Fig. 3: Epigenetic regulation of TROP2 expression in breast cancer cells.

A Effects of epigenetic modulators on expression of E-cad and TROP2. Breast cancer cell lines BCX-010CL, BCX-011CL, and SUM159 cells were treated with decitabine, azacytidine, panobinostat, vorinostat, tazemetostat at 1, 1, 0.02, 1, 1, and 10 µM respectively for 3 days, followed by immunoblotting for E-cad and TROP2 with β-actin as control. B, C Effects of epigenetic modulators on expression of E-cad and TROP2 in ZEB1 knockdown cells. BCX-010CL cells with or without ZEB1 knockdown were treated with decitabine at 1 µM or panobinostat at 20 nM for 3 days. Protein levels of E-cad and TROP2 were determined by immunoblotting (B) and TROP2 mRNA levels were determined by quantitative PCR (C) (mean ± SEM). D Immunocytochemistry of TROP2. SUM159 cells were treated with decitabine at 1 µM for 3 days. After cell fixation, cell pellet blocks were processed and probed with anti-TROP2 antibody, followed by hematoxylin staining. E Methylation-specific PCR (MSP). Genomic DNA was extracted from BCX-010CL and BCX-011CL cells treated decitabine at 1 µM for 3 days, or from ZEB1 knockdown cells, and then subjected bisulfite CT conversion, followed by MSP using methylation specific primers specific to a CpG region in the TROP2 promoter. GAPDH was used as a control. F Immunoblotting of DNMTs. Breast cancer cell lines were treated with decitabine at 1 µM for 3 days. DNMT isoforms were detected by immunoblotting with antibodies against DNMT1, DNMT3A, and DNMT3B.