Anti e cadherin

Soluble CD58 (sCD58), AKT inhibitor (LY294002), and Akt activator (SC-79) were acquired from Med Chem Express (MCE, Shanghai, China). Anti-Oct4 (11263-1-AP), anti-Sox2 (66411-1-Ig), anti-CD24 (18330-1-AP), anti-vimentin (10366-1-AP), and anti-c-Myc (10828-1-AP) were purchased from Proteintech (Wu Han, China). Anti-EPCAM (#2626S), anti-E-cadherin (#3195S), anti-AKT (#9272S), anti-GSK-3β (#5676S), anti-Phospho-GSK-3β (Ser9) (5558S), anti-β-Catenin (#8480S), anti-non-phosphorylated (active) β-catenin (S33/37/T41) (#8814S), anti-phospho-β-Catenin (Ser552) (#9566S), and anti-cyclin D1 (#55506S) were acquired from Cell Signaling Technology. Anti-CD58 (A0806) and anti-phospho-AKT (Ser473) (AP0140) were purchases from ABclonal (Wu Han, China).

Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA), emodin was obtained from Shanghai future industry Limited by Share Ltd (Shanghai, China) and BLM was acquired from Nippon Kayaku (Tokyo, Japan). The primary antibodies described in the study include: anti-E-cadherin, anti-vimentin, anti-cleaved caspase-3, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2, anti-Smad3, anti-phospho-Erk1/2 and anti-Erk1/2 (Cell Signaling Technology, CA, USA); anti-caspase-3, anti-Bax (Santa Cruz Biotechnology, CA, USA); anti-fibronectin (Proteintech, Chicago, USA); anti-caspase-8, anti-Bcl-2 (Absci, MD, USA); anti-TGF-β1, anti-FSP-1, anti-α-SMA (Abcam, USA); and anti-GAPDH (Beyotime Institute of Biotechnology, Haimen, China). Other reagents were obtained from Beyotime Institute of Biotechnology unless otherwise indicated.

Western blots were performed as previously described [11] (link). Briefly, as soon as the harvested cells were lysed and the supernatant was collected, 60 µg protein was loaded onto SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). Membranes were blocked with 5% skimmed milk for 1 hour and incubated overnight with one of the following primary antibodies: anti-PSMD10 (diluted at 1∶1,000; Sigma, St. Louis, MO, USA), anti-Twist2 (diluted at 1∶200; Abcam, Cambridge, UK), anti-Vimentin (diluted at 1∶500; Cell Signaling Technology, Beverley, MA, USA), anti-β-catenin (diluted at 1∶500; Cell Signaling Technology), anti-E-cadherin (diluted at 1∶500; Cell Signaling Technology) and anti-cyclin D1(diluted at 1∶500; Cell Signaling Technology) rabbit monoclonal antibody, followed by 1 hour of incubation with the appropriate secondary antibody (1∶5,000). The anti-GAPDH (Epitomics) rabbit monoclonal antibody was diluted to 1∶1,000 for use as a sample loading control.

B16-F10 cells (2 × 10⁶ cells per well) were seeded into a 10 cm dish for 24 h, and then cells were treated with different concentrations of PSS (12.5, 25, 50, 100 μg/mL) for 24 h. The medium was removed, and the cells were washed with PBS three times. Cells were then lysed in 200 μL of lysis buffer on ice. The total protein was determined using the Bicinchoninic Acid (BCA) Kit (Solarbio, Beijing, China). Equal amounts of protein in the cell extracts were fractionated by 10% SDS-PAGE, and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with TBST (20 mM Tris-buffered saline and 0.1% Tween) containing 5% nonfat dry milk for 1 h at room temperature, the membranes were incubated for 2 h with monoclonal antibodies, such as anti-MMP-9, anti-MMP-2, anti-E-cadherin, anti-Vimentin, anti-ERK 1/2, anti-p-ERK 1/2, anti-AKT, anti-p-AKT (Ser473), anti-p38, anti-p-p38, anti-p-p38, anti-NF-κB, anti-p-NF-κB, and anti-β-actin, which were purchased from Cell Signaling Technology. The membranes were then washed three times and incubated with HRP-conjugated secondary antibodies (Abcam, Cambridge, Massachusetts, USA). The proteins were then detected using chemiluminescence agents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).

Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.

Primary antibodies used were anti-γH2AX (#2577, Cell Signaling), anti-GAPDH (MAB374, Millipore), anti-CAD (PA5-19913, Thermo Fisher), anti-STING (#13647, Cell Signaling), anti-phospho-STING (#19781, Cell Signaling), anti-ICAD (#9732, Cell Signaling), anti-cGAS (#79978, Cell Signaling), anti-E-cadherin (#3195, Cell Signaling) and anti-α-tubulin(T9026, Sigma–Aldrich). Secondary antibodies were (Western blotting) anti-rabbit IgG(H + L)-HRP (A6667, Sigma-Aldrich), anti-mouse IgG(H + L)-HRP (115-035-166, Dianova), (immunofluorescence) anti-mouse IgG(H + L)-Cy5 (715-175-151, Dianova) and anti-rabbit IgG(H + L)-Alexa488 (711-545-152, Dianova).