Fig. 6: ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB. | Cell Death & Disease

Fig. 6: ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB.

From: m5C-dependent cross-regulation between nuclear reader ALYREF and writer NSUN2 promotes urothelial bladder cancer malignancy through facilitating RABL6/TK1 mRNAs splicing and stabilization

Fig. 6

A Integrative- genomics-viewer tracks representing the methylated level of m5C sites in NSUN2 when NSUN2 was silenced (the methylated level is 0.213 for siCTRL and empty for siNSUN2, respectively). The triangle represents the m5C site in NSUN2 (chr5: 6600023). B The m5C level of NSUN2 in 36 UCBs and in 29 adjacent normal tissues from SYSUCC. A two-tailed unpaired Student’s t-test was applied to determine the P-value. C Pearson correlation analysis showing the association between NSUN2 mRNA expression and its m5C level in 36 UCBs of SYSUCC. Shaded regions represent the 95% confidence interval. D Luciferase reporter assay showing the luciferase mRNA (Left) and activity (Right) level of NSUN2-wild-type m5C site containing plasmid and NSUN2 mutated m5C site containing plasmid in T24 cells. Data represent the mean ± S.D., n = 3. The P-value was determined by a two-tailed unpaired Student’s t-test. E The relative enrichment of m5C level in wild-type NSUN2 containing m5C-site compared with m5C-site mutant NSUN2. Data represent the mean ± S.D., n = 3, and a two-tailed unpaired Student’s t-test was applied to determine the P-value. F Integrative- genomics-viewer tracks representing the read coverage of NSUN2 in ALYREF-Flag RIP-seq data and the m5C levels of 36 UCB and 29 adjacent non-neoplastic tissues from SYSUCC. The triangle indicates the m5C site (chr5: 6600023) in NSUN2. G Upper panel: Western blotting shows Flag IP efficiency between ALYREF WT and K171A mutant. Bottom panel: Relative enrichment representing NSUN2 mRNA levels associated with ALYREF compared to an input control. IgG antibody used as a control. Data show the mean ± S.D., n = 3. The P-values were calculated by a two-tailed unpaired Student’s t-test. H Western blotting assays showing the expression level of NSUN2 and ALYREF in control and shALYREF#3 T24 cells, which expressing WT ALYREF and the K171A mutant and were normalized by α-tubulin expression. I IHC staining assays of mice bladder slices from orthotopic xenograft models showing the effect of ALYREF with a WT m5C site on the restoration of the ALYREF (Left column) and NSUN2 (Right column) expression in ALYREF-knockdown cells relative to ALYREF with K171A mutant. Scale bars, 100 μm. J Pearson correlation analysis showing the association between NSUN2 and ALYREF mRNA expression in the SYSUCC cohort (Left, n = 36) and TCGA cohort (Right, n = 430). Shaded regions showed the 95% confidence interval.

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