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. 2021 May 3;11(13):6592–6606. doi: 10.7150/thno.59816

Figure 5.

Figure 5

MYOCD recruits PRMT5/MEP50 methyltransferase complex to epigenetically silence TGFBR2 transcription. (A) PRMT5 and WDR77 reduced TGFBR2 luciferase activity in A549 cells in does dependent manner. A549 cells were transfected with pGL3-600 reporter plasmid (0.8 μg), increased amounts of PRMT5 or WDR77 expression plasmid (0, 0.5, 1.5 μg) and 100 ng renilla luciferase plasmid, followed by monitoring luciferase 48 hours later. (B-C) PRMT5 and WDR77 inactivation promoted TGFBR2 transcription in A549 and H460 cells. RNA was extracted from indicated cell lines; qPCR was performed to check the expression of TGFBR2. (D-E) PRMT5 and WDR77 knockdown promoted sphere forming ability and this ability was suppressed by LY2109761 in A549 (D) and H460 (E) cells. Cells were treated with DMSO or LY2109761 (300 nM) for 10 days before quantification. Representative images of sphere assay (left) and statistics of sphere formation (right). (F) PRMT5/WDR77 associates with TGFBR2 is dependent on MYOCD. ChIP-PCR analysis of TGFBR2 promoters using indicated antibodies in A549 cells. (G) PRMT5/WDR77 failed to suppress TGFBR2 transcription in A549 MYOCD inactivation cells. A549-Teton-shMYOCD cells were transfected with indicated plasmids, then the cells were left treated or untreated with DOX for 2 days, RNA was extracted and qPCR was performed to check the expression of TGFBR2. (H) PRMT5 methyltransferase activity was important for MYOCD mediated suppression of TGFBR2 transcription. The A549 cells were firstly transfected with a control or shPRMT5 plasmid (2 μg). cells were selected with puromycin (1 μg/ml) for 24 h, then cells were re-transfected with PRMT5 or PRMT5 MT-dead expression plasmid (0.3 μg each). RNA was extracted to analysis TGFBR2 mRNA level 48 hours after re-transfection. (I) PRMT5 methyltransferase mutation failed to activate TGFBR2 reporter activity. The A549 cells were firstly transfected with a control or shPRMT5 plasmid (2 μg). Cells were selected with puromycin (1 μg /ml) for 24 h and then re-transfected with the PGL-600 promoter reporter and PRMT5 or PRMT5 MT-dead expression plasmid (0.3 μg each). Luciferase assays were performed 48 h after re-transfection.