FIG 6.

Piwil2 inhibits apoptosis by impairing p53 phosphorylation. (A) Increased apoptosis in Piwil2 knockdown cells rescued by p38 inhibitor. NC (shNC) and Piwil2 knockdown (shPiwil2) cells were mock treated or treated with 5 μM SB203580 or 5 μM SB202190 for 6 h and then collected for flow cytometry using annexin double staining. Error bars indicate SE (*, P < 0.05). (B) Piwil2 suppresses interaction between p38 and p53. Cell lysates were prepared and subjected to IP assays. (C) Piwil2 overexpression rescued the increase of p53 in Piwil2 knockdown cells. (D) P38 is involved in Piwil2 knockdown-induced p53 phosphorylation. For p38 inhibition, HeLa cells were transfected with shNC or shPiwil2 vector and treated with 5 μM SB203580 or 5 μM SB202190 for 1 h as indicated. (E) Colocalization between p-p38 and p-p53. (F) The impact of Piwil2 knockdown on p38 nuclear localization was determined by Western blotting by cytosolic (C)/nuclear (N) fractionation followed by Western blotting. (G and H) Piwil2 knockdown increased p-p53 fluorescence intensity and p-p38 nuclear localization. HeLa cells were transfected with shNC or shPiwil2 vector and harvested for immunofluorescence assay with anti-p-p53 or anti-p-p38 antibodies. Scale bars, 5 μm.