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. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2019 Oct 17;40(1):112–127. doi: 10.1161/ATVBAHA.119.312976

Fig.3. AIP1B transcription was epigenetically regulated by RIF1/H3K9 methylation.

Fig.3

A-B. TNF induced downregulation of RIF1/H3K9me3. HUVECs were untreated or treated with TNF (10 ng/ml) for 24 h. AIP1, EZH2/H3K27me3, and RIF1/H3K9me3 were detected by western blotting with the respective antibodies (A). AIP1 protein bands were quantified by densitometry and fold changes are presented by taking untreated group as 1.0 (B). n=3. C-E. RIF1/H3K9me3 repressed AIP1B expression. HUVECs were transfected with control or RIF1 siRNA for 48 h (C), or treated with the H3K9 methyltransferase inhibitor, BIX-01294 (5 μM for 24 h (D). AIP1, RIF1/H3K9me3, and total H3 were detected by western blotting with the respective antibodies. AIP1 protein bands were quantified by densitometry and fold changes are presented by taking control group as 1.0. n=3. F. RIF1 repressed AIP1B transcription. HUVECs were transfected with control or RIF1 siRNA for 48 h. The 5’ RACE was performed with AIP1-specific nested PCR with two sets of internal reverse primers (R837 and R605). The PCR products were analyzed by agarose gel electrophoresis, followed by extraction for DNA sequencing. G. Diagram of the AIP1 transcripts. Exon sequences from the 5’ RACE are depicted in red. Potential translational start sites for AIP1A and AIP1B proteins are indicated by blue arrows. Paired primers near the TSS used for H3K4me3 and Pol II ChIP assays (H-I) are indicated by red and green arrows, respectively. H-I. ChIP assay for the promoter occupancy by H3K4me3 and Pol II phosphor-Ser5 (Pol II S5). HUVECs were untreated or treated with TNF (10 ng/ml) for 24 h and genomic DNAs were used for ChIP assays with antibodies against H3K4me3 and Pol II S5 followed by qPCR with selected primers near the TSS regions of AIP1 transcripts. GAPDH transcript was used as an internal control. The specific binding was quantified by normalization with each input and fold changes are presented by taking untreated group as 1.0. n=3. All data are presented as the mean ± SEM. ** P<0.01, ** P<0.001, unpaired, two tailed t-test.