Figure 6. Regulation of p57 in epithelial and cancer cell lines.
A, B. Quantitation of p57 by HCIF in (A) MCF-12A mammary epithelial cells and (B) PWR-1E prostate epithelial cells treated with the indicated growth factors and inhibitors of MEK (PD) or PI3K-mTOR (BEZ) for 24 hours. The percentage of p57-positive cells was determined as in Fig. 2. Error bars represent the SD of triplicate wells.
C, D. Quantitation of p57 by HCIF in T47D (C) and MD-MBA-468 (D) cells cultured in the presence of the indicated inhibitors of MEK (PD), PI3K and mTOR (BEZ), histone deacetylases (trichostatin A TSA), or DNA methyltransferases (5′-aza-deoxycytidine - AZA) for 24 hours. Bars represent the average p57 intensity of triplicate wells +/− SD. Under some conditions (marked by †) a large percentage of cells committed apoptosis; p57 fluorescence values were derived from surviving cells.
E. HCIF images of shRNA-mediated p57 depletion in PIK3CA-mutant cells. MCF-10A cells containing mutant alleles of PIK3CA (E545K or H1047R) at the endogenous locus and stably expressing non-targeting (NT) or p57-specific shRNA were cultured in the absence or presence of EGF for 2 days.
F. HCIF images of Rb phosphorylation in PIK3CA mutant cells. Cells were cultured as in (E).
G. Quantitation of pRb-positive cells for the conditions in (E). Bars represent the average of three independent wells, +/− SD.
H. Growth curve analysis of p57-depleted PIK3CA mutant cells. Cells were cultured as in (E), and cell counts were determined by HCIF analysis of DAPI-stained nuclei. Error bars represent the SD of triplicate wells.
All data shown are derived from individual experiments representative of at least three independent replicates.