Fig 1: RT-PCR analysis of ATP7B exon12 exclusion by siRNAs targeting SRp20 (siSRp20), SRSF1 (siSRSF1), and hnRNP A1 in CHO-K1 cells. (A) RT-qPCR analysis of relative SRp20 mRNA expression levels. (B) RT-qPCR analysis of relative SRSF1 mRNA expression levels. (C) RT-qPCR analysis of relative hnRNP A1 mRNA expression levels. (D) RT-PCR analysis of various kinds of splicing factors on ATP7B exon11-12-13 mini-gene splicing using RNA interference. Controls are cells co-transfected with siNC and the ATP7B exon11-12-13 mini-gene. (E) Ex12 exclusion ratio analysis of various kinds of splicing factors on ATP7B exon11-12-13 mini-gene splicing. Quantification was carried out using SynGene Gene Tools. The ex12 exclusion ratio was expressed as previously described.
Fig 2: ATP7B exon12 exclusion pattern of compounds from Schizonepeta: ATP7B exon11-12-13 mini-gene vector. (A) RTPCR analysis of various kinds of compounds on ATP7B exon11-12-13 mini-gene splicing. CHO-K1 cells were transfected with the ATP7B exon11-12-13 mini-gene vector, held for 24 h, and then treated with various kinds of compounds for another 24 h. Cells were harvested, total RNAs were extracted, and ATP7B splicing products were analyzed by RT-PCR. Controls are cells transfected with the ATP7B exon11-12-13 mini-gene vector without any compound treatment. Amiloride-treated cells were used for the inhibition of ex12 exclusion controls. (B) Ex12 exclusion ratio analysis of various kinds of compounds or Amiloride on ATP7B exon11-12-13 mini-gene splicing. Quantification was carried out using SynGene Gene Tools. The ex12 exclusion ratio was expressed as: (PCR product (-ex12))/(PCR product (-ex12) + PCR product (+ex12)). Data are expressed as previously described. (C) Western blot analysis of SRp20, SRSF1, and hnRNP A1 protein expression levels modulated by compounds derived from Schizonepeta. CHO-K1 cells were respectively treated with apigenin, luteolin, caffeic acid, and p-coumaric acid for 24 h at 37°C. Cells were harvested, washed, and lysed. The lysates were resolved by 12% SDS-PAGE and western blotting. Briefly, the membrane was incubated with primary antibodies, and then incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich). Signals were visualized using chemiluminescence following the manufacturer’s protocol (Chemicon).
Fig 3: SRSF3 acts as an oncogenic role in NPC cells.A S26 and 5–8 F cells were transiently transfected with SRSF3 siRNAs (si-SRSF31 and si-SRSF3-2) and control siRNA (si-NC). Western blotting was performed to evaluated the knockdown efficiency of SRSF3, with ACTIN as control. B Colony formation assay was performed with cells described in A, and the statistical analysis was shown at the right. C Representative images of EdU staining for S26 cells described in A (left) and the corresponding statistical analysis was exhibited at the right. D Western blotting assay demonstrated the protein levels of PARP1, cleaved-PARP1, CCND1, and C-MYC in S26 and 5–8 F cells described in A. ACTIN was used as control. E Representative images of transwell assay in NPC cells described in A. Corresponding statistical analysis was presented at the right. F The growth curves of xenograft tumors derived from S26 cells stably expressing SRSF3 shRNAs (sh-SRSF3-1 and sh-SRSF3-2) or control shRNA (sh-Luci) lentiviruses. G Tumor size (left) and weight (right) for the xenograft tumors excised from F were presented. H Immunofluorescence staining of Ki-67 in paraffin-embedded xenograft tumors presented in F. I S26 and 5–8 F cells were infected with lentivirus expressing SRSF3 or empty vector. The overexpression level of SRSF was confirmed by qRT-PCR assay. J Colony formation assay was performed with cells described in I and the statistical analysis was shown at the right. K EdU staining was performed with the cells described in I and the percentage of EdU (+) cells in each group was presented at the right panel. L Transwell assay was performed to evaluated the migration ability of S26 and 5–8 F cells described in I with statistical analysis showing at the right. Scale bar, 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: Global landscape of SRSF3-regulated AS events in NPC cells.A RNA-seq was performed with S26 cells transiently transfected with siRNAs against SRSF3 or control siRNA to analyze the differential AS events influenced by SRSF3 knockdown. Venn diagram showed the total number of AS events altered in both two SRSF3 siRNAs, compared with control group. B Quantification of AS events described in A, which were classified into five categories: Cassette, retained intron (IR), alternative 5'-splice site (A5SS), alternative 3'- splice site (A3SS), and mutually exclusive exon (MXE). C Heatmap showed the global alteration of SRSF3-affected AS events demonstrated by the PSI. D Gene ontology pathways analysis of SRSF3-regulated splicing events. E The cassette splicing events described in B were classified into exon inclusion and exclusion. F, G RT-PCR was performed to validate the SRSF3-regulated AS events described in E. Representative graphs illustrated the exon exclusion (F) and exon inclusion (G) with NPC cells transfected with SRSF3 siRNAs. The PSI of each gene was presented on the corresponding bottom lane. The boxes on the bottom represents the exons, of which red boxes indicate alternative exons. H The correlation between the ?PSI of the RNA-seq analysis and RT-PCR validation presented in F, G was shown.
Fig 5: SRSF3 is highly expressed and associated with worse prognosis in NPC.A Heatmap results showed the transcriptome expression levels of 12 classical SRSFs in NPC tissues (n = 87) and control tissues (n = 10). B RNA-seq data presented as TPM described in A showed the expression of SRSF3 in tumor and control tissues. C qRT-PCR was performed to detect the mRNA expression of SRSF3 in NPC cell lines compared with immortalized non-cancerous nasopharyngeal epithelial cells (NP69). D Western blotting assay showed the protein levels of SRSF3 in another NPC cohort. ACTIN was used as an internal control. E IHC staining was performed to evaluate the protein expression of SRSF3 in NPC samples (n = 95). F Kaplan–Meier survival analysis showed the correlation between the protein expression of SRSF3 and disease-free survival of NPC patients described in F. Scale bar, 200 µm. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-SRSF3 antibody