Fig. 2: ALYREF is upregulated in UCB and enhances UCB cell proliferation and invasion as an m5C reader in vitro. | Cell Death & Disease

Fig. 2: ALYREF is upregulated in UCB and enhances UCB cell proliferation and invasion as an m5C reader in vitro.

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. 2

A Western blotting showing ALYREF expression in 10 pairs of UCB and adjacent non-neoplastic tissues. The expression was normalized by α-tubulin expression. T, tumor tissue; N, nonneoplastic bladder tissues. B IHC staining assays and representative images of ALYREF in nonneoplastic bladder tissues (Left) and UCB tissues (Medium and Right). Scale bars, 100 μm. C Kaplan–Meier analysis showing that upregulated ALYREF predicts poor OS in the SYSUCC cohort. The P-value was calculated by a log-rank test. D Kaplan–Meier analysis showed patients with high mRNA expression level of ALYREF predicted poorer OS. The P-value was calculated by a log-rank test. The group cutoff was 50%, which was the expression threshold for splitting the high-expression and low-expression cohorts. The data were from the public database http://gepia2.cancer-pku.cn/#index. E Organoid model showing the growth effect transfected with shCTRL and shALYREF#3 (Left). Statistical analysis of organoid size after 7 days of infection with shCTRL and shALYREF#3 (Right). Scale bars, 100 μm. 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 Representative Hematoxylin-eosin staining images of organoids after infection with shCTRL (Left) and shALYREF#3 (Right). Scale bars: 100 μm. G Colony forming assay showing the effect of ALYREF with a WT m5C site on the restoration of cell growth in ALYREF-knockdown cells relative to ALYREF with K171A mutant. Left: representative images of cell colonies in T24 (Top) and UM-UC-3 (Bottom) cells; Right: histograms of colony numbers. Data represent the mean ± S.D., n = 3. A two-tailed unpaired Student’s t-test was applied to calculate the P-value. H Migration assay showing the effect of ALYREF with a WT m5C site on the restoration of cell migration in ALYREF-knockdown cells relative to ALYREF with K171A mutant. Left: representative images of migration cells in T24 (Top) and UM-UC-3 (Bottom) cells; Scale bars, 100 μm. Right: histograms of the number of migration cells. Data represent the mean ± S.D., n = 3. A two-tailed unpaired Student’s t-test was applied to calculate the P-value.

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