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. 2023 Apr 6;108(9):2487–2502. doi: 10.3324/haematol.2022.282016

Figure 4.

Figure 4.

Knockdown of EZH2 induced abnormal nuclear cells and impaired enucleation during terminal erythropoiesis. (A) Schematic diagram of experiment method. The day of getting CD34+ cells was recorded as day 0. Lentivirus transduction human CD34+ at day 2. Doxycycline (DOX) or EPZ6438 was added at day 7. (B) Quantitative real-time polymerase chan reaction (qRT-PCR) results showing EZH2 expression in erythroblasts infected with lentivirus containing scramble-small hairpin RNA (shRNA) and EZH2-shRNA on days 11 and 15. (C) Representative western blot images showing the knockdown efficiency of scramble-shRNA and EZH2-shRNA on days 11 and 15. (D) Quantitative analysis the knockdown efficiency of EZH2 from 3 independent experiments. (E) Representative images of scramble-shRNA, EZH2-shRNA, diemthyl sulfoxide (DMSO) control, EPZ6438-0.5 mM, and EPZ6438-5 mM on day 15. Red arrow pointed to cells with abnormal nuclear morphology; scale bar, 5 mm. Statistical analysis of abnormal nuclear cells from 3 independent experiments. (F) Flow cytometry analysis showing the enucleation efficiency of scramble-shRNA, EZH2-shRNA, DMSO control, EPZ6438 0.5 mM, and EPZ6438 5 mM on days 11 and 15. Statistical analysis of the enucleation efficiency from 3 independent experiments. Statistical analysis is from 3 independent experiments, and the bar plot represents mean ± standard deviation of triplicate samples. ns: not significant; *P<0.05, **P<0.01, ***P<0.001.