Activation of p53 Pathway and Cell Cycle Arrest by Silencing of PYCR1 and PYCR2.
(A) HFF-hTERT cells were transduced with pSUPER.retro.puro retroviruses (left panel) expressing shRNAs that silenced both PYCR1 and PYCR2 or with pLKO.1 lentiviruses (right panel) expressing shRNAs that silenced PYCR1 or PYCR2 alone or both. Non-silencing (NS) shRNA was used as control for each vector. Four days later, cell lysates were immunoblotted with antibodies to PYCR1, PYCR2, p53, RRM2B and p21CIP1. (B) HFF-hTERT cells transduced with pLKO.1 lentiviruses were seeded in 6-well plates (n = 3) four days after infection. Cell numbers were scored 48 and 72 hours later. Data were plotted as % of shNS control for each time point. (C) HFF-hTERT cells transduced with either pLKO.1 (top panel) or pSUPER.retro.puro (bottom panel) were seeded at low density in 60 mm-dishes four days after infection. Colonies were fixed and stained 2 weeks later. (D) HFF-hTERT cells transduced with pLKO.1 lentiviruses expressing shNS (left panel) or shPYCR1/2-A (right panel) were stained with PI solution and analyzed by FACS 6 days post-infection.