Fig 2: RNF144A negatively affects the stability of YY1 protein. A MDA-MB-231 and Hs578T cells stably expressing pCDH and Flag-RNF144A were subjected to immunoblotting analysis with the indicated antibodies. B MCF-7 and MDA-MB-231 cells stably expressing shNC and shRNF144A were subjected to immunoblotting analysis with the indicated antibodies. C HEK293T cells were transfected with HA-YY1 alone or in combination with increasing doses of Flag-RNF144A. After 48 h of transfection, cells were collected for immunoblotting analysis with the indicated antibodies. D Hs578T, MDA-MB-231, and MCF-7 cells were treated with DMSO or 10 µM of MG-132 for the indicated times, and then subjected to immunoblotting analysis with the indicated antibodies. E MDA-MB-231 and Hs578T cells stably expressing pCDH and Flag-RNF144A were treated with DMSO or 10 µM of MG-132 for 6 h, and then subjected to immunoblotting analysis with the indicated antibodies. F and G MDA-MB-231 and MCF-7 cell lines stably expressing shNC and shRNF144A were treated with DMSO or 100 µg/ml of CHX for the indicated times, and then subjected to immunoblotting analysis with the indicated antibodies. The representative images of immunoblotting and corresponding quantitative results (YY1/Vinculin) are shown in F and G, respectively

Fig 3: Silencing of YY1 repressed FAM3C-induced HSF1 up-regulation and proliferation of MDA-MB-231 cells. A, Silencing of YY1 repressed FAM3C-induced increase in HSF1 protein and Akt activation. Representative gel images were shown in upper panel, and quantitative data shown in lower panel. B, Silencing of YY1 repressed FAM3C-induced increase in HSF1 and Cyclin D1 mRNA levels. C, Representative images of cell density after YY1 inhibition. D, Cell number counting assays after YY1 inhibition. E, Cell viability assays after YY1 inhibition. N = 3-5, *P < 0.05 vs scramble + Ad-GFP group of cells, #P < 0.05 vs scramble + Ad-FAM3C group of cells. FAM3C, family with sequence similarity three member C; YY1, Ying-Yang 1; HSF1, heat shock factor 1; Akt, protein kinase B

Fig 4: 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

Fig 5: Schematic representation of proposed mechanism of action of miR-302b and cisplatin in TNBC cells. Upper scheme represents the biological activity of miR-302b in monotherapy. Exogenous expression of miR-302b does not affect ITGA6, since YY1 can interact with E2F2 to maintain ITGA6 transcription. Lower scheme represents the effect of combined intervention of miR-302b and cisplatin, that results in YY1 and E2F2 down-modulation impeding their activity over ITGA6 and leading to TNBC cell death. This proposed mechanism acts together with the well-known E2F1 down-modulation, another predicted ITGA6 TF, by miR-302b.