Figure 5(A) Immunoblots analysis was performed to determine CEP55 levels in MDA-MB-231 cells treated with multiple inhibitors targeting the EGFR/HER2 pathway for 24 hour. The following inhibitors were used: MEK1/2i (AZD6244 (1 µM)), the AKT, PI3K/mTORi (BEZ235 (0.5 µM), AKTi VIII (1 µM)), EGFRi and HER2i (Erlotinib (1 µM), Afatinib (0.25 µM), Lapatinib (1 µM), Trastuzumab (10 µg/ml) or the pan VEGFR, PDGFR and RAF kinases (Sorafinib, (1 µM)). COX-IV as a loading control.(B) Immunoblots analysis of MDA-MB-231 cells treated with three different MEK1/2 inhibitors at various concentrations (Selumetinib AZD6244, Trametinib GSK1120212 and Binimetinib MEK162) after 24h. COX-IV as a loading control.(C) Relative CEP55 promoter luciferase activity in MDA-MB-231 cells either treated with AZD6244 (1 µM) for 6 hours or ERK1/2 knockdown for 24 hours. PGL basic reporter plasmid was used to normalize basal CEP55 promoter activity. Graph represents the mean±SEM of two independent experiments.(D) Immunoblot analysis of MDA-MB-231 cells cultured in 0.1% fetal bovine serum for 24h and stimulated with EGF (100 ng/mL) for the indicated time points. Phosphorylation of EGFR, ERK1/2 along with CEP55 and MYC were determined. COX-IV as a loading control.(E) Relative CEP55 promoter luciferase activity upon MYC siRNA was determined using DualGlo assay in MDA-MB-231 cells similar to experiment in panel C. Graph represents the mean±SEM of two independent experiments.(F) Immunoblots analysis showing MYC and CEP55 expression in MDA-MB-231 cells upon transfection with siRNA against MYC, and ETS1 (10 nM), with and without EGF (100 ng/mL) stimulation as indicated time points. COX-IV served as a loading control.(G) Immunoblots analysis showing CEP55 and MYC levels following 4-hydroxytamoxifen (4OHT) (0.5 µM) induction in MCF10A MYCER cells cultured in 0.1% fetal bovine serum contained media at indicated time points. COX-IV served as a loading control.(H) Analysis of Pearson correlation coefficients (PCC) of CEP55 vs. MYC levels with R=0.9982, p=0.0016. Protein band intensities as shown in H were measured using ImageJ software.(I) Analysis of Pearson correlation coefficients (PCC) of CEP55 vs. MYC transcriptional activity in breast cancer TCGA tumor data with R=0.4157, p<0.0001.(J) Both control and CEP55 knockdown MDA-MB-231 cells were exposed with different concentrations of PLK1 (BI2536) alone (i) or in combination with AZD6244 (1 µM) (ii-iv), and cell viability was determined after 6 days. The dose-response curve was generated by calculating cell viability relative to untreated control and plotted against drug concentration. Graph represents the mean±SEM of three independent experiments.(K) Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors after 96 h. Graph represents the mean±SEM of two independent experiments.(L) Immunoblots analysis of both control and CEP55 knockdown MDA-MB-231 cells treated with single and combination treatments after 96h and cleaved PARP, Caspase-3 along with MYC, ERK1/2 and CEP55 were determined. COX-IV as a loading control.