FIGURE 6.
pX activates ATR via the p38MAPK pathway leading to p53 transcriptional activation. A, Western blot analyses of phospho-ATR using WCE from apoptotic 4pX-1 and 4pX-1-p38MAPKαkd cells grown with (+) or without (-) pX, as indicated. ATR is the internal control. B, WCE (2 mg) from 4pX-1, 4pX-1-ATRkd, and 4pX-1-p38MAPKαkd cells grown with (+) or without (-) pX, isolated at 2 h following the onset of apoptosis, were immunoprecipitated with p53 and actin antibodies added in the same reaction. Immunoprecipitates were analyzed by Western blots using antibodies for p53, phospho-Ser18, phospho-Ser23, and phospho-Ser389 of p53. Actin and IgG are used as immunoprecipitation controls. Quantification for A and B is by the Scion software. A representative assay is shown from three independent experiments. C, real-time PCR quantification of Bax, Fas, and Noxa mRNAs employing RNA isolated from 4pX-1, 4pX-ATRkd, and 4pX-1-p38MAPKαkd cell lines grown in apoptotic conditions for 12 h as a function of pX. Results, expressed as pX-dependent induction, -Tet/+Tet ratio, are from three independent RNA isolations, each PCR reaction performed in identical triplicates. Quantification is relative to 18 S rRNA. The pX-dependent induction of Bax, Fas, and Noxa mRNAs in 4pX-1 cells relative to the knockdown cell lines is significant (p < 0.005).