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. 2023 Sep 22;8(18):e162893. doi: 10.1172/jci.insight.162893

Figure 6. 3′UTR shortening of MORC2 amplifies promoter methylation and downregulation of DAPK1 via enhancing DNMT3A recruitment.

Figure 6

(A and B) qPCR (A) and immunoblotting (B) were performed to evaluate DAPK1 expression in KIRC cells transfected with Flag vector or Flag-MORC2 with silencing of indicated gene respectively (n = 3). (C and D) qPCR (C) and immunoblotting (D) were performed to evaluate DAPK1 expression in KIRC cells transfected with Flag vector or Flag-DNMT3A (n = 3). (E and F) qPCR (E) and immunoblotting (F) were performed to evaluate DAPK1 expression in KIRC cells transfected with control or DNMT3A-specific siRNA (n = 3). (G) MeDIP-qPCR was performed to evaluate promoter methylation level of DAPK1 in WT and MORC2-depleted Caki-1 cells transfected with Flag vector or Flag-DNMT3A (n = 3). (H) Co-IP was performed to detect the interaction between MORC2 and DNMT3A in KIRC cells. (I) ChIP-qPCR was performed to determine the abundance of DNMT3A at the DAPK1 promoter in Caki-1 cells transfected with control or MORC2-specific siRNA (n = 3). (J and K) qPCR (J) and immunoblotting (K) were performed to evaluate DAPK1 expression in KIRC cells transfected with vector, short 3′UTR, or long 3′UTR MORC2 plasmid (n = 3). (L and M) qPCR (L) and immunoblotting (M) were performed to evaluate DAPK1 expression in WT and S-KO Caki-1 cells (n = 3). (N) MeDIP-qPCR was performed to evaluate promoter methylation level of DAPK1 in WT and S-KO Caki-1 cells (n = 3). (O) ChIP-qPCR was performed to determine the abundance of DNMT3A at the DAPK1 promoter in WT and S-KO Caki-1 cells (n = 3). All data represent the mean ± SD. Two-tailed t test, 1-way ANOVA with Tukey multiple-comparison test, or 2-way ANOVA with Tukey multiple-comparison test analyses were performed. *P < 0.05; **P < 0.01; ***P < 0.001.