Figure 2.
Induction of p53 and p21WAF1/CIP1 is dependent on HEY1 expression and associated with cell cycle arrest. A, TC252 cells were transfected with either a nontargeting shRNA or EF30, in the absence or presence of a control nontargeting siRNA pool (siCo) or a siRNA to HEY1 (siHEY1), as indicated. Protein expression of EWS-FLI1, p53, PSer15-p53, p21, and β-actin for loading control was monitored on the Western blot; HEY1 expression was followed on the RNA level by RT-PCR. B, nuclear accumulation of p53 in TC252 cells upon EWS-FLI1 silencing (EF30) and ectopic HEY1 expression (HEY1), monitored by immunostaining with the p53-specific mAb DO1. For control, cells were tansfected with a nontargeting shRNA. Left, p53 staining; right, 4′,6-diamidino-2-phenylindole staining of nuclei. C, HEY1-mediated induction of p21WAF1/CIP1 expression is dependent on the presence of p53. TC252 cells were transfected with either 4-μg pSPORT empty vector (Co) or pSPORT-based HEY1 expression vector in the absence or presence of 1.5 μg of shp53. p53, PSer15-p53, p21, and β-actin protein expression were followed on the Western blot 96 h after transfection. Expression of the HEY1 transgene was monitored by RT-PCR. Note that in contrast to EWS-FLI1 silencing (Fig. 1), HEY1 does not induce p53 phosphorylation. D, cell fate determination after EWS-FLI1 silencing or ectopic HEY1 expression compared with control transfection. TC252 cells and STA-ET-7.2 cells were transfected with the indicated EWS-FLI1 silencing vectors (EF30 or EF22) or with a nontargeting shRNA in duplicates, subjected to puromycin selection 24 h after transfection, and the fraction of surviving cells incorporating the nucleoside analogue EdU within a 45-min incubation period was monitored in 24 h intervals over 4 d starting 48 h after transfection. EdU incorporation was measured by flow cytometry, exclusively gating on living cells. At 96 h posttransfection, protein extracts were prepared from all puromycin-resistant cells and tested for EWS-FLI1, p53, and PARP expression. The reduction of the p113 full length PARP band and the occurrence of a p89 PARP cleavage band were taken as an indication for apoptosis.