Fig. 7
TRPM2-AS enhances PABPC1-mediated inhibition of NUMB expression. A The complementary pairing sequence of TRPM2-AS and NUMB mRNA 3'UTR. B, C RT-qPCR (B) and western blot (C) measuring the expression level of NUMB in the control group, TRPM2-AS overexpression group, and TRPM2-AS knockdown group. D, E The inhibitory effects of PABPC1 on NUMB were identified using RT-qPCR (D) and western blot (E). F RAP analysis of the relative enrichment of NUMB mRNA by biotin-labeled short DNA probe specifically complemented with TRPM2-AS with/without protease K treatment in substrate. G The direct interaction between PABPC1 and NUMB mRNA was confirmed using RIP analysis. H-J Negative control shRNA (sh-NC) and shRNA against PABPC1 (PABPC1-sh1/sh2) were transfected into HUVECs with/without TRPM2-AS overexpression. RAP followed by western blot/RT-qPCR analysis revealing the TRPM2-AS, PABPC1 protein and NUMB mRNA level pulled down by biotin-labeled short DNA probe specifically complemented with TRPM2-AS in different groups. K Western blot showing the inhibitory effects of TRPM2-AS on NUMB expression with/without PABPC1 knockdown. L-O RIP and RT-qPCR were used to quantify NUMB mRNA pulled down by PABPC1 in HUVECs with/without TRPM2-AS knockdown (L, M) and overexpression (N, O). P-S RT-qPCR and western blot analysis detecting the potentiating effect of TRPM2-AS on the inhibition of PABPC1 on NUMB. T Western blot showing the expression levels of NOTCH1 signaling pathway related proteins in control group cells and in PABPC1 knockdown group cells with/without TRPM2-AS overexpression/knockdown. Data were assessed with unpaired Student’s t test or one-way ANOVA and presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance