Primary antirabbit antibodies

The total protein of tissues and cells was extracted by high-efficiency RIPA lysis buffer (R0010, Solarbio). After centrifugation at 15,000 rpm/min for 15 min and lysis at 4°C for 15 min, the supernatant was harvested, and the protein concentration was evaluated employing the bicinchoninic acid kit (20201ES76, Yeasen Biotechnology, Shanghai, China). Following separation utilizing polyacrylamide gel electrophoresis, the protein was transferred onto PVDF membranes which were sealed at 5% BSA for 1 h at ambient temperature and probed with the diluted primary antirabbit antibodies (all from Abcam) against Sesn2 (ab178518, 1: 10000), Srx1 (ab203613, 1: 10000), Trx1 (ab273877, 1: 10000), Beclin-1 (ab210498, 1: 10000), LC3A/B (ab128025, 1: 1000), Ki67 (ab92742, 1: 10000), Aggrecan (ab3778, 1: 10000), MMP3 (ab39012, 1: 10000), GRP94 (ab238126, 1: 10000), APOB (ab20737, 1: 10000), and GAPDH (ab8245, 1: 10000) overnight at 4°C. The next day, the membrane was reprobed with HRP-labeled goat antirabbit IgG (ab205718, 1: 20000, Abcam) for 1 h at ambient temperature. The developing solution was added for development. ImageJ 1.48 software (National Institutes of Health) was employed for protein quantitative analysis, and protein quantitative analysis was implemented with the gray value ratio of each protein to the internal reference GAPDH.

Cells were incubated on uncoated glass coverslips for duration of 24 hours, and then fixed by means of 4% paraformaldehyde for duration of 15 minutes at room temperature. The adherent cells were permeated in PBS containing 0.1% Triton X‐100, followed by blocking with 1% bovine serum albumin (BSA)/PBS containing 10% normal goat serum for 1 hours and incubation overnight with primary anti‐rabbit antibodies (Abcam, Cambridge, UK) against α‐SMA (1:500, ab32575), FAP (1:500, ab53066), FSP‐1 (1:250, ab124805) and vimentin (1:500, ab193555) at 4°C. Afterwards, cells were incubated with Alexa Fluor^® 647‐conjugated secondary donkey anti‐rabbit immunoglobulin G (IgG) antibody (ab150075, 1:500) for 1 hours. Finally, cells were fixed with Vectashield containing 4′6‐diamidino‐2‐phenylindole (DAPI) and observed under a LSCM.