Luc) or p65BTK-specific (BTK) siRNA and used to produce cells blocks. b IHC using BN30 on slides from cells blocks; bar: 50 μM. 40X magnification. c Western blot analysis of lysates from SW480 cells harvested 48hs after transfection with control (Luciferase) or p65BTK-specific (BTK) siRNA and from B-cell lymphoblastic leukemia cell line Nalm-6, which expresses both p65 and p77BTK. BD#611117: anti-BTK antibody from Becton Dickinson raised against the N-term of the protein and not cross-reacting with p65BTK (PDF 1492 kb)

Figure S1. BN30 antibody characterization. a Western blot analysis of lysates from HCT116p53KO cells harvested 48hs after transfection with control (Luc) or p65BTK-specific (BTK) siRNA and used to produce cells blocks. b IHC using BN30 on slides from cells blocks; bar: 50 μM. 40X magnification. c Western blot analysis of lysates from SW480 cells harvested 48hs after transfection with control (Luciferase) or p65BTK-specific (BTK) siRNA and from B-cell lymphoblastic leukemia cell line Nalm-6, which expresses both p65 and p77BTK. BD#611117: anti-BTK antibody from Becton Dickinson raised against the N-term of the protein and not cross-reacting with p65BTK (PDF 1492 kb)

Table S1. Clinicopathological characteristics of NSCLC patients (n = 383). LGT: Lepidic growth type; AdC: adenocarcinoma; LC, large cell AdC; SCC: squamous cell carcinoma; AdC/SCC, mixed adeno-squamous carcinoma; R, rearranged. pTx or pNx, this information could not be established. (PDF 72 kb)

Figure S4. p65BTK is overexpressed and active in NSCLC cell lines scarcely responsive to EGFR inhibition. a Immunofluorescence staining of phosphorylated p65BTK (pBTK) in untreated human p53-null NSCLC cell lines. Nuclei were counterstained with DAPI. b Western blot analysis of phosphorylated p65BTK in NSCLC cell lines. 100 pg of purified activated form p77BTK (#B4312, Sigma-Aldrich) were also loaded as a positive control. Expression levels of total p65BTK were assessed by BN49. Vinculin was used as a loading control. c Immunofluorescence staining of pBTK after 2 h treatment of SK-Lu-1 cells with BTK inhibitors (IBRU20 = Ibrutinib 20 μM; AVL10 = AVL-292 10 μM; RN10 = RN486 10 μM). d Western blot analysis of phosphorylated p65BTK in SK-Lu-1 cells after 2 h treatment with BTK inhibitors (IBRU20 = Ibrutinib 20 μM; AVL10 = AVL-292 10 μM; RN10 = RN486 10 μM. Expression levels of total p65BTK were assessed by BN49. Vinculin was used as a loading control. (PDF 3221 kb)

Table S1. Clinicopathological characteristics of NSCLC patients (n = 383). LGT: Lepidic growth type; AdC: adenocarcinoma; LC, large cell AdC; SCC: squamous cell carcinoma; AdC/SCC, mixed adeno-squamous carcinoma; R, rearranged. pTx or pNx, this information could not be established. (PDF 72 kb)

Figure S5. Cell death triggered by the combination of BTK inhibitors and target therapy or SOC chemotherapy is apoptosis. a Caspase-3/7 activation after 24 hs treatment of SK-Lu-1 and NCI-H2228 cells with the indicated drugs, as assessed by luminometric assay. Error bars represent mean ± SEM. n = 3. b Cell viability of SK-Lu-1 and NCI-H2228 cell lines in response to combinations of BTK inhibitor RN486 and EGFR inhibitor Gefitinib or Cisplatin. (T0 = time 0; NT = untreated; RN10 = RN486 10 μM; QVD 10 = Q-VD-OPh 10 μM; GEF20 = Gefitinib 20 μM; CIS10 = Cisplatin 10 μM). X-axis crosses in correspondence of T0 values (before starting the treatment); 72 h values are then expressed as the percentage variation relative to the initial cell number. Data are presented as mean ± SEM. n ≥ 3 independent experiments. (PDF 301 kb)

Figure S2. p65BTK expression in NSCLC or AdC non-smoker patients stratified by ALK translocation (T) (PDF 247 kb)

Figure S3. p65BTK expression in T cells. FACS-purified CD3 cell lysate was tested for p65BTK expression by BN49 antibody and for p77BTK expression using the anti-BTK (#611117, from Becton Dickinson. 100 pg of purified p77BTK (#B4312, Sigma-Aldrich) were also loaded as a positive control. (PDF 469 kb)

Figure S2. p65BTK expression in NSCLC or AdC non-smoker patients stratified by ALK translocation (T) (PDF 247 kb)

Figure S4. p65BTK is overexpressed and active in NSCLC cell lines scarcely responsive to EGFR inhibition. a Immunofluorescence staining of phosphorylated p65BTK (pBTK) in untreated human p53-null NSCLC cell lines. Nuclei were counterstained with DAPI. b Western blot analysis of phosphorylated p65BTK in NSCLC cell lines. 100 pg of purified activated form p77BTK (#B4312, Sigma-Aldrich) were also loaded as a positive control. Expression levels of total p65BTK were assessed by BN49. Vinculin was used as a loading control. c Immunofluorescence staining of pBTK after 2 h treatment of SK-Lu-1 cells with BTK inhibitors (IBRU20 = Ibrutinib 20 μM; AVL10 = AVL-292 10 μM; RN10 = RN486 10 μM). d Western blot analysis of phosphorylated p65BTK in SK-Lu-1 cells after 2 h treatment with BTK inhibitors (IBRU20 = Ibrutinib 20 μM; AVL10 = AVL-292 10 μM; RN10 = RN486 10 μM. Expression levels of total p65BTK were assessed by BN49. Vinculin was used as a loading control. (PDF 3221 kb)