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. 2014 Aug 25;9(8):e104776. doi: 10.1371/journal.pone.0104776

Figure 2. Ezh2 SET domain depletion impaired liver growth.

Figure 2

A: Schematic diagram of Ezh2 SET domain KO system. Black triangle, square, and black narrow triangle are indicted as loxP sequence, exon, and primers respectively. F1, R1, and R2 mean forward primer 1, reverse primer 1 and 2. B: Deletion of Ezh2 in liver cells of fetal mouse ED 13.5 obtained from pregnant mouse injected with tamoxifen (TAM) at ED 8.5–10.5 detected by genomic PCR. Cre: Cre allele; Floxed: Floxed Ezh2 allele; and Flox del: floxed Ezh2 allele after removal of exons 18 and 19 by Cre recombinase, respectively. C: Efficiency of deletion of Ezh2 SET domain in fetal mouse at ED 8.5, 9.5, 10.5, and 11.5 (TAM administrated from ED 8.5) by genomic PCR. The bands for Ezh2 SET domain depletion (Flox del) appeared completely clear at ED 10.5. D: Immunofluorescence staining for H3K27me3, CK8/18 (epithelial marker) and DAPI in the liver tissues of control and Ezh2 KO at ED 13.5 after 3 days TAM injection (ED 8.5–10.5). E: frequency of H3K27me3 expressing cells in CK8/18 positive cells at indicated points. Data are mean ± SD (n = 3). P values (asterisks) are from the Mann–Whitney U-test. *P<0.05. F: Macroscopic images of livers in the control (CreER(T2)−/−;Ezh2F/F mouse) and Ezh2 KO (Ezh2 SET domain depleted mouse; Rosa26::CreER(T2)+/−;Ezh2F/F mouse) at ED 13.5 after 3 days TAM injection (ED 8.5–10.5). Scale bar = 100 µm. G: Left panel: liver weight of the control and Ezh2 KO at ED 13.5 (TAM; ED 8.5–10.5). Right panel: Absolute number of TER119− CD45− liver cells per a liver of the control and Ezh2 KO at ED 13.5 (TAM; ED 8.5–10.5). Data are mean ± SD (n = 3). P values (asterisks) are from the Mann–Whitney U-test. *P<0.05. H: Appearance of livers in the control and Ezh2 KO at ED 18.5 after 3 days TAM injection (ED 12.5–14.5). Scale bar = 100 µm. I: Left panel: liver weight of the control and Ezh2 KO at ED 18.5 (TAM; ED 12.5–14.5). Right panel: Absolute number of TER119− CD45− cells per a liver of the control and Ezh2 KO at ED 18.5 (TAM; ED 12.5–14.5). Data are mean ± SD (n = 3). *P<0.05. N.S.; not significant (P>0.05).