Fig 1: Expression of SNRNP200, SRRM1 and SRSF3 in a highly aggressive cohort of prostate cancer (PCa) samples. (a) SNRNP200, SRRM1 and SRSF3 expression levels in a battery of highly-aggressive PCa samples, with or without the presence of metastasis (n = 42). represent the mean ± SEM of mRNA expression levels. b-f) Correlation between SNRNP200, SRRM1 and SRSF3 expression levels and Gleason score (b), expression levels of AR-v7 (c), AR (d) MKI67 (e) and KLK3 (f). mRNA levels were determined by a microfluidic-based qPCR array and adjusted by normalization factor calculated from ACTB and GAPDH expression levels. Asterisk (* p < 0.05) indicates statistically significant differences between groups.
Fig 2: SRSF3 regulates CD44 AS in breast cancer cells.A Semi-quantitative RT-PCR (CD44 RT-PCR) of CD44 isoforms after SRSF3 KD in HCC1806 and HCC1937 cells. B, C CD44v/s analysis by qPCR after SRSF3 KD. D CD44 splicing events verified by semi-quantitative RT-PCR (primers vx-F and v-R). E, F CD44v/s analysis by qPCR upon SRSF3 OE. G mRNA expression of CD44 (total) in MDA-MB-231 and HCC1806 cells upon SRSF3 OE. H qPCR analysis of changes in CD44v/s under various SRSF3 concentrations in MDA-MB-231 cells. Data are means ± SD.
Fig 3: Molecular consequences of SNRNP200, SRRM1 and SRSF3 silencing in 22Rv1 cell line. a) Basal phospho-AKT, phospho-ERK1/2 and phospho-JNK levels in SNRNP200-, SRRM1- and SRSF3 silenced 22Rv1 cells (si-SNRNP200, si-SRRM1 and si-SRSF3, respectively; 24 h; n = 3). Protein levels were normalized by total AKT, ERK and JNK protein levels. Representative images are shown in right panel. Protein data were represented as percent of scramble cells. b) Expression levels of selected transcripts in response to SNRNP200 (upper panel), SRRM1 (central panel) and SRSF3 (bottom panel) silencing (24 h) in 22Rv1 cells. Ratio between the expression of splicing variants is shown in bars with dotted pattern. c) Expression levels of KHDRBS1, SFPQ and U2AF2 in response to SNRNP200- and SRRM1-silencing in 22Rv1 cells. d) Expression levels of C-MYC, PTEN and TP53 in response to SNRNP200-, SRRM1- and SRSF3-silencing in 22Rv1 cells. mRNA levels were determined by qPCR and adjusted by normalization factor calculated from ACTB and GAPDH expression levels. Data were represented as percent of scramble-treated control cells (mean ± SEM). Asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001) indicate statistically significant differences between groups.
Fig 4: SRSF3-regulated splicing of SRF. (A) qPCR assays for analyzing the effects of SRSF3 knockdown on SRF mRNA expression in HCT-8 cells (n=6). (B) Western blotting to analyze the effects of SRSF3 knockdown on SRF protein expression in HCT-116 and HCT-8 cells (n=2). (C) RIP assays for analyzing the binding of SRSF3 protein with SRF pre-mRNA in HCT-116 cells. GAPDH was used as a negative control, while TP53 and CCDC50S were used as positive controls (upper panels: RT–PCR for SRF mRNA, lower panels: immunoblotting for SRSF3 protein). (D) The schematic diagram of SRF mRNA and minigene. The SRF mRNA contains seven exons, and the predicted binding sites of SRSF3 were enriched on exons 5 to 7. The exons 5–7 were constructed into pcDNA3.1 vectors. Two forward primers (FP1 and FP2) and one reverse primer (RP1) were designed to amplify the transcripts of minigenes. (E) RT–PCR assays for investigating the transcripts of the SRF minigene in HCT-116 cells (n=2). (F) Schematic diagram and sizes of SRF minigene transcripts analyzed by DNA sequencing. (G) RT–PCR assays for investigating the effects of SRSF3 knockdown or overexpression on SRF minigene transcripts in HCT-116 cells (n=2). (H) Schematic diagram of three SRF minigene mutant plasmids. (I) RT–PCR assays for investigating the effects of SRSF3 overexpression on transcripts of SRF minigene mutant plasmids in HCT-116 cells (n=2). (J) Schematic diagram and sizes of SRF Mut3 plasmid transcripts analyzed by DNA sequencing. Data represent mean ± SD. Significance was assessed by two-sided t test. ***P < 0.001.
Fig 5: Immunohistochemical analysis of SNRNP200,SRRM1 and SRSF3 in prostate cancer (PCa) samples. a) Comparison of SNRNP200,SRRM1 (b) and SRSF3 (c) protein levels by immunohistochemistry (IHC) between a representative set of PCa samples (n = 47), prostatic intraepithelial neoplasia (PIN; n = 6) and benign prostatic hyperplasia (BPH; n = 7). Association of protein levels with clinically significant PCa (SigPCa; defined as Gleason score higher than 7) and the presence of metastasis at diagnosis (central panel and right panel, respectively). Representative images of BPH, PIN, PCa with Gleason score = 6 and SigPCa stained with SNRNP200 (400× magnification), SRRM1 (400× magnification) and SRSF3 (200× magnification) antibodies are showed below a, b and c panels, respectively. Scale bar indicates 100 µm. Data are expressed as mean ± SEM of IHC staining scaled from low [1] to high [3] intensity. Asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001) indicate statistically significant differences between groups.
Supplier Page from Abcam for Anti-SRSF3 antibody [EPR16976]