Fig. 3

PME‐1 promotes anoikis resistance in PTEN‐deficient prostate cancer cells. (A) siCtrl‐ and siPME‐1‐transfected PC‐3 cells were plated 72 h post‐transfection on normal cell culture or low‐attachment plates for 24 h, before collection and lysis, and subsequently analyzed by western blotting for cleaved PARP‐1 (cPARP (p25)). Shown is a representative result from six independent experiments. (B) Apoptosis induction, as measured by PARP cleavage, in CRISPR/Cas9 generated PC‐3 cells, with and without non‐targeting gRNA (lanes 1–3), a pool of PME‐1 gRNA transfected cells (lane 4), and a single cell subclone of PME‐1 targeted cells (lane 5), after 24 h detachment. Shown is a representative result from three independent experiments. (C) The effect of stable PME‐1 overexpression in mouse embryonic fibroblasts (MEFs) from a PTEN/p53KO mouse model, after 24 h of detachment, was assessed by western blotting for cleaved PARP. Shown is a representative result from two independent experiments. (D) Mechanosensitive PARP cleavage in PME‐1‐depleted PC‐3 cells. The cells were cultured on soft (0.5 kPa) or stiff (50 kPa) polyacrylamide hydrogel for 24 h before being scraped into PBS, spun down, lyzed and analyzed for protein expression. Shown is a representative result from four independent experiments. (E) The number of Annexin V positive objects, a marker for apoptosis, in cultures of siCtrl‐ and siPME‐1‐transfected PC‐3 cells upon varying degrees of cell‐matrix interaction. The cells were grown on PEG (Poly(ethylene glycol))‐coated surfaces with 0–20% biotinylated compound bound to integrin ligand, fibronectin fragment FNIII(7–10). STS = staurosporine. Mean ± SD of three independent experiments. (F) Annexin V positive objects in PC‐3 cultures with no or high levels of integrin ligand, after 60 h of incubation. Mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, one‐way ANOVA with Bonferroni's multiple‐comparison test.