Fig 2: GSK3β promotes H/R-induced cardiomyocyte apoptosis and oxidative stress through downregulation of FTO and KLF5: (a) RT-qPCR was used to detect the mRNA level of FTO and KLF5 in cardiomyocytes with siFTO or siKLF5 transfection; (b) Western blot analysis was used to detect the protein of FTO and KLF5 in cardiomyocytes with siFTO or siKLF5 transfection; (c) TUNEL detected the apoptosis of cardiomyocytes with siGSK3β, siFTO, or siKLF5 transfection following H/R treatment; the scale bar was 25 μm; (d) the activity of SOD and the content of MDA in cardiomyocytes with siGSK3β, siFTO, or siKLF5 transfection following H/R treatment; (e) MitoSOX Red staining fluorescence intensity statistics graph of cardiomyocytes with siGSK3β, siFTO, or siKLF5 transfection following H/R treatment; (f) flow cytometry was used to detect JC-1 staining of cells with siGSK3β, siFTO, or siKLF5 transfection following H/R treatment. Measurement data were expressed as the mean ± standard deviation. Comparisons among multiple groups were conducted by one-way or repeated measurement ANOVA with Tukey's post hoc test. p < 0.05 was indicative of statistical significance. The cell experiment was repeated three times.

Fig 3: Metapristone inhibited the tumor growth through regulating miR-492 and its downstream target genes Klf5 and Nrf1 in vivo. a The tumor xenograft model of RL95-2 cells and Ishikawa cells. (n = 6 per group). b The expression of Ki-67 and Cleaved-caspase 3 in the tumor xenograft of RL95-2 cells and Ishikawa cells with or without metapristone treatment were detected by immunohistochemistry. Magnification: × 200. c The expression of Klf5 and Nrf1 in the tumor xenograft of RL95-2 cells and Ishikawa cells with or without metapristone treatment were detected by immunohistochemistry. Magnification: × 50. d The mRNA expression of miR-492, Klf5 and Nrf1 in the tumor xenograft of RL95-2 cells and Ishikawa cells with or without metapristone treatment were measured by real-time PCR (n = 6 per group). Values are means ± SD. e The protein expression of Klf5 and Nrf1 in the tumor xenograft of RL95-2 cells and Ishikawa cells with or without metapristone treatment were measured by western blot (n = 6 per group). Values are means ± SD. f The schematic representation of metapristone inhibitory effect on endometrial cancer cells through regulating cell proliferation and apoptosis and miR-492/Klf5/Nrf1 axis

Fig 4: Schematic map of GSK3β-mediated FTO and KLF5 in MI/R injury. In the case of MI/R injury, the expression of GSK3β in cardiomyocytes was increased, which promoted the phosphorylation of FTO and KLF5, thereby promoting their ubiquitination and degradation. The low expression of FTO promoted the m6A modification of Myc mRNA and resulted in the inhibition of Myc translation, while the low expression of KLF5 inhibited the transcription expression of Myc. Therefore, the expression of Myc in cardiomyocytes was reduced, leading to MI/R-induced cardiomyocyte apoptosis and increased ROS, thereby exacerbating MI/R injury.

Fig 5: FTO and KLF5 promote Myc expression in cardiomyocytes: (a) RT-qPCR detection of Myc expression in the myocardial tissue of sham-operated mice and MI/R mice; (b) RIP analysis using FTO specific antibody in cardiomyocytes; (c) MeRIP adopted to analyze the m6A modification of Myc mRNA in the cardiomyocytes of each group; (d) bioinformatics analysis of the top three binding sites of KLF5 and Myc promoter; (e) ChIP detected the enrichment of KLF5 in the Myc promoter region in cardiomyocytes; (f) dual-luciferase assay detected the targeting relationship between KLF5 and Myc; (g) RT-PCR was employed to detect the expression of Myc mRNA in the cardiomyocytes of each group; (h) Western blot analysis was used to detect the protein of FTO, KLF5, and Myc in the cardiomyocytes of each group. Measurement data were expressed as the mean ± standard deviation. Data in compliance with normal distribution and homogeneity of variance between two groups were compared using an unpaired t-test. Comparisons among multiple groups were conducted by one-way ANOVA or repeated measurement ANOVA with Tukey's post hoc test. p < 0.05 was indicative of statistical significance. The cell experiment was repeated three times.