Additional file 1 of Complement inhibitor CSMD1 modulates epidermal growth factor receptor oncogenic signaling and sensitizes breast cancer cells to chemotherapy

Additional file 1: S.Figure 1 Expression of mRNA coding for (A) EGF, (B) TGF-α and (C) AREG in MDA-MB-231 CTRL and CSMD1 clonal cells. EGFR gene expression (FPKM) plotted against CSMD1 (FPKM) gene expression in (D) all BC patients and in (E) TNBC patients of SCAN-B cohort (F-G) Protein extracts of MDA-MB-231 BCCs were immunoprecipitated with anti-EGFR. Eluted proteins were analyzed by immunoblotting with (F) anti-phosphotyrosine (pTyr) or anti-EGFR antibody and (G) anti-phosphoserine (pSer) or anti-EGFR antibody, as indicated. (H & I) Densitometric western blot analysis of total phosphorylation tyrosine and serine residues of EGFR. Bars display mean ± SD. Mann–Whitney comparison test was used (*<0.05). (J) Binding assays with 125I-labeled EGF in CTRL and CSMD1 MDA-MB-231 BCCs. All experiments were repeated at least 3 times with bars indicating mean ± SD, grey circles correspond to independent data points for CTRL and CSMD1 groups, respectively. S.Figure 2. (A) Ubiquitinated EGFR was examined via EGFR immunoprecipitation followed by immunoblotting with anti-ubiquitin antibody in denaturing lysates. Representative blots from three independent experiments are presented in CTRL and CSMD1 MDA-MB-231 BCCs. (B) EGFR internalization kinetics using 125I-EGF in MDA-MB-231 BCCs. The amounts of internalized and surface 125I-EGF (cpm) where plotted against time upper panel, while the ratio of internalized/surface EGF against time was used to calculate the internalization rate constant ke. (C) Fractionation analysis in cytosol and membrane of CTRL and CSMD1 MDA-MB-231 BCCs upon stimulation with EGF (25 ng/mL) for 2h. Representative blots are shown. The fractions were blotted for CSMD1, EGFR, EEA1, LAMP1, β-tubulin and NA/K ATPase (D) Ratio of cytosolic to membrane EGFR was calculated. Bars display mean ± SD. S. Figure 3 Validation of the major findings in BT-20 TNBC cell line (A) Cell lysates were immunoprecipitated using antibodies against CSMD1 or corresponding IgG control followed by immunodetection of EGFR and CSMD1 in BT-20 cells. (B) Immunoblot analysis of phosphorylated EGFR at the residue Y1068, total EGFR and β-tubulin used as a loading control. (C) Densitometry of pEGFR Y1068 normalized to total EGFR. (D) Densitometry of pEGFR Y1068 normalized to β-tubulin, and (E) densitometry of total EGFR normalized to β-tubulin. (F) Immunoblot analysis of phosphorylated Akt at the residue Ser473 (pAkt Ser473), total Akt and GAPDH used as a loading control in BT-20 cells. (G) Densitometry of pAkt Ser473 normalized to total Akt and (H) densitometry of pAkt Ser473 normalized to GAPDH in BT-20 cells. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing 3 or more groups with 2 variables (*<0.05). (I) EGFR degradation: representative immunoblots of EGFR levels in lysates of BT-20 CTRL and CSMD1 overexpressing cells, pre-treated with translation inhibitor cycloheximide (100 g/mL) for 2 h, followed by EGF stimulation (25 ng/ml) for 0, 2, 4, 8 hours (h). (J) Quantification of EGFR levels in MDA-MB-231 cells plotted against time. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing CTRL and CSMD1 groups (*<0.05, **<0.01). (K) BT-20 CTRL and CSMD1 cells were treated with different chemotherapy agents (doxorubicin and epirubicin) for 48h, and apoptosis was monitored using annexin V staining while live/dead cell discrimination was performed with Zombie Aqua staining, both using flow cytometry. Bar graphs showing percentages of late apoptotic cells. All experiments were repeated at least 3 times with bars indicating mean ± SD, grey circles correspond to independent data points for CTRL and CSMD1 groups, respectively. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing CTRL and CSMD1 groups in different concentration of chemotherapy drugs or treatments. S. Figure 4 Validation of the major findings in MDA-MB-468 TNBC cell line (A) Immunoblot analysis of phosphorylated EGFR at the residue Y1068, total EGFR and β-tubulin in MDA-MB-468 cells. (B) Densitometry of pEGFR-Y1068 normalized to total EGFR. (C) Densitometry of pEGFR-Y1068 normalized to β-tubulin, and (D) densitometry of total EGFR normalized to β-tubulin. (E) Immunoblot analysis of phosphorylated Akt at the residue Ser473 (pAkt-Ser473), total Akt and GAPDH in MDA-MB-468 cells. (F) Densitometry of pAkt-Ser473 normalized to total Akt and (G) densitometry of pAkt-Ser473 normalized to GAPDH. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing 3 or more groups with 2 variables (*<0.05). (H) EGFR degradation in MDA-MB-468 breast cancer cell line: detection of EGFR in lysates of MDA-MB-468 CTRL and CSMD1-overexpressing cells, pre-treated with translation inhibitor cycloheximide (100 g/mL) for 2 h followed by EGF stimulation (25 ng/ml) for 0, 2, 4, 8 hours (h). (I) Quantification of EGFR levels in MDA-MB-468 cells plotted against time. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing CTRL and CSMD1 groups (*<0.05, **<0.01). (J) MDA-MB-468 CTRL and CSMD1-overexpressing cells were treated with different chemotherapy agents (doxorubicin and epirubicin) for 48h, and apoptosis was monitored using annexin V staining while live/dead cell discrimination was performed with Zombie Aqua staining, both using flow cytometry. All experiments were repeated at least 3 times with bars indicating mean ± SD, grey circles correspond to independent data points for CTRL and CSMD1 groups, respectively. Bar graphs show percentages of late apoptotic cells. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing CTRL and CSMD1 groups in different concentration of chemotherapy drugs or treatments. S. Figure 5 (A) Drug efflux activity in MDA-MB-231 CSMD1-overexpressing cells and CTRL cells. Representative histograms are shown. (B) Bar graphs showing gMFI of intracellular doxorubicin content in MDA-MB-231 CSMD1 and CTRL cells. Student’s t-test was used when comparing CTRL and CSMD1 (C) Representative immunoblots of autophagy markers p62 and LC3B in lysates of MDA-MB-231 CTRL and CSMD1 cells upon treatment with GEF and CQ and combination of them for 24h. All experiments were repeated at least 3 times with bars indicating mean ± SD, grey circles correspond to independent data points for CTRL and CSMD1 groups, respectively. S. Figure 6 Frequency (%) of CSMD1 expression in (A) all BC patients and in (B) TNBC patients of SCAN-B cohort. (C) Box plot of EGFR gene expression for tumor samples of SCAN-B cohort subset of TNBC stratified according to basal and non-basal like properties. Table S1 List of antibodies used in this study.