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. 2021 Oct 30;12(4):1913–1927. doi: 10.1016/j.apsb.2021.10.025

Figure 4.

Figure 4

PHF6 and SUV39H1 cooperatively regulate the methylation of H3K9 and rDNA transcription in an interdependent manner. (A) and (B) ChIP analysis of H3K9me3 binding to the rDNA loci in PHF6-silenced (A) or PHF6-overexpressed (B) HeLa, Jurkat and U937 cells. Immunoblot of PHF6 or Flag-PHF6 in the indicated HeLa, Jurkat or U937 cells. (C) and (D) ChIP analysis of H3K9me3 binding to the rDNA loci in SUV39H1-silenced (C) or PHF6-overexpressed (D) HeLa, Jurkat and U937 cells. Immunoblot of SUV39H1 or V5-SUV39H1 in the indicated HeLa, Jurkat and U937 cells. (E) Confocal images of HeLa cells silenced using shNC, shSUV39H1, and then pulsed with FUrd for 15 min to stain with anti-BrdU (red). Scale bar, 10 μm. (F) and (G) qPCR analysis of 47S pre-rRNA from SUV39H1-silenced (F) or overexpressed (G) HeLa, Jurkat and U937. Immunoblot of SUV39H1 in the indicated HeLa, Jurkat and U937 cells. GAPDH was used as loading control. (H) PHF6-silenced (shPHF6) or control (shNC) HeLa cells were transfected with the V5-SUV39H1 plasmid (+) or a control without the plasmid (−), and then (I) ChIP analysis of H3K9me3 binding to the rDNA loci, and (J) qPCR analysis of 47S pre-rRNA were performed. (K) SUV39H1-silenced (shSUV39H1) or control (shNC) HeLa cells was transfected with the Flag-PHF6 plasmid (+) or a control without the plasmid (−), and then (L) ChIP analysis of H3K9me3 binding to the rDNA loci, and (M) qPCR analysis of 47S pre-rRNA was performed. (N) HeLa cells were co-transfected with Flag-PHF6 and V5-SUV39H1 (R235H). Cell lysates were prepared to analyze Flag/V5-SUV39H1 (R235H) levels using Western blotting. (O) ChIP analysis of the binding of H3K9me3 to the rDNA loci in (N) cells. (P) qPCR analysis of 47S pre-rRNA from (N) cells. All values are expressed as the mean ± SD, n = 3; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.