Fig 2: SNW1 interacts with NUDT21/CPSF6 to regulate SRPK1 splicing(A) Proteins interacting with SNW1 were immunoprecipitated with anti-SNW1 antibody and identified by LC‒MS/MS. Table lists the top 10 SNW1-associated proteins in T24 cells.(B) Pearson correlation analysis showing associations between SNW1 and NUDT21, and SNW1 and CPSF6, in the TCGA cohort.(C and D) CoIP validation of interactions between endogenous SNW1, NUDT21, and CPSF6.(E and F) Western blot analysis of SRPK1 protein following NUDT21 (E) or CPSF6 (F) knockdown.(G–J) RT-qPCR analysis of SRPK1-L (G, I) and SRPK1-S (H, J) transcript levels. Data are presented as mean ± SD, ∗∗p < 0.01, ∗∗∗p < 0.001 (one-way ANOVA).(K–N) Transwell assays show reduced migration and invasion of T24 (K, L) and UM-UC-3 (M, N) cells after NUDT21 or CPSF6 knockdown.(O and P) RIP assays with anti-NUDT21 (O) or anti-CPSF6 (P) antibodies showing reduced SRPK1 pre-mRNA enrichment in SNW1 KD cells relative to Control KD cells. Data are presented as mean ± SD, ∗∗∗p < 0.001 (one-way ANOVA).

Fig 3: SNW1 regulates SRPK1 splicing(A) Quantification of AS events in T24 cells treated with three independent SNW1-targeting siRNAs. AS events were classified as skipped exon (SE), alternative 3′ splice site (A3SS), mutually exclusive exon (MXE), alternative 5′ splice site (A5SS), or retained intron (RI).(B) Volcano plot of differentially expressed transcripts following SNW1 knockdown (fold change ≥2, FDR <0.01).(C) Venn diagram shows overlap of upregulated and downregulated transcripts.(D) Schematic of SRPK1 transcripts shows primer pairs distinguishing the long (SRPK1-L) and short (SRPK1-S) isoforms.(E and F) RT-qPCR validation of SRPK1-L (E) and SRPK1-S (F) levels after SNW1 knockdown. Data are presented as mean ± SD, ∗∗p < 0.01 (one-way ANOVA).(G and H) RT-qPCR validation of SRPK1-L (G) and SRPK1-S (H) levels after SNW1 overexpression. Data are presented as mean ± SD, ∗p < 0.05, ∗∗∗p < 0.001 (unpaired t test).(I and J) Western blot analysis of SRPK1 protein levels following SNW1 knockdown (I) or SNW1 overexpression (J).(K) Quantification of SRPK1 pre-mRNA enrichment in RIP assays using an anti-SNW1 antibody. RNA enrichment was normalized to IgG. Data are presented as mean ± SD, ∗∗∗p < 0.001 (unpaired t test).

Fig 4: SNW1 positively correlates with SRPK1 expression and predicts LN metastasis in BCa(A) IHC staining of SNW1 and SRPK1 expression in BCa tissues. Scale bars: upper panel = 200 μm; lower panel = 50 μm.(B) Pearson correlation analysis of SNW1 and SRPK1 levels in 104 clinical bladder tissues (r2 = 0.6836, p < 0.0001).(C and D) AOD values of SNW1 (C) and SRPK1 (D) were compared across three tissue groups: normal adjacent tissues (NAT, n = 32), bladder cancer tissues without lymph node metastasis (LN−, n = 52), and with lymph node metastasis (LN+, n = 20). One-way ANOVA was performed to compare the differences among groups. Data are presented as mean ± SD, ∗∗∗p < 0.001.

Fig 5: SNW1 promotes bladder cancer metastasis(A–D) Bladder cancer cells were transfected with SNW1-specific siRNAs or control siRNA. SNW1 knockdown significantly inhibited the migration of T24 (A) and UM-UC-3 (B) cells in wound healing assays, as well as their migratory and invasive potential in transwell assays (C, D). Data are presented as mean ± SD, ∗∗∗p < 0.001 (one-way ANOVA).(E and F) SNW1 overexpression enhanced T24 cell migration in wound healing assays (E), and increased migration and invasion in transwell assays (F). Data are presented as mean ± SD, ∗∗∗p < 0.001 (unpaired t test).(G) Schematic representation of the popliteal LN metastasis mouse model.(H) Representative bioluminescent images of LN metastasis in mice injected with SNW1 knockdown (SNW1 KD) or control knockdown (Control KD) T24 cells.(I and J) Representative images of dissected popliteal LNs (I), and quantitative analysis of LN weights (n = 7 per group) (J). Data are presented as mean ± SD, ∗∗∗p < 0.001 (unpaired t test).(K) HE staining of LNs from Control KD and SNW1 KD mice. Scale bars: upper panel = 500 μm, lower panel = 50 μm.