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. 2017 Apr 13;8(30):49093–49109. doi: 10.18632/oncotarget.17095

Figure 5. JMJD2A inhibits P21(WAF1/Cip1) via JMJD2AΔ.

Figure 5

(A) Super-EMSA(gel-shift) with biotin-P21WAF1/Cip1 promoter probe and anti-H3K9me3 antibody. The intensity of the band was examined by Western blotting with anti-Bioton.HistoneH3 as internal control. (B) Chromatin Immunoprecipitation(CHIP) with anti-H3K9me3 followed by PCR with P21 WAF1/Cip1 promoter primers in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, rLV-JMJD2AΔ, pCMV6-AC-GFP-JMJD2A plus miR372 inhibitor, rLV-JMJD2AΔ plus miR372 inhibitor, respectively. IgG CHIP as negative control. P21 WAF1/Cip1 promoter as INPUT. (C) Chromatin Immunoprecipitation(CHIP) with anti-RNApolII followed by PCR with P21 WAF1/Cip1 promoter primers in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, rLV-JMJD2AΔ, pCMV6-AC-GFP-JMJD2A plus miR372 inhibitor, rLV-JMJD2AΔ plus miR372 inhibitor, respectively. IgG CHIP as negative control. P21 WAF1/Cip1 promoter as INPUT. (D) The assay of P21 WAF1/Cip1 promoter luciferase activety. Each value was presented as mean ± standard error of the mean (SEM).**P < 0.01. (E) Western blotting analysis with anti- P21WAF1/CIP1 in liver cancer cells Hep3B cell lines transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-JMJD2A, rLV-miR372, rLV-JMJD2AΔ, pCMV6-AC-GFP-JMJD2A plus miR372 inhibitor, rLV-JMJD2AΔ plus miR372 inhibitor, respectively. β-actin as internal control. Antisense-mature miR372 as miR372 inhibitor.