Fig 2: ALYREF promotes UCB pathogenesis and metastasis as an m5C reader in vivo.A The orthotopic xenograft model showing the effect of ALYREF with a WT m5C site on the restoration of submucosal lesions in ALYREF-knockdown cells relative to ALYREF with K171A mutant. Left: representative bioluminescence images; Right: statistical results for the bioluminescence signals. Data show the mean ± S.D. The P-values were calculated by a two-tailed unpaired Student’s t-test. n = 5, ns: no significance. B Representative Hematoxylin-eosin staining images in different groups of orthotopic xenograft models. Scale bars, 100 µm. C The subcutaneous xenograft model showing the effect of ALYREF with a WT m5C site on the restoration of subcutaneous tumor formation in ALYREF-knockdown cells relative to ALYREF with K171A mutant. D The lung metastasis model showing the effect of ALYREF with a WT m5C site on the restoration of tumor metastasis in ALYREF-knockdown cells relative to ALYREF with K171A mutant. Left: representative bioluminescence images are shown at 0 and the 6th week after injection; Right: statistical results for the mean bioluminescence signals in different groups at the 6th week. Data show the mean ± S.D. The P-values were calculated by a two-tailed unpaired Student’s t-test. n = 5, ns: no significance. E Left: Hematoxylin-eosin staining and metastatic nodules (indicated by arrows) in lung tissues from different groups at the 6th week. Scale bars: 400 µm; Right: Statistical results for the number of metastatic nodules in the lung among different groups at the 6th week. Data show the mean ± S.D, The P-values were calculated by a two-tailed unpaired Student’s t-test. n = 5, ns: no significance.

Fig 3: ALYREF is upregulated in UCB and enhances UCB cell proliferation and invasion as an m5C reader in vitro.A Western blotting showing ALYREF expression in 10 pairs of UCB and adjacent non-neoplastic tissues. The expression was normalized by a-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.

Fig 4: m5C regulators are predominantly upregulated in UCB.A, B Heat maps of m5C regulator mRNA expression profiles in paired UCB tumor tissues and adjacent normal tissues (22 pairs from SYSUCC cohort [A] and 19 pairs from TCGA cohort [B]). C, D The expression levels of ALYREF and NSUN2 (fold change > 1.1, P <0.05) in the SYSUCC cohort (22 pairs) (C) and TCGA cohort (19 pairs) (D). Data represent the mean ± S.D., and a two-tailed paired Student’s t-test was applied to determine the P-value. E The signal pathways in which m5C regulators may be involved from RNA-seq on SYSUCC UCB cohort. Positive correlations are shown in red; negative correlations are shown in blue. F WGCNA showing the networks among m5C regulators in the hierarchical cluster tree.

Fig 5: ALYREF enhances UCB pathogenesis in an m5C-dependent manner.A The m5C level of RABL6 mRNA in 36 UCBs and in 29 adjacent non-neoplastic tissues from SYSUCC. Data represent the mean ± S.D. The P-values were calculated by a two-tailed unpaired Student’s t-test. B m5C-RIP-qRT-PCR showing the relative enrichment of m5C level of RABL6 and TK1 was upregulated in UCB. Left: m5C-RIP-qRT-PCR showing the relative enrichment of m5C level of RABL6 in 5 pairs of UCB and normal tissues. Right: m5C-RIP-qRT-PCR showing the relative enrichment of m5C level of TK1 in 5 pairs of UCB and normal tissues. Data represent the mean ± S.D., n = 5, and a two-tailed paired Student’s t-test was applied to determine the P-value. C Colony forming assay showing the effect of RABL6 with a WT m5C site on the restoration of cell growth in RABL6-knockdown cells relative to RABL6 with a mutated m5C site. Top: representative images of cell colonies; Bottom: histograms of colony numbers. Data show the mean ± S.D., n = 3. The P-values were calculated by a two-tailed unpaired Student’s t-test. D Colony forming assay showing the effect of TK1 with a WT m5C site on the restoration of cell growth in TK1-knockdown cells relative to TK1 with a mutated m5C site. Top: representative images of cell colonies; Bottom: histograms of colony numbers. Data show the mean ± S.D., n = 3. The P-values were calculated by a two-tailed unpaired Student’s t-test. E Colony forming assay showing the effect of RABL6 with a WT m5C site on the restoration of cell growth in ALYREF-knockdown cells. Top: representative images of cell colonies; Bottom: histograms of colony numbers. Data show the mean ± S.D., n = 3. The P-values were calculated by a two-tailed unpaired Student’s t-test. F Left: The subcutaneous xenograft model showing the effect of RABL6 with a WT m5C site on the restoration of subcutaneous tumor formation in ALYREF-knockdown cells. Right: statistical results for the mean tumor weight in different groups. Data show the mean ± S.D. The P-values were calculated by a two-tailed unpaired Student’s t-test. n = 5. G Colony forming assay showing the effect of TK1 with a WT m5C site on the restoration of cell growth in ALYREF-knockdown cells. Top: representative images of cell colonies; Bottom: histograms of colony numbers. Data show the mean ± S.D., n = 3. The P-values were calculated by a two-tailed unpaired Student’s t-test. H Left: The subcutaneous xenograft model showing the effect of TK1 with a WT m5C site on the restoration of subcutaneous tumor formation in ALYREF-knockdown cells. Right: Statistical results for the mean tumor weight in different groups of the subcutaneous xenograft model. Data indicates the mean ± S.D. The P-values were calculated by a two-tailed unpaired Student’s t-test. n = 5. I Migration assay showing the effect of TK1 with a WT m5C site on the restoration of cell migration in ALYREF-knockdown cells. Top: representative images of migration cells; Bottom: histograms of migration cell numbers. Data show the mean ± S.D., n = 3. The P-values were calculated by a two-tailed unpaired Student’s t-test. J The lung metastasis model showing the effect of TK1 with a WT m5C site on the restoration of tumor metastasis in ALYREF-knockdown cells. Representative bioluminescence images are shown at 0 and the 6th week after injection. K Hematoxylin-eosin staining and metastatic nodules (indicated by arrows) in lung tissues from different groups at the 6th week. Scale bars: 100 µm.