PAPSS2 depletion-mediated premature senescence occurs through the p53-p21 pathway. (a–c) MCF7 cells were transfected with Con Si, p53 Si, or p21 Si (first transfection) 6 h before transfection with Con Si or PAPSS2 Si (second transfection). (a) After 2 days, cells were harvested and subjected to WB analysis using the indicated antibodies. (b) Quantification of relative cell number and (c) SA-β-gal positivity were performed 3 days after transfection. (d–f) HCT116 parental, p53−/−, and p21−/− cells were transfected with Con Si or PAPSS2 Si, and experiments were performed as described in a–c. Cell numbers are relative to that of Con Si-transfected cells. Error bars indicate the S.D. of three independent experiments. ***P<0.001 and #P>0.05