Fig. 7: circATXN1 reduced SLUG mRNA stability by Preventing ALKBH5 Nuclear Translocation.
From: Regulation of partial endothelial-to-mesenchymal transition by circATXN1 in ischemic diseases
a RT-qPCR of circATXN1 and SLUG mRNA expression in HCMECs with or without circATXN1 overexpression (n = 4 biological replicates). b Western blot of SLUG and ALKBH5 in HCMECs infected with different constructs (n = 3 biological replicates). c RT-qPCR of SLUG mRNA half-lives in HCMECs with Act D stimulation, and infected with Ad Scr-sh or Ad ALKBH5-sh (n = 3 biological replicates). d MeRIP-qPCR analysis of m6A levels of circATXN1 in HCMECs infected with Ad Scr-sh or Ad ALKBH5-sh (n = 4 biological replicates). e Dual FISH/IF of circATXN1 (red) and ALKBH5 (green) in HCMECs with DAPI staining (blue) (n = 3 biological replicates, scale bar: 10 µm). f Western blot of ALKBH5 in nuclear and cytoplasmic fractions of HCMECs, with GAPDH and LMNB as controls (n = 3 biological replicates). g RIP-qPCR of pre- and mature SLUG mRNA in HCMECs using anti-IgG or anti-ALKBH5 (n = 3 biological replicates). h Structural predictions of ALKBH5 binding to Mature-SLUG mRNA (top) and Pre-SLUG mRNA (bottom). ALKBH5 is shown in blue, while SLUG mRNA is represented in pink. Magnified views highlight the binding interface, showing that pre-mRNA forms more extensive interactions with ALKBH5 compared to mature mRNA. i The distribution of interaction sites along the mRNA sequence for both mature (cyan) and pre-mRNA (yellow). The curves represent the relative binding affinity, while the bars indicate the number of interactions at each position. j Phosphorylated tyrosine residues of ALKBH5 protein in HCMECs with or without circATXN1 knockdown (n = 3 biological replicates). k Phosphorylated tyrosine residues of ALKBH5 protein in HCMECs with different constructs (n = 3 biological replicates). l Immunoprecipitation of ALKBH5 and CRM1 in HCMECs with or without circATXN1 knockdown (n = 3 biological replicates). m Western blot of ALKBH5 protein expression in nuclear and cytoplasmic extracts of HCMECs infected with different constructs (n = 3 biological replicates). Statistical analysis: two-Way ANOVA with multiple comparisons test for (a, c, g); unpaired two-tailed Student t test for (d).
