Fig. 4: circE7 epigenetically downregulates galectin-9 expression mediated by ACC1.

A RNA pulldown analysis was performed using biotin-labeled circE7 probe to detect the binding between circE7 and ACC1 protein in CAL27 and HN30 cells. Non-biotin-labeled circE7 probe was used as a control. B. Binding of ACC1 protein with circE7 was evaluated by RIP assay using anti-ACC1 antibody in CAL27 and HN30 cells overexpressing circE7, or in HPV-positive SCC090 and SCC154 cells, followed by quantification using RT-qPCR. C RNA immunoprecipitation using anti-Flag antibody to evaluate the binding between ACC1 protein and its truncation mutants with circE7, followed by RT-qPCR analysis of circE7. D RNA pulldown using biotin-labeled circE7 probe and western blot using anti-Flag antibody to detect the binding between circE7 and ACC1 and its truncation mutants. E RNA immunoprecipitation using anti-ACC1 antibody to evaluate the direct binding between ACC1 protein and circE7 and its truncation mutants, followed by RT-qPCR analysis of circE7 and its truncation mutants. F RNA pulldown using biotin-labeled circE7 probe to investigate the binding between circE7 and its truncation mutants with ACC1 protein. circE7 were overexpressed in CAL27 and HN30 cells, or si-circE7 was transfected into SCC090 and SCC154 cells. G Western blot to detect the expression levels of total ACC1 protein and p-ACC1 protein. H Acetyl-CoA content in cell lysate was measured using the Acetyl-CoA detection kit. I Western blot to assess the expression levels of global acetylation. J Western blot to assess the expression levels of H3K27 Ac, H3K9 Ac, H3K14 Ac, and H3K23 Ac. as shown in Supplementary Fig. S7L. K Overexpression of circE7 in the HN30 cells, ChIP-seq peaks show the levels of H3K27ac at the LGALS9 promoter region. The horizontal axis represents genomic coordinates, and the vertical axis represents ChIP-seq signal intensity. L ChIP-qPCR to investigate the changes in H3K27 acetylation level in the LGALS9 promoter region in CAL27 and HN30 cells overexpressing circE7 or SCC090 and SCC154 cells with circE7 knocked down. Correlations were calculated using unpaired two-tailed Student’s t test for (B, C, E, H, L), *p < 0.05, **p < 0.01, and ***p < 0.001. These experiments were derived from three independent repetitions. Each western blots were reproduced three times with similar results (A, D, F, G, I, J). Data are presented as mean ± SD. Source data are provided as a Source Data File.