Chemi xt 4

Total proteins were extracted from transfected cells using RIPA lysis buffer containing PMSF (Solarbio) and protease inhibitor cocktail (Promega). The protein concentration was determined by a BCA Protein Assay Kit (Multi Sciences). After the samples were mixed with loading buffer and heated at 99 °C for 5 min, the protein lysates were separated by 12% SDS and transferred onto PVDF membranes (Millipore). The transferred membranes were blocked with 5% skim milk for 1 h at room temperature and incubated overnight with specific primary antibodies at 4 °C. Subsequently, the membranes were incubated at room temperature with horseradish peroxidase–conjugated goat anti-rabbit IgG (KPL) for 1 h. The bands were visualized with enhanced chemiluminescence (ECL) detection reagent (Multi Sciences) by Chemi XT 4 (Syngene). The primary antibodies used were anti-HOXD10 (RRID: AB_2049745), anti-E-cadherin (RRID: AB_2660958), anti-vimentin (RRID: AB_2492284), anti-β-catenin (RRID: AB_2811667), and anti-β-actin (RRID: AB_1873358).

The RIPA lysis buffer and PMSF (Solarbio) were used to extract proteins from cells. BCA Protein Assay Kit (Multi Sciences, Hangzhou, China) was used to detect the protein concentration. The protein lysates were transferred to PVDF membranes (Millipore, Sigma, Burlington, MA, USA) after separated by 10% polyacrylamide gel electrophoresis. The enhanced chemiluminescence detection reagent (Multi Sciences) was used to detect the protein bands by Chemi XT 4 (Syngene). The main antibodies were listed as follows: anti-β-actin (ZenBioScience, Cat# 380624), anti-E-cadherin (ZenBioScience, Cat# R22490), anti-N-cadherin (ZenBioScience, Cat# 380671), anti-Vimentin (ZenBioScience, Cat# R22775), anti-MMP2 (ZenBioScience, Cat# 380817), anti-RBBP4 (ZenBioScience, Cat# 385565), anti-HDAC1 (Proteintech, Cat# 10197-1-AP).

Cultured macrophages were washed in ice-cold phosphate-buffered saline and lysed for 20 min on ice. The cell lysates were centrifuged at 13,000 rpm for 15 min at 4 °C to collect the supernatants. Total protein in the supernatant was quantified using the bicinchoninic acid assay (BCA1, Sigma-Aldrich). The volumes of cell lysates were standardized to an equal amount of protein (20 μg), mixed with 5X SDS sample containing 50 mM dithiothreitol, separated by 10% SDS-PAGE gels, and transferred onto nitrocellulose membranes. The membranes were blocked in 5% dried milk in TBS-T and 0.1% Tween 20, washed, and incubated with anti-IL-8 (ab18672, Abcam, Cambridge, UK, 0.1 μg/mL), anti-IL-1β (12242, CST, USA, 1:1000), anti-TGF-β1 (ab179695, Abcam, Cambridge, UK, 1:1000), anti-CD163 (ab182422, Abcam, Cambridge, UK, 1:1000), anti-CD206 (ab125028, Abcam, Cambridge, UK, 1:2000), anti-Scavenging Receptor SR-BI (ab217318, Abcam, Cambridge, UK, 1:2000), anti-Arginase-1 (9819, CST, USA, 1:1000), anti-OATP1A2 (ab221804, Abcam, Cambridge, UK, 1:1000) and β-actin (Sigma, Germany, 1:1000). After washing to remove excess antibodies, the membranes were incubated with appropriate secondary antibodies followed by chemiluminescence and fluorescence detection agent (Chemi XT4, Syngene, UK). Specific proteins were quantified by densitometry using the ImageJ analysis program (NIH, USA).