Fig 2: Tuberculous pleural effusion increased SRSF6 and COL1A2 protein in pleural mesothelial cells.(A–C) Cells were collected from tuberculous pleural effusion (TBPE) (n = 8) or transudative pleural effusion (n = 4). Relative SRSF6 protein levels and mRNA were investigated by Western blotting (A and B) and quantitative real-time PCR (qRT-PCR) (C). (D–G) Human pleural mesothelial cells (PMCs) were incubated with TBPE (5%), TPE (5%) for 24 or 48 hours, after which intracellular protein levels of SRSF6, COL1A2, and ACTA2 were measured by Western blotting. (D) n = 12; (F) n = 6 (SRSF6), n = 11 (COL1A2), n = 12 (ACTA2). (G) mRNA levels of SRSF6, COL1A2, and ACTA2 were detected by RT-qPCR at 48 hours; n = 4. (H) Human PMCs were incubated with TBPE (5%), bleomycin (0.2 µg/mL), or TGF-ß1 (5 ng/mL). After 24 hours, SRSF6 protein was detected by immunofluorescence staining and nuclei with DAPI staining. Scale bar: 100 µm. Original magnification, ×200. Data are shown as mean ± SEM of n individual experiments. P values were determined by unpaired Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001 (versus TPE).

Fig 3: Confirmation of SRSF6 regulating mRNAs.(A) After transfection with human SRSF6 siRNA2 or negative control (NC) siRNA for 48 hours, PMCs were incubated with bleomycin (0.2 µg/mL) for 24 hours, after which whole RNA sequencing was performed. The heat map shows representative differentially expressed genes in bleomycin plus SRSF6 siRNA-treated cells compared with bleomycin-treated cells. (B) PMCs were treated as described for A, after which indicated mRNA levels were measured by RT-qPCR and normalized to the housekeeping gene GAPDH. Data are shown as mean ± SEM of n individual experiments. n = 3, *P < 0.05, **P < 0.01, ****P < 0.0001 (unpaired Student’s t test).

Fig 4: Bleomycin and TGF-ß1–induced pleural fibrosis via SRSF6/WNT5A signaling pathway.(A–H) PMCs were incubated with bleomycin (0.2 µg/mL) or TGF-ß1 (5 ng/mL) in the presence or absence of SRSF6 siRNA for 24 or 48 hours, after which intracellular WNT5A protein and mRNA levels were measured as described in Methods. (A and E) Representative Western blots and changes of density of WNT5A protein at 24 and 48 hours. (A) n = 6; (E) n = 5. (B and F) Changes of WNT5A mRNA at 24 and 48 hours, n = 3. (C and G) Representative Western blots and changes of density of WNT5A protein at 48 hours. (C) n = 3; (G) n = 6. (D and H) Changes of WNT5A mRNA, n = 3. Data are shown as mean ± SEM of n individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA followed by the Dunnett’s test in A, B, E, and F or the Bonferroni’s test in C, D, G, and H). (I) Mouse pleural fibrosis model was induced by intrapleural injections of bleomycin plus carbon particles. IHC staining for WNT5A protein was performed on lung sections. Brown, WNT5A protein positive. Scale bar: 50 µm. Original magnification, ×400.

Fig 5: SRSF6 knock down by siRNA suppressed TBPE-, bleomycin- and TGF-ß1–induced COL1A2 and ACTA2 synthesis in PMCs.After transfection with SRSF6 siRNA or negative control siRNA (siRNA NC), PMCs were incubated with or without (A) TBPE (5%), (B) bleomycin (0.2 µg/mL), or (C) TGF-ß1 (5 ng/mL) for 48 hours, after which COL1A2 and ACTA2 protein was detected by immunofluorescence staining. Red, COL1A2; green, ACTA2; and blue, DAPI. Scale bar: 50 µm. Original magnification, ×630.