Srsf3

Immunohistochemical (IHC) staining was performed incubating 4 µm FFPE sections in 10 mmol/L citrate buffer (pH 6.0) at 120°C for 5 minutes for antigen retrieval. Endogenous peroxidase was neutralized using EnVision FLEX Peroxidase‐Blocking Reagent (Dako) for 10 minutes. Tissue sections were blocked with 3% bovine serum albumin and incubated with the primary antibodies overnight at 4°C. Primary antibodies used were FRMD6 (Sigma‐Aldrich), ZEB1 (Sigma‐Aldrich), HTR2B (Sigma‐Aldrich), AE1AE3 (Thermo Scientific), CDX2 (Novus Biologicals), SRSF3 (Abcam) and SERPINA1 (Sigma‐Aldrich). After incubation with the EnVision FLEX + mouse or rabbit linker (Dako), EnVision FLEX/HRP (Dako) was used as the secondary antibody for 1 hour at room temperature, followed by 3,3'‐diaminobenzidine (DAB) staining (Dako). CMS molecular classification was performed according to Thrin et al,14 analysing the intensity and content of FRMD6, ZEB1, HTR2B, AE1AE3 and CDX2. SRSF3 and SERPINA1 tumoral epithelial expression was categorized as high (intense staining) and low expression (moderate and negative staining). Individual cores were scored by trained pathologists (CVP and SGL).

The RIP assay was performed as described previously [10 ]. In brief, SW480 cells were collected and washed twice with ice-cold PBS and lysed in RIP lysis buffer on ice for 30 min. The cell lysis supernatant was collected and incubated with SRSF3 (Abcam, Cambridge, UK) or IgG (Beyotime, Shanghai, China) antibodies against the RNA-binding protein of interest at 4 °C overnight. Then, protein-A/G beads were added and incubated for 4 h. The protein-A/G beads were then washed with RIP buffer five times to discard unbound material. RNA was purified for RT–PCR, and protein was isolated for WB assays.