Fig. 4: WIP1 loss triggers cellular senescence through p53 activation and ectopic expression of WIP1 reverses the senescent phenotypes observed in cells depleted of CDK5RAP2.
From: CDK5RAP2 loss-of-function causes premature cell senescence via the GSK3β/β-catenin-WIP1 pathway
A BJ cells transfected with WIP1 siRNA were analyzed by SDS-PAGE and immunoblotting for WIP1, pSer15-p53, p53, and p21CIP1. Actin was used as a loading control. Representative blots are from one of three independent experiments (n = 3) showing similar results. The numbers under the WIP1, pSer15-p53, and p21CIP1 bands represent the ratios of the densitometric levels of WIP1 vs actin, pSer15-p53 vs total p53, and p21CIP1 vs actin, with values from cells transfected with control siRNA normalized to 1.0. B BJ cells transfected with WIP1 siRNA were analyzed by SA-β-gal staining. Representative images of SA-β-gal staining (left panel) are from one of three independent experiments (n = 3) showing similar staining patterns. The number of SA-β-gal positive cells (B, right panel) was assessed in ~200 cells per treatment group in each of the 3 independent experiments (n = 3). *p = 0.00016. C, D Lysates of BJ cells co-transfected with CDK5RAP2 siRNA #2 and pReceiver-M02 carrying WIP1 were analyzed by SDS-PAGE and immunoblotting for CDK5RAP2 and WIP1 (C) and SA-β-gal staining (D). Actin was used as loading control. Representative blots are from one of three independent experiments (n = 3) showing similar results. The numbers under the CDK5RAP2 and WIP1 bands represent the ratios of the densitometric levels of CDK5RAP2 or WIP1 vs actin, with values from cells co-transfected with control siRNA and empty vector normalized to 1.0. Representative images of SA-β-gal staining in D (left panel) are from one of three independent experiments (n = 3) showing similar staining patterns. The number of SA-β-gal positive cells (D, right panel) was assessed in ~100 cells per treatment group in each of the 3 independent experiments (n = 3). *p < 0.001.
