Fig. 2. Ex vivo inducible knockout of PDCD2 in ESCs results in loss of S phase entry and increased p53.(A) Growth curve of inducible knockout and WT ESCs.

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Fig. 2. Ex vivo inducible knockout of PDCD2 in ESCs results in loss of S phase entry and increased p53.(A) Growth curve of inducible knockout and WT ESCs in presence or absence of Tamoxifen. Ex vivo inducible knockout of PDCD2 in ESCs results in loss of S phase entry and increased p53.(A) Growth curve of inducible knockout and WT ESCs in presence or absence of Tamoxifen. Tam-treated Pdcd2flox/lacZ ESCs exhibit a cessation of growth 96–120 h following Tam treatment. (B) Cell cycle analysis of Pdcd2+/+ and Pdcd2flox/lacZ ESCs after 120 h after Tam treatment. Tam treatment of Pdcd2flox/lacZ ESCs reduced percentages of cells in S phase and G2/M, with increased numbers of cells in G1. Histograms summarize raw cell cycle data, shown in supplementary material Fig. S6A. (C) qPCR analysis of p53 target gene RNA levels. Results represent the fold change of Pdcd2flox/lacZ (with and without Tam) compared to Pdcd2+/+ cells (+Tam) 96 h after Tam treatment. (D) Western blot analysis of PDCD2, p53, PERP and p21 protein levels in Pdcd2flox/lacZ cells 24 to 96 h after Tam treatment. β-actin protein levels are shown as a loading control. Additional controls are shown in supplementary material Fig. S6C. Bottom panel shows quantification of PDCD2 and p53 protein level normalized to β-actin levels. (E) Immunofluorescence for p53 protein 72 h after Tam treatment of WT and Pdcd2flox/lacZ ESCs. Left panel: confocal acquisitions of ESCs stained for DNA (Hoechst, blue), cortical actin (Phalloidin, red) and p53 (green); right panel: quantification of percentage of cells with nuclear p53 cells per ESC colony (TCN = total cell number, quantified based on Hoechst staining). Significantly more cells with nuclear p53 are present in Pdcd2flox/lacZ ESCs (average 55.35%, N = 5 colonies) than WT ESCs (average 16.02%, N = 6 colonies), (p = 0.001, t test) 72 h after Tam treatment. Scale bars: 50 µm. Celine J. Granier et al. Biology Open 2014;bio.20148326 © 2014. Published by The Company of Biologists Ltd