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. 2020 Jul 9;9:e56008. doi: 10.7554/eLife.56008

Figure 4. The effects of small molecule treatment on genomic stability in mouse ESCs.

(A) Representative images of γH2AX immunostaining in mouse ESC E14 treated with indicated doses of small molecules. (B) Analysis of relative intensity of γH2AX in mouse ESCs in (A). The results were normalized to those in control group. Each dot represents the relative fluorescence intensity of a single nucleus. (n ≥ 180 single nucleus). (C) Representative images of 53BP1 immunostaining in mouse ESC E14 treated with indicated doses of small molecules. (D) Quantification of 53BP1 foci numbers in (C). At least 50 cells were included for each group. (E) Representative images of alkaline comet assay of mouse ESC E14 treated with indicated doses of molecules for 24 hr (left panel). The tail moments of at least 50 cells for each group were quantified using Cometscore software (Sumerduck, VA, USA) (right panel). (F) Karyotyping analysis indicated that 48 hr farrerol treatment post spCRSIPR/Cas9 mediated editing did not greatly affect karyotypes (40, XY) of the Oct4-ΔPE-GFP transgenic C57BL/6 × PWK ESCs. Error bars in (B), (D) and (E) represent the s.e.m. Error bars in (F) represent the s.d. *p<0.05, **p<0.01, ***p<0.001, n.s., not significant, t-test.

Figure 4—source data 1. The genotoxicity of small compounds treatment.

Figure 4.

Figure 4—figure supplement 1. The effects of small molecule treatment on genomic stability in HCA2-hTERT cells.

Figure 4—figure supplement 1.

(A) Representative images of γH2AX immunostaining in HCA2-hTERT cells treated with indicated doses of small molecules. (B) Quantification of the percentage of cells with more than 10 γH2AX foci per nucleus in (A). At least 50 cells were included for each group. (C) Representative images of 53BP1 immunostaining in HCA2-hTERT cells treated with indicated doses of small molecules. (D) Quantification of the percentage of cells with more than 10 53BP1 foci per nucleus in (C). At least 50 cells were included for each group. Error bars represent the s.d. *p<0.05, **p<0.01, ***p<0.001, n.s., not significant, t-test.
Figure 4—figure supplement 2. The effects of small molecule treatment on cell proliferation.

Figure 4—figure supplement 2.

Proliferation of mouse ESC E14 treated with indicated doses of molecules was analyzed using EdU incorporation assay. Error bars represent the s.d., n.s., not significant, t-test.