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. 2020 Mar 12;9:e54756. doi: 10.7554/eLife.54756

Figure 2. ATR and CHK1-mediated RSR triggers activation of key 2C-specific genes in ESCs.

(a) FACS analysis on pZscan4-Emerald ESCs showing the number of Em+ cells upon treatment with APH and specific ATR and ATM inhibitors. (b) Immunoblot for the phosphorylation status of key DDR kinases (CHK1 and CHK2) and the ZSCAN4 protein level upon treatment with APH and ATM/ATR inhibitor in ESCs. (c) RT-qPCR analysis of two ESCs lines for the expression of MERVL upon treatment with APH and ATRi. (d) Plot showing the scaled expression of 2C-specific markers and the percentage of cells expressing 2C-related genes in CNTL, APH-treated and APH+ATRi conditions. Fisher's exact test was used to determine p-values. (e) Immunoblot for ZSCAN4, ATR and the phosphorylation status of CHK1 upon APH treatment of AtrSec/Sec and Atr+/+ ESCs. (f) Immunoblot showing the expression of ZSCAN4 and p-CHK1 in Chk1+/- and Chk1+/+ ESCs upon treatment with APH. (g–k) RT-qPCR for 2C-specific genes in AtrSec/Sec and Atr+/+ ESCs treated with APH. (l) FACS analysis of pZscan4-Emerald ESCs showing the number of Em+ cells upon treatment with APH and a specific CHK1 inhibitor. Statistical significance compared to CNTL unless otherwise indicated. All bar plots show mean with ± SD (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA). For western blots quantification refer to Figure 2—source data 1.

Figure 2—source data 1. FACS, qPCR and Western quantification.

Figure 2.

Figure 2—figure supplement 1. ATR-mediated RSR triggers activation of key 2C-specific genes in ESCs.

Figure 2—figure supplement 1.

(a) FACS analysis of pZscan4-Emerald ESCs upon treatment with ATM and ATR inhibitors after induction of RS upon HU treatment. (b) RT-qPCR analysis of Dux mRNA upon APH treatment and ATR KD (c,d) FACS analysis of pZscan4-Emerald ESCs for DNA damage markers γH2AX and p-CHK1 (Em+ and Em- correspond to Emerald-GFP positive and negative populations, respectively). (e, f) Immunoblot showing the expression of ZSCAN4, p-CHK1 and p-CHK2 proteins in ESCs upon treatment with UV or APH for 8 hr. (g) RT-qPCR analysis for Zscan4d gene upon treatment with a specific ATRi in two distinct ESC lines (E14 and R1). (h) Immunoblot showing the absence of ATM kinase in ATM KO cells. (i,j) RT-qPCR analysis for Dux and Zscan4 mRNA upon APH treatment in ATM WT and KO ESCs. (k) RT-qPCR analysis for Dux mRNA upon APH and two different concentrations of p38 inhibitors (1 and 2). (l-n) Box plot showing the expression level of 2C-related genes (within the subpopulation of cells expressing the specific marker) at the single-cell level in CNTL, APH and APH+ATRi condition. Statistical significance compared to CNTL unless otherwise indicated. All bar plots show mean with ± SD (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA). For western blots quantification refer to Figure 2—source data 1.
Figure 2—figure supplement 2. ATR and CHK1-mediated RSR triggers activation of key 2C-specific genes in ESCs.

Figure 2—figure supplement 2.

(a, b) Immunostaining of AtrSec/Sec, Atr+/+ ESCs and Chk1-/+, Chk1+/+ ESCs for the canonical pluripotency markers POU5F1 and NANOG (bar = 25 µm). (c–f) RT-qPCR analysis for 2C-related genes in Atrsec /sec and Atr+/+ ESCs upon treatment with APH. (g) FACS analysis of Chk1+/- and Chk1+/+ ESCs for the basal expression level of ZSCAN4 protein (h–n) RT-qPCR analysis for 2C-related genes in Chk1+/- and Chk1+/+ ESCs upon treatment with APH. (o) RT-qPCR analysis for Trp53 expression upon siRNA-mediated KD. CNTL sample was transfected with sc siRNA. (p) Immunoblot showing ZSCAN4 expression and phosphorylation status of P53 and CHK1 upon APH treatment in CNTL and Trp53 KD ESCs. (q,r) RT-qPCR analysis for Trp53 and Dux expression in Trp53 WT and KO ESCs. Statistical significance compared to CNTL unless otherwise indicated. All bar plots show mean with ± SD (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA). For western blots quantification refer to Figure 2—source data 1.