Tm4sf1

Cells were collected and lysed in RIPA buffer (Beyotime, China) with 10 nM PMSF for 20 min, qualified and then performed for SDS-PAGE. The protein on the SDS gels was transferred to PVDF membranes. Antibodies against TM4SF1 (1:2000 dilution, Abcam, USA), DDR1 (1:1000 dilution, CST, USA), MMP2 (1:1000 dilution, Santa Cruz Biotechnology, USA) and MMP9 (1:500 dilution, Abcam, USA) were used to detect the protein level. β- tubulin (1:1000, Beyotime, China) was used as control to equal protein loading. The signals were detected by an enhanced chemiluminescence detection kit (Millpore, USA). For co-IP, PANC-1 and AsPC-1 cells at 80–90% confluence were washed with ice-cold DPBS three times before being lysed in IP lysis buffer. Then the lysates were incubated with antibodies overnight at 4 ^oC. Protein A/G PLUS Agarose beads were added for 10 h at 4 ^oC. The beads were collected and washed with lysis buffer for three times. The precipitated proteins were eluted and denatured in 2 × SDS loading buffer and analyzed by western blot.

The cellular fractions were obtained by utilizing the nuclear protein extraction kit (R0050, Solarbio Life Science). Typically, cells were initially broken down using cytoplasmic protein extraction buffer, and then nuclear extraction buffer was used to isolate the nucleus fraction. The analysis employed antibodies against PD-L1, Tubulin, Lamin B1, NUP43, IPO5, GAPDH, TM4SF1, JAK2, p-JAK2, STAT3, and p-STAT3 from Abcam (UK). Additionally, Flag antibodies from Sigma (St. Louis, MO, USA) and secondary antibodies against mouse or rabbit from Cell Signaling Technology were used. In immunoprecipitation experiments, protein lysates were subjected to incubation with anti-PD-L1, anti-IPO5, anti-Flag, anti-Myc, or the normal IgG antibody at a temperature of 4 °C for an extended period of time overnight, with continuous rotation. On the second day, the mixture was also cultured with protein A/G beads or M2 anti-Flag resin at room temperature for a duration of 2–3 h. After undergoing three rounds of washing with lysis buffer, the beads were subjected to boiling and thereafter proceeded with a western blot assay.

The CRC cell lines LoVo and NCM460 were cultured in DMEM (Boster, China). The HCT116, SW480, RKO, FHC, and DLD1 cell lines were cultured in RPMI 1640 medium (HyClone, USA). All of the cell lines were cultured in medium supplemented with 10% foetal bovine serum (FBS, ScienceCell). Primary antibodies against the proteins TM4SF1, N-cadherin, vimentin, SOX2, MMP9, CD133, Par3 and β-catenin were purchased from Abcam (USA), while those against E-cadherin, c-Myc, and ZO1 were purchased from Santa Cruz (USA), and those against Smad2 and CD44 were purchased from Sigma (USA). The second antibody, affinity-purified, biotinylated rabbit anti-rat IgG, was purchased from Sigma (USA). Accession numbers are available in Table S1.