Skip to main content
. 2020 Mar 12;9:e54756. doi: 10.7554/eLife.54756

Figure 4. ETAA1-mediated activation of ATR induces 2C-like cells in a RS-free context.

(a) FACS analysis for γH2AX and Emerald-GFP in Dox-iETAA1 ESCs in the presence or absence of Dox and upon treatment with ATRi or ATMi. (b) RT-qPCR results for Dux expression in Dox-iETAA1 ESCs upon Dox induction in the presence or absence of ATRi or ATMi. (c) Immunoblot showing the expression of ETAA1-AAD, ZSCAN4, MERVL-GAG and the phosphorylation status of CHK1, CHK2 and H2AX in Dox-iETAA1 ESCs upon treatment with Dox, ATRi or ATMi. (d) Immunoblot showing the expression of ETAA1-AAD, ZSCAN4, MERVL-GAG in Dox-iETAA1 ESCs upon treatment with Dox and Dux knock down. (e–g) RT-qPCR analysis of Dox-iETAA1 ESCs for expression of 2C-related genes (Dux, MERVL and Zscan4) upon treatment with Dox and Dux knock down. 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 4—source data 1.

Figure 4—source data 1. qPCR and Western quantification.

Figure 4.

Figure 4—figure supplement 1. Characterization of ETAA1-AAD inducible ESCs.

Figure 4—figure supplement 1.

(a) FACS analysis for γH2AX and Emerald upon overexpression of ETAA1-AAD by Dox in two different lentivirus clones with respect to empty vector (EV)-infected ESCs. (b) FACS analysis of CASPASE-3 in Dox-iETAA1 ESCs. (c) RT-qPCR analysis for the expression of ETAA1-AAD in Dox-iETAA1 ESCs (E14 and R1) upon Dox treatment. (d, e) RT-qPCR results for Dux and Zscan4 expression in Dox-iETAA1 ESCs after 24, 48 and 72 hr of Dox administration (f) RT-qPCR results for MERVL element in Dox-iETAA1 ESCs upon Dox administration in the presence or absence of ATRi or ATMi. (g–h) RT-qPCR results for 2C-related genes, Zscan4 and Dux in Dox-iETAA1 ESCs upon Dox and CHK1i treatment. (i–j) RT-qPCR analysis for Zscan4 and Dux expression in Dox-iETAA1 ESCs in the absence or presence of Dox and CHK1i (LY2603618). (k–m) RT-qPCR analysis for the expression of 2C-related genes in Dox-inducible Dux KO ESCs in comparison with WT ESCs upon treatment with Dox. Statistical significance compared to CNTL unless otherwise indicated. (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, one-way ANOVA).
Figure 4—figure supplement 2. ETAA1-mediated activation of ATR induces 2C-like cells in a RS-free context.

Figure 4—figure supplement 2.

(a,b) RT-qPCR analysis for ETAA1 and Dux genes expression in siRNA-mediated Etaa1 KD ESCs upon treatment with APH. CNTL ESCs were treated with sc siRNA. (c) Immunoblot for expression of ETAA1, ZSCAN4 and p-CHK1 in siRNA-mediated ETAA1 KD ESCs upon treatment with APH. (d) Immunostaining showing the ETAA1 expression in ZSCAN4 positive cells. (e-h) RT-qPCR analysis of 2C-related genes expression in Dox-iETAA1 ESCs (R1 and E14) upon Dox treatment and/or siRNA mediated Dux KD. For western blots quantification refer to Figure 4—source data 1.