Fig. 3

PEG10 inhibition suppresses EMT and overcomes palbociclib resistance. A, B Immunoblot showed changes in the ZEB1 and E-cadherin expression after the knockdown of PEG10 using PEG10 siRNA and PEG10-ASO in MCF7-PR and T47D-PR cells. C Representative images from a migration and invasion assay of parental (MCF7 and T47D) versus palbociclib-resistant cells (MCF7-PR and T47D-PR), respectively. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. D Representative image from a migration and invasion assay of palbociclib-resistant (MCF7-PR and T47D-PR) cells after the transient PEG10 knockdown by PEG10 siRNA. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: *p < 0.05, **p < 0.01, ***p < 0.001. E Cell cycle analysis using PI staining after PEG10 knockdown in MCF7-PR and T47D-PR cells. The cell cycle was initially synchronized (syn) at G0/G1 with a double-thymidine block and then released and analyzed at the indicated time points. The bar represents the cell population distribution in each phase of the cell cycle. F Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with PEG10 siRNA or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of siRNA for 72 h. Three independently repeated experiments were performed with similar results. G Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with PEG10-ASO or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of ASO for 72 h. Three independently repeated experiments were performed with similar results. H Immunoblot showed induction of cleaved caspase-3 in the combination treatment of PEG10 siRNA and palbociclib. I Immunoblot showed ZEB1 inhibition by ZEB1 shRNA for 48 h. J Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with ZEB1 shRNA or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of ZEB1 shRNA for 72 h. Three independently repeated experiments were performed with similar results