Penicillin streptomycin

RAW264.7, HEK293T cells, RAW-Lucia™ ISG cells (rawl-isg, Invivogen); RAW-Lucia™ ISG-KO-TBK1 cells (rawl-kotbk, Invivogen) and BMDMs were cultured in DMEM (G4512, Servicebio) with 10% (vol/vol) FBS (10099141, Gibco) and 100 units/mL penicillin/streptomycin (15140122, Gibco). HeLa cells were cultured in MEM (G4551, Servicebio) with 10% FBS and 100 units/mL penicillin/streptomycin. Cell lines were verified to be free of mycoplasma contamination, and the identities were authenticated by STR profiling. All cells were cultivated in a 37 °C incubator with 5% CO₂.
Regarding the in vitro culture of PBMCs, cells were cultured in complete RPMI 1640 medium (G4532, Servicebio) with 100 units/mL penicillin, 100 μg/mL streptomycin, and 20% serum from six randomly selected patients with sepsis or age- and sex-matched healthy control (HCs) and incubated at 37 °C. For experiments involving HET0016 treatment, 5 μM HET0016 was added to PBMCs, and PBMCs were cultured for 24 h before further analysis.

The human ovarian cancer cell lines SK-OV-3 and ES-2 were purchased from American Type Culture Collection (ATCC) and were cultured in McCoy’s 5 A medium (Sigma-Aldrich) supplemented with 10 % fetal bovine serum (FBS) (Gibco) and 100 units/ml penicillin/streptomycin (Servicebio, Wuhan). All cells were maintained at 37 ℃ and 5 % CO₂.

SCs were obtained from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China) to evaluate the impact of composite conduits on nerve regeneration. The SCs were cultivated on the nanofibers using high‐glucose DMEM (Gibco, USA) complete medium, supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin‐streptomycin (Servicebio, Wuhan, China). The SCs were cultured at a temperature of 37 °C in a CO₂ culture incubator.

PHL (purity>98%) was commercially purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorogenic probe, Torin2 (SML1224), and Chloroquine (CQ, C6628) were obtained from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from ExCell Biological Technology (Shanghai, China). Dulbecco's Modified Eagle Medium F-12 (DMEM/F-12), penicillin/streptomycin, Dulbecco's Phosphate-Buffered Saline (DPBS), and pancreatin were obtained from Servicebio Technology (Wuhan, China). CCK-8 assay kit, Hoechest33258, and JC-1 stain were bought from Beyotime Biotechnology (Shanghai, China). AnnexinV/propidium iodide was purchased from Solarbio Science & Technology (Beijing, China). Antioxidant capacity assay kits were obtained from Nanjing jiancheng Bioengineering Institute (Nanjing, China). Primary antibodies against LC3 (A19665) and p62 (A7758) were purchased from ABclonal. Bax (50599-2), Bcl-2 (12789-1-AP), and cleaved Caspase-3 (19677-1-AP) were from proteintech. Goat anti-rabbit IgG-HRP (A21020) and Goat anti-mouse IgG-HRP (A21010) conjugated secondary antibody were purchased from Abbkine. Other reagents were analytically pure.

We isolated synovial tissues from Wistar rats (8 weeks, males) to isolate the primary synoviocytes. After mincing, synovial tissues were placed into DMEM/F12 medium with 0.2% collagenase II for 4 h at 37 °C. Synoviocytes were collected by filtration and centrifugation and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (Gibco, NY, America, 10270-106) and 100 U/mL of penicillin–streptomycin (Servicebio, Wuhan, China, G4003) in an incubator at 37 °C with 5% CO₂. For the multiple passaged senescent synoviocytes model, synoviocytes were subcultured for 6–8 passages till the growth rate decreased significantly [55 (link)]. For the H₂O₂-elicited senescent synoviocytes model, synoviocytes were treated with 500 μM H₂O₂(Aladdin, Shanghai, China, 7722-84-1) for 4 h [56 (link)].

A549, H838, HCC827 and H1299 NSCLC cells were obtained from the ATCC and characterized using short tandem repeat markers (Genetic Testing Biotechnology Corporation, Suzhou, China). The cells were cultivated in DMEM comprising penicillin‐streptomycin (Servicebio, Wuhan, China) and 10% FBS in a 5% CO₂ incubator at 37°C. The cisplatin‐resistant A549 and H1299 cell lines were established by ourselves and stored in our laboratory.³³, ³⁴ Cisplatin‐resistant cells were cultured in complete medium supplemented with 1 μg/ml DDP. Lipofectamine 2000 (Invitrogen, USA) was employed to transfect these cells based on provided directions.