Fig. 2: Specific knockdown of PHF8 not only exhibits similar effects as HER3 depletion to induce cell cycle G1 arrest and upregulatep27^kip1, but also abrogates activation of HER3-mediated downregulation of p27^kip1 in TNBC cells.

TNBC cell lines (HCC1806, HCC70, HCC1937, and MDA-MB-468) were transiently infected with lentivirus containing either control shRNA (sc) or specific shRNAs targeting PHF8 (sh1 and sh2). A Cells were seeded onto 6-well plates for colony formation assays. Representative images of the cell colonies were captured using a digital camera after crystal violet staining (left panel), and the colony numbers were quantified using ImageJ software (right panel). Error bars represent the standard deviation (SD). *, p < 0.05; **, p < 0.01; #, p < 0.005. B Cell cycle progression was examined by flow cytometry analysis. C Western blot was assays were conducted to detect PHF8, E2F1, Cyclin D1, p27^kip1, p21^waf1, and β-actin. D, RT-qPCR was performed to measure PHF8 and p27^kip1 mRNA expression. E, F HCC1806 and HCC1937 cells were infected with lentivirus containing either control shRNA (-) or HER3-specific shRNA (+). After 48 h, cells were collected and subjected to western blot analysis of HER3, PHF8, p27^kip1, and β-actin (E). RT-qPCR analysis was performed to quantify the mRNA expression levels of HER3, PHF8, and p27^kip1 (F). G HCC1806 and HCC1937 cells with ectopic expression of HER3 were subjected to western blot analysis of HER3, PHF8, p27^kip1, and β-actin. H TNBC cells infected with lentivirus containing either control shRNA (-) or PHF8-specific shRNA (+) were stimulated with HRG-β1 (25 ng/ml) for 24 h. Cells were collected and examined by western blot analysis of p-HER3, PHF8, p27^kip1, and β-actin.