Supplementary Figure 6: NSUN2 and YBX1 target the m5C of HDGF mRNA.
From: 5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs
a,b, Spearman’s correlation analysis demonstrating positive correlation between the expression level of NSUN2 (a) or YBX1 (b) and HDGF mRNA expression in UCB tissues from SYSUCC cohorts and TCGA, respectively. The centre line and the shaded regions indicate the regression line and 95% confidence interval, respectively. Spearman’s correlation coefficient and P value were calculated using the function “cor.test” in the statistical language R. c, Reduced HDGF mRNA half-life by silencing NSUN2 or YBX1 in T24 cells. Data are mean ± s.d. (n = 3 independent experiments). d, Western blotting showing the protein expression of NSUN2 and HDGF in NSUN2-knockdown UCB cells, which stably expressing NSUN2-WT, NSUN2- C321A, or NSUN2-DM. GAPDH was used as loading control. e, Western blotting showing the protein expression of YBX1 and HDGF in YBX1-knockdown UCB cells, which stably expressing YBX1-WT or YBX1-W65A. ACTIN was used as loading control. f, Relative luciferase mRNA expression of luciferase reporter gene with HDGF-m5C WT site (HDGF-WT) or mutant site (HDGF-Mut) in control, NSUN2-knockdown (top) or YBX1-knockdown (bottom) T24 cells. Data are mean ± s.d. (n = 3 independent experiments). g, Relative luciferase activity of luciferase reporter gene with HDGF-m5C WT site (HDGF-WT) or mutant site (HDGF-Mut) in control or NSUN2-knockdown (top) or YBX1-knockdown (bottom) T24 cells (n = 3 independent experiments). h, Knockdown of HDGF through injection of shRNA lentivirus strongly inhibited the subcutaneously growth of PDX tumour in vivo. Left: statistical analysis of xenograft tumour weight of each group. Right: volume-tracking plots. Data are mean ± s.d. (n = 5 animals). All P values were determined by a two-sided unpaired Student's t-test. The experiments were performed twice independently with similar results for d and e. Unprocessed gels for d and e are provided in Supplementary Figs. 8–10. The precise P values (two-sided unpaired Student’s t-test) and source data for c, f-h are presented in Supplementary Table 10.
