Fig 2: Correlations of SNRPC with immune infiltration levels in HCC. (A) Correlations of SNRPC expressions and immune infiltration levels in HCC. (B) The prognostic value of SNRPC expression and abundance of immune cell infiltration.

Fig 3: Divergent levels of U1 snRNP proteins and U1 snRNA during phenotypic modulation. (A) Western blots for different splicing factors and RNA binding proteins in mouse aorta and bladder samples, comparing differentiated (D) and proliferative (P) samples. Acta2 and Myh-11 antibodies are markers of smooth muscle differentiation. Rpb1, Tubulin and Gapdh are loading controls. snRNA expression levels in mouse (B) and rat PAC1 cells (C) measured by qPCR. Primers for snRNA levels were normalized against the same set of genes as in Figures 5 and 6. Error bars represent standard deviation of the mean (n = 3). Statistically significance was calculated using Student's t-test, and is shown *P < 0.05. (D) RT-PCR for Snrp70 (qPCR with primers as in Figure 6) and Actn1 and Tpm1 (with primers as in Figure 2) in P and D Rat PAC1 cells. Error bars represent mean and standard deviation, (n = 3). (E) Left panels: RNA-FISH for U1 snRNA in rat PAC1 D and P cells. Right panels show DAPI staining for nuclei. (F) Immunofluorescence in rat PAC1 cells for SNRNP70 and U1C. Sm Actin is a marker of SMC differentiation.

Fig 4: SRSF5 interacted with U1A to promote binding of U1 snRNP to M pre-mRNA. A) A549 cells were infected with PR8 virus at 2.0 MOI for 24 h, followed by co-IP with anti-SRSF5 or control IgG and immunoblotting with anti-U1-70K, U1A, and U1C antibody in the presence or absence of RNase A. B) Association of hSRSF5-Flag with U1 snRNP withstands high salt washes. Added SRSF5-Flag, which was produced and purified in vitro, to cell lysates of srsf5-/- HEK293 for 1 h at 4 °C. Flag-M2 affinity beads were added to the reaction and left for 1 h at 4 °C. The washes were done, by increasing salt concentration, from 200 to 400 mm of NaCl. Bound proteins were eluted with Laemmli and immunoblotted with antibodies directed against U1A and U1-70K. C) SRSF5 interacts with U1A in vitro. Purified hSRSF5-Flag was incubated with glutathione-agarose bound GST-U1A-His, GST-U1-70K-His, GST-U1C-His. Following washes, the beads were washed five times in binding buffer and the bound proteins eluted with Laemmli and immunoblotted using anti-Flag or anti-His antibodies. D) HEK293 cells were transfected with Flag-tagged U1A, Flag-tagged U1C, or Flag-tagged U1-70K for 24 h. Cell lysates were incubated with biotin-labeled M mRNA and immunoprecipitated with streptavidin beads. Immuno-detection was performed with indicated Flag antibodies. Empty beads were used as control. E) Purified GST-U1A-His, GST-U1C-His, GST-U1-70K-His, or GST-His was incubated with in vitro transcribed M mRNA for 6 h at 4 °C. M mRNA eluted from His immunoprecipitates were quantified by RT-qPCR. Data presented as means ± SD. F) Colocalization of endogenous U1A (green) and viral M mRNA (red) in PR8 virus-infected srsf5 +/+ and srsf5 -/- HEK293 cells at 0, 6, and 12 hpi as detected by RNA FISH. Nuclei were stained with DAPI (blue). Quantification of colocalized SRSF5 and M mRNA in cells (right). Means ±SD from 3 biological samples. G) srsf5 -/- HEK293 cells were transfected with increasing amount of SRSF5-Flag expression vector or with control Flag vector, as indicated, for 24 h. Cell lysates were then immunoprecipitated with anti-U1A or control IgG. Bound-RNA was extracted for M mRNA detection by RT-qPCR. Data presented as means ± SD. H) RNA pull-down of biotinylated M mRNA or mutant M mRNA bound to U1A protein and purified Flag-SRSF5. For A–H) data are representative of three independent experiments. Statistical significance in E) and J) was determined by unpaired two-tailed Student's t-test.

Fig 5: SNRPC promotes EMT and motility of HCC cells in vitro. The efficiency of SNRPC overexpression and knockdown was measured by (A) real-time PCR and (B) western blotting assays. Cell growth of SNRPC overexpression and knockdown cells measured by (C) Cell Counting Kit-8 assays and (D) colony formation assay is shown. Results were obtained from three independent assays. Cell motility of SNRPC overexpression and knockdown cells measured by (E) Transwell and (F) wound healing assay. Error bar presented as mean ± standard deviation (from triplicates), and significance is determined by the Student’s t test (*P < 0.05). Scale bars: 100 µm (E); 200 µm (F). (G) EMT markers in stably knocked down or overexpressed SNRPC cells examined by western blotting assays.