Ab215975

Cell proteins were extracted with RIPA lysis buffer (Thermo Fisher, USA). The protein was extracted by incubation, vortex, and centrifugation (15,000 ×g, 4°C for 25 min). A BCA reagent kit (Beyotime, China) was used to measure the protein concentrations. Total protein was separated by SDS-PAGE gel (100 V, 1.5 h) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) (50 V, 80 min). After blocking in 5% nonfat milk for 1 hour, the membranes were incubated overnight at 4°C with the indicated primary antibodies, including anti-CD63 (Abcam, ab68418), anti-TSG101 (Abcam, ab133586), anti-E-cadherin (Abcam, ab76055), anti-N-cadherin (Abcam, ab76011), anti-vimentin (Abcam, ab20346), anti-JAK1 (Abcam, ab138005), anti-STAT3 (Abcam, ab68153), IL-10 (Abcam, ab215975), arginase-1 (ab133543) and anti-GAPDH (Abcam, ab9485). They were then incubated with secondary antibodies for 1 hour at room temperature and visualized by the ECL chemiluminescence reagent (Millipore, USA).

Cells were first fixed using 4% paraformaldehyde (PFA). This was followed by a wash and permeabilization step using 1% Triton X-100 (Carl Roth) for 30 min at room temperature. Possible unspecific antibody binding sites were blocked using 2% BSA (Sigma Aldrich). CD163 and CD80 staining was used to validate the macrophage polarized phenotypes. For CD163, CD80, and IL-10 staining, the cells were incubated with specific primary antibodies (rabbit anti-human CD163 mAb, Abcam ab182422; mouse anti-human CD80 mAb, Invitrogen, 16080985; and rabbit anti-human IL-10 mAb, Abcam ab215975) overnight at 4°C. The following day, the wells were washed and a staining solution, which included secondary antibodies (goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody AlexaFluor 568 (A-11004, 1:1000, Thermo Fisher) for CD80; goat-anti rabbit IgG (H+L) cross-adsorbed secondary antibody AlexaFluor 488 (A-11008, 1:500, Thermo Fisher) for CD163, phalloidin (to visualize F-actin; A-30105, 1:400), and DAPI (0.1 µg/ml, D8417-5mg, Sigma-Aldrich) was applied in DPBS for two hours. Then, fresh DPBS was supplied, and microscopical images, captured from random fields of view within each well, with a 10x magnification were taken with the Axio Observer Z1 microscope (Zeiss Oberkochen, Germany).