Chemiluminescence analyzer

Human primary HCC tissues and cells were lysed with RIPA buffer (Thermo Scientific, MA, USA) supplemented with a protease inhibitor cocktail and phosphatase inhibitor (Roche, Welwyn Garden, Swiss). After the proteins were quantified and denatured, samples were separated by SDS-PAGE electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Massachusetts, USA). The membranes were blocked with 5% nonfat milk solution for 1 h at room temperature, incubated with primary antibody overnight at 4 °C and then reacted with HRP-conjugated secondary antibody for 1.5 h at room temperature. The protein bands on the membranes were visualized by a Pierce ECL development system (Thermo Scientific, MA, USA) via a chemiluminescence analyzer (Bio-Rad, CA, USA). β-actin was used as an internal loading control for all the western blot experiments. The antibodies used are listed in Table S2. The CXCL12 levels in the cultured supernatants of HCC cells were measured by ELISA (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions.

The western blot technique was used to detect the protein expression of HaCaT cells. At the end of the treatment, the cells were washed with PBS 3 times and lysed with RIPA buffer for 30 min. The concentration of protein was determined by the BCA Protein Assay Kit (Elabscience, Wuhan, China). The protein samples were subjected to SDS-PAGE gel electrophoresis and then transferred to PVDF membranes. The blots were incubated overnight at 4 ℃ with the following primary antibodies: GADPH (ab8245, Abcam, 1: 5000), p53 (ab32389, Abcam, 1: 5000), MPO (ab208670, Abcam, 1: 1000), TNF-α (ab183218, Abcam, 1: 2000), IL-1β (ab254360, Abcam, 1: 1000), MMP-1 (ab134184, Abcam, 1: 1000) and MMP-3 (ab52915, Abcam, 1: 5000). The membranes were visualized using a chemiluminescence analyzer (Bio-Rad, Hercules, CA, USA). The relative density of protein bands was analyzed using the ImageJ software, and optical density values were normalized to GADPH.

Total proteins extracted from the cells were lysed with RIPA buffer (Thermo Scientific, USA) and separated on 8%–12% SDS-PAGE gels, transferred onto polyvinylidene fluoride membranes (Millipore, USA), incubated with primary antibodies overnight at 4°C, probed with horseradish peroxidase-conjugated secondary antibodies, and visualized using enhanced chemiluminescence reagent (Pierce, USA) via a chemiluminescence analyzer (Bio-Rad, USA). Information on the antibodies is listed in Table S3.