Deferiprone dfp

All the compounds, coming from our in-house MBC library,³² (link) were prepared with a stock concentration of 25 or 10 mM in DMSO. The final % of DMSO in cell culture was not higher than 0.1%. Bafilomycin A1 (Baf1) (Enzo Life Sciences – BML-1100–0100) and okadaic acid (OA) (Sigma – O9381) were also prepared in DMSO. Deferiprone (DFP) (Sigma − 379409) was dissolved in water. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma, C2920) was dissolved in ethanol.

COS-7, HeLa or HEK 293T cells were seeded and transfected as indicated in (24 (link)). In the case of monocistronic vectors, 200 ng of the RLuc plasmid was co-transfected with 200 ng of musFluc, msFLuc, musFLucAAC or msFLucAAC. For transfection of bicistronic plasmids, cells were transfected with 200 ng of bicistronic DNA together with 50 ng of pcDNA 3.1-LacZ plasmid, encoding the β-galactosidase, which was used as a control for the transfection efficiency. At 24 h post-transfection, the culture medium was removed and the cells were harvested using Passive Lysis Buffer supplied with the DLR™ Assay System (#E194A; Promega) according to manufacturer’s protocols. For the poliovirus HRV 2A protease experiments COS-7 cells were cotransfected with 200 ng of the dl sHBZ 5′UTR plasmid and 500 ng of the HRV p2A-wt or HRV p2A-mut per well in a 24-well plate. Deferiprone (DFP) (CAS: 30652–11-0; #379409; Sigma-Aldrich) was used as described in (23 (link)). In these experiments 200 ng of dl sHBZ 5′UTR, dl HTLV-1 IRES or dl PV IRES were co-transfected with 50 ng of pcDNA3.1-LacZ in COS-7 cells. DFP was added 6 h post-transfection at 50 100 or 250 μM and 24 h post-transfection, the culture medium was removed and the cells were harvested.

Roxadustat (FG-4592) was purchased from Selleck (Shanghai, China) and dissolved in a mixed solvent containing 5% DMSO, 40% PEG-300, 5% Tween-80 and 50% ddH₂O according to Selleck in vivo usage instructions for mouse intraperitoneal injection. Rox powder was stored at −20 °C in silver paper to avoid light and ensure dryness, with its solvent prepared immediately prior to use. Rox was dissolved in solvent to reach a concentration of 100 mM and then diluted to concentrations of 5–100 μmol/L with cell culture medium for cell assays. METH was obtained from Shanghai Standard Biotech Co., Ltd. (Shanghai, China) and diluted with saline for i.p. injection (2.0 mg/kg). Morphine was purchased from Shenyang First Pharmaceutical Factory (Shenyang, China) and dissolved in saline for i.p. injection (5.0 or 20.0 mg/kg). Deferiprone (DFP) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phenylmethanesulfonyl fluoride (PMSF) was purchased from Cell Signaling Technology (Danvers, MA, USA). Other common reagents were purchased from Sinopharm (Beijing, China).