S. 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, -tubulin and NA/K ATPase (D) Ratio of cytosolic to membrane EGFR was calculated. Bars display mean SD. S. Physique 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. Alendronate sodium hydrate (B) Immunoblot analysis of phosphorylated EGFR at the residue Y1068, total EGFR and -tubulin used as a loading control. (C) Densitometry of pEGFR Y1068 normalized to total EGFR. (D) Densitometry of pEGFR Y1068 normalized to -tubulin, and (E) densitometry of total EGFR normalized to -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 Alendronate sodium hydrate Ser473 normalized to total Akt and (H) densitometry of pAkt Ser473 normalized to GAPDH in BT-20 cells. A Alendronate sodium hydrate two-way ANOVA Bonferronis 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 ICAM4 levels in MDA-MB-231 cells plotted against time. A two-way ANOVA Bonferronis 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 brokers (doxorubicin and epirubicin) for 48h, and apoptosis was monitored using annexin V staining while live/lifeless 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 impartial data points for CTRL and CSMD1 groups, respectively. A two-way ANOVA Bonferronis multiple comparisons test was used when comparing CTRL and CSMD1 groups in different concentration of chemotherapy drugs or treatments. S. Physique 4 Validation of the major findings in MDA-MB-468 Alendronate sodium hydrate TNBC cell line (A) Immunoblot analysis of phosphorylated EGFR at the residue Y1068, total EGFR and -tubulin in MDA-MB-468 cells. (B) Densitometry of pEGFR-Y1068 normalized to total EGFR. (C) Densitometry of pEGFR-Y1068 normalized to -tubulin, and (D) densitometry of total EGFR normalized to -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 Bonferronis multiple comparisons test was used when comparing 3 or more groups Alendronate sodium hydrate 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 Bonferronis 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 brokers (doxorubicin and epirubicin) for 48h, and apoptosis was monitored using annexin V staining while live/lifeless 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 Bonferronis multiple comparisons test was used when you compare CTRL and CSMD1 organizations in different focus of chemotherapy medicines or remedies. S. Shape 5 (A) Medication efflux activity in MDA-MB-231 CSMD1-overexpressing cells and CTRL cells. Representative histograms are demonstrated. (B) Pub graphs displaying gMFI of intracellular doxorubicin content material in MDA-MB-231 CSMD1 and CTRL cells. College students.