Fig. 3: p53 inactivation limits ATRX loss-induced G4 formation and RS in TP53 wt NGP cells.

A Left panel, for p53 inactivation, C-terminally V5-tagged dominant-negative p53 mutants (p53_R273H or p53_R175H) or a control empty vector (ShCtrl) were stably introduced by lentiviral infection into Ctrl or ATRX KO NGP cells. Western blotting using anti-V5 and anti-p53 antibodies analyzed the expression levels of V5-tagged p53 mutants. β-Actin was used as a loading control. Right panel, immunoblots for the expression of DSB checkpoint proteins in cell lysates prepared from the indicated samples. p53 inactivation in ATRX KO NGP cells results in the loss of the DDR and p53-ATM checkpoint proteins. β-Tubulin was used as a loading control. As a positive control (P+), parental NGP cells were treated with doxorubicin (0.5 μg/mL) for 24 h. B, C γH2AX IF indicates decreased DDR after p53 inactivation in ATRX KO NGP cells. NGP cells treated with doxorubicin (0.5 μg/mL, 24 h) were used as the positive control. C Quantification of γH2AX+ cells among 100 cells analyzed in (B). D, E IF staining of G4 (1H6) in the indicated cells. As a positive control (P+), parental NGP cells were treated with the DNA G-quadruplex stabilizer CX-5461 (50 nM) for 24 h and stained with the anti-G4 (1H6) antibody. Nuclei are counterstained with DAPI (blue). E Quantification of G4+ cells among 100 cells analyzed in (D). F The RS markers p-KAP1, p-Chk1, and p-RPA32 were not induced in p53-inactivated ATRX KO NGP cells, representing a reduction in RS. β-Tubulin was used as a loading control.