Journal: Cell Death & Disease

Article Title: piR-26441 inhibits mitochondrial oxidative phosphorylation and tumorigenesis in ovarian cancer through m6A modification by interacting with YTHDC1

doi: 10.1038/s41419-025-07340-6

Figure Lengend Snippet: a Overlapping analysis (Venn diagram) revealing the m6A regulators that were pulled down by biotin-labelled piR-26441 from the lysates of OVCAR3 cells in mass spectrometry (MS) assays with differentially expressed genes in OC cells (GSE36668) and m6A regulators. b RNA Pulldown assay showed an interaction between piR-26441 and YTHDC1. c RIP and qRT-PCR assays revealed an interaction between piR-26441 and YTHDC1. d Schematic representation of the interaction between piR-26441 and YTHDC1. e FISH and immunofluorescence staining showed piR-26441 (red) and YTHDC1 (green) in cultured CAOV3 cells positioning with DAPI nuclear staining (blue). f Western blotting showed YTHDC1 protein levels increased in CAOV3 and OVCAR3 cells after piR-26441 overexpression, which was contrary to that after knockout. g , h MG132 (20 μM) treatment reduced the regulatory effect of piR-26441 on YTHDC1 protein levels in OC cells. i After treatment with cycloheximide (CHX) (80 μg/ml), the degradation rate of YTHDC1 protein in piR-NC group and sh-piR-26441 group was accelerated. j Western blot results showed a significant reduction in the ubiquitination levels of YTHDC1 after piR-26441 overexpression. k RNA Pulldown assay showed an interaction between piR-26441 and TRIM56. l Co-IP assays revealed an interaction between YTHDC1 and TRIM56. m Co-IP assays revealed reduced binding of YTHDC1 to TRIM56 after piR-26441 overexpression. n Western blot results showed that TRIM56 protein levels decreased in CAOV3 and OVCAR3 cells after piR-26441 overexpression, which was contrary to that after knockout. All independent experiment repeated three times. Values are presented as the mean ± SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: The following antibodies were used: rabbit anti-YTHDC1 (1:5000, Proteintech), rabbit anti-TSFM (1:1000, Proteintech), rabbit anti-nicotinamide adenine dinucleotide+hydrogen:ubiquinone oxidoreductase subunit B8 (NDUFB8; 1:5000, Proteintech), anti-tripartite motif containing 56 (TRIM56; 1:1000, Zenbio), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, Proteintech), and anti-β-actin (1:2000, Proteintech).

Techniques: Mass Spectrometry, Quantitative RT-PCR, Immunofluorescence, Staining, Cell Culture, Western Blot, Over Expression, Knock-Out, Co-Immunoprecipitation Assay, Binding Assay