Skip to main content
. 2014 Mar 12;3:e02313. doi: 10.7554/eLife.02313

Figure 7. ZNF304 also directs transcriptional silencing of INK4-ARF in hESCs.

(A) Immunoblot analysis showing ZNF304 levels in undifferentiated (DMSO) or retinoic acid (RA)-treated hESCs. (B and C) ChIP analysis monitoring binding of ZNF304, KAP1, SETDB1, and DNMT1 to INK4-ARF in undifferentiated or RA-treated hESCs (B) or in hESCs expressing a NS or ZNF304 shRNA (C). (D) qRT-PCR analysis monitoring INK4-ARF expression in hESCs expressing a NS, ZNF304, KAP1, SETDB1, or DNMT1 shRNA. (E) Bisulfite sequencing analysis of the p14ARF and p16INK4A promoters in H9 hESCs and DLD-1 cells. (F and G) ChIP analysis monitoring enrichment of H3K27me3, H3K9me3, and H3K4me3 (F) and EZH2 and BMI1 (G) at INK4-ARF or an irrelevant DNA region (NC) in H9 hESCs and DLD-1 cells. (H and I) ChIP analysis monitoring binding of EZH2 and BMI1 (H) and H3K27me3, H3K9me3 and H3K4me3 (I) at INK4-ARF in H9 hESCs and DLD-1 cells expressing a NS or ZNF304 shRNA. (J) qRT-PCR analysis monitoring INK4-ARF expression in H9 hESCs and DLD-1 cells expressing an NS, EZH2, or BMI1 shRNA. Data are represented as mean ± SD. *p≤0.05, **p≤0.01. Control experiments related to Figure 7 are shown in Figure 7—figure supplements 1,2.

DOI: http://dx.doi.org/10.7554/eLife.02313.029

Figure 7.

Figure 7—figure supplement 1. Knockdown efficiencies in H9 hESCs.

Figure 7—figure supplement 1.

qRT-PCR analysis monitoring knockdown efficiency of ZNF304, KAP1, SETDB1, and DNMT1 in H9 hESCs. The results were normalized to that obtained with the NS shRNA, which was set to 1. Data are represented as mean ± SD. *p≤0.05, **p≤0.01.
DOI: http://dx.doi.org/10.7554/eLife.02313.030
Figure 7—figure supplement 2. Assessment of the role of EZH2 and BMI1 in INK4-ARF transcriptional silencing in H9 hESCs and DLD-1 cells.

Figure 7—figure supplement 2.

(A and B) qRT-PCR analysis monitoring knockdown efficiency of two unrelated EZH2 and BMI1 shRNAs in H9 hESCs and DLD-1 cells. The results were normalized to that obtained with the NS shRNA, which was set to 1. (C) qRT-PCR analysis monitoring INK4-ARF expression in H9 hESCs and DLD-1 cells expressing an NS shRNA, or an shRNA against EZH2 or BMI1 that is unrelated to that used in Figure 7J. Data are represented as mean ± SD. *p≤0.05, **p≤0.01.
DOI: http://dx.doi.org/10.7554/eLife.02313.031