Ba410e

Cells were seeded on glass coverslips and incubated with the corresponding serum. Then, the coverslips were fixed with 4% paraformaldehyde for 30 min at 37°C, rinsed three times with PBS, treated with 0.1% Triton X-100 at 37°C for 30 min and subsequently washed three times with PBS. Following blocking with 5% bovine serum albumin for 1 h at 37°C, the coverslips were incubated with diluted primary antibody [CD206 (1:100), CD54 (1:100)] overnight at 4°C. After washing three times with PBS, the coverslips were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG and goat anti-rat IgG H&L for 1 h at 37°C in the dark. The nuclei were stained with DAPI, and the cells were imaged under a fluorescence microscope (Motic, BA410E).

Synovial tissue samples were obtained from RA patients who had undergone joint replacement surgery. Briefly, the samples were cut into small fragments and cultured in Dulbecco's modified Eagle medium (DMEM) medium containing penicillin (100 U/ml) and streptomycin (0.1 mg/ml). The immortalized cell line of RA‐FLS was obtained by using lentivirus vector‐mediated SV40+ T antigen transfection.¹⁷ The second generation of the cells was used for identification, and the level of Vimentin protein was identified by immunofluorescence (IF) cytochemistry. RA‐FLS were thereafter fixed by paraformaldehyde and incubated with Triton X‐100 for 0.5 h. The primary anti‐vimentin (Bs‐8533R; Bioss) was then added and incubated at 4°C for 8 h. The secondary antibody (1:400, goat anti‐rabbit) was then added to all the sections and incubated for 1 h. After DAPI staining, the tablets were sealed and observed under a fluorescence microscope (BA410E; Motic). Based on this preliminary study, 3–5 generations of cells were selected for the subsequent experiments. FLS for NC (NC‐FLS, HUM‐iCell‐s010; iCell Bioscience Inc.) were cultured in DMEM medium containing 10% FBS (sh30256.01; Hyclone).

RA‐FLS was fixed in the cold acetone for 10 min and then in 0.1% Triton X‐100 permeable membrane for 0.5 h. Thereafter, the goat serum was added to the block for 0.5 h. After removing the goat serum, anti‐Nrf2 (1:400, SC‐365949; Santa Cruz), and anti‐HO‐1 (1:400, SC‐390991; Santa Cruz) antibodies were added and incubated at 37℃ for 1 h. All the sections were stained with corresponding secondary antibodies (1:400) and incubated for 20 min followed by DAPI (C1005; Beyotime) staining for 5 min. Finally, the fluorescence images of cells were captured under a fluorescence microscope (BA410E; Motic).