Fig. 7

The YY1/HDAC2 complex downregulates the expression of YTHDC1 in ccRCC. A, The PROMO web tool predicted the potential transcriptional factors of YTHDC1. B, The Ominer web tool predicted the potential transcriptional factors of YTHDC1. C-E, 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were collected for western blot analysis (C), RT-qPCR assay (D), and ChIP-qPCR assay (E). Data presents as mean ± SD with three replicates. ***, P < 0.001. F, a diagram demonstrated the sequence and position of the YY1 binding peak in the YTHDC1 promoter. TSS transcriptional start site, WT wild type, MUT mutant type. G, 786-O and A498 cells were transfected with empty vector, GV592-YTHDC1 plasmids WT, MUT1, or MUT2 for 48 h. Cells were harvested and the activity of YTHDC1 promoter was measured. Data present as mean ± SD with three replicates. Ns, not significant; **, P < 0.01; ***P < 0.001. H, 786-O cells were transfected with indicated siRNAs for 24 h. Then, cells were transfected with EV, GV592-YTHDC1 plasmids WT another 24 h. Cells were harvested and the activity of YTHDC1 promoter was measured. Data present as mean ± SD with three replicates. Ns, not significant; ***P < 0.001. J and K, 786-O and A498 cells were transfected with indicated siRNAs for 48 h. Cells were collected for western blot analysis (J) and RT-qPCR assay (K). Data presents as mean ± SD with three replicates. ***, P < 0.001. L and M, 786-O and A498 cells were transfected with empty vector, 1 ng HDAC2 plasmids, or 5 ng HDAC2 plasmids for 24 h. Cells were harvested for western blot analysis (L) and RT-qPCR assay (M). Data presents as mean ± SD with three replicates. ***, P < 0.001. N. the ChIP-qPCR was performed by using the IgG or HDAC2 antibodies in 786-O and A498 cells. Data presents as mean ± SD with three replicates. ***, P < 0.001. O, The ChIP was firstly performed by using the YY1 antibodies. Then, the ChIP-re-ChIP assay was performed by using the IgG or HDAC2 antibodies in 786-O and A498 cells. Data presents as mean ± SD with three replicates. ***, P < 0.001. P, 786-O and A498 cells were transfected with the indicated siRNAs for 48 h. Cells were collected for western blot analysis