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. 2022 Dec 6;23(23):15402. doi: 10.3390/ijms232315402

Figure 1.

Figure 1

Ino80 loss leads to extension of G1-phase and apoptosis in embryonic stem cells cultured in serum-media (serum–ESCs). (A) Result of PCR genotyping. ROSA26-CreER allele was detected only in Ino80 iKO ESCs. After treating the ESCs with 4-OHTM, homozygous conditional allele was detected in CONT ESCs and Ino80 knockout allele was detected in Ino80 iKO ESCs. Ino80 iKO ESCs, inducible Ino80 knockout ESCs. 4-OHTM, 4-hydroxytamoxifen. ESCs, embryonic stem cells. CONT ESCs, control ESCs. (B) Expression levels of Ino80 using mIno80 primer (detecting exon 6) in both CONT and Ino80 iKO ESCs maintained in 2i- or serum-media was examined by qRT-PCR (N = 4). mIno80, mouse Ino80. (C) Immunofluorescence analysis of pluripotent markers (Nanog, Sox2, and SSEA1) in CONT and Ino80 iKO ESCs cultured in 2i-media containing 4-OHTM. Scale bar, 200 μm. (D) Representative images of CONT and Ino80 iKO ESCs cultured in either the 2i- or serum-media. ESCs were treated with 4-OHTM for 3 days and adapted to 2i- and serum-media for at least 2 days. Scale bar, 200 μm. (E) Cell proliferation assay. Cell numbers of the CONT and Ino80 iKO ESCs were counted at days 2, 3, and 4 after seeding 20,000 ESCs cultured in either 2i- or serum-media (N = 4). (F) Representative images of apoptosis analysis by flow cytometry and quantification. Annexin V(+) cells were measured in the CONT and Ino80 iKO ESCs. The difference in apoptosis rates between CONT and Ino80 iKO ESCs cultured in 2i-media is 3.40% on average, and in serum-media is 7.14% on average. p-value was calculated by one-way ANOVA test (N = 3). (G) Representative histograms of cell cycle and quantification. Percentages of G1, S, and G2/M phases of the CONT and Ino80 iKO ESCs measured by flow cytometry were plotted (N = 3). (H) Relative expression of Cdkn1a, by using qRT-PCR in both 2i-media and serum-media, the expression levels of Cdkn1a in CONT and Ino80 iKO ESCs were investigated (N = 4). Statistical significance was determined at a p-value lower than 0.05 (*), 0.01 (**) or 0.0001 (****).