Pacritinib

Type-I JAK inhibitors rux (CAT#S1378), BMS-911543 (CAT#S7144), AZD-1480 (CAT#S2162), fedratinib (CAT#S2736), momelotinib (CAT#S2219), and pacritinib (CAT#S8057) were all purchased from Selleckchem. Type-II JAK inhibitor, CHZ-868 (CAT#HY-18960) was purchased from MedChemExpress. Inhibitor stocks (10 mM) were diluted in DMSO so that the final concentration of DMSO in culture media and assays was 0.05–0.1%.

All the chemicals employed in this study were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Anticancer agents, vinorelbine ditartrate (S4269), picropodophyllin (S7668), pacritinib (S8057), and SKLB610 (S6526) were obtained from Selleck Chemicals (Houston, TX, USA).

PMA (Sigma-Aldrich) was used at 0.4-100 ng/ml and ionomycin (Sigma-Aldrich) was used at a concentration of 100 ng/ml. 3-azido-3-deoxythymidine (zidovudine; AZT) (Sigma-Aldrich) was used at 10 µM, raltegravir at 5 µM and efavirenz at 0.32 µM were obtained from the NIH AIDS Research and Reference Reagent Program. TNFα (Merck) was used at a concentration of 0.08-2 ng/ml and LPS at 100 ng/ml (Merck). Fedratinib, TG101209, AZD1480, AZ960, gandolitinib, WP1066, XL109, ruxolitinib, momelotinib, JQ1 were used at a concentration of 0.2-5 µM, pacritinib at 40 nM-1 µM and Q-VD-Oph at 10 µM (all from Selleckchem). Vorinostat (SAHA) was used at a concentration of 0.2-5 µM (Prochifar srl, Italy) and panobinostat at 3.2-400 nM (LC Laboratories).

In this study, the Chinese Academy of Science cell bank (Shanghai, China) provided the mouse microglial cell line, BV2 cells and cultured in high‐glucose Dulbecco's modified eagle medium (DMEM) containing foetal bovine serum (10%, FBS; Gibco, Carlsbad, CA, USA) and penicillin–streptomycin solution (100 U/ml and 100 mg/ml, respectively, Invitrogen, Carlsbad, CA, USA). Cells were maintained with 5% CO₂ at 37°C. The plasmids pcDNA3.1‐TLR4 and pcDNA3.1‐DDX3X were constructed by Asia‐Vector Biotechnology (Shanghai, China). The siRNA sequences and negative control siRNA/shRNA were provided by Sangon Biotech (Guangzhou, China) (Table S1). The plasmids and siRNAs were transfected into microglial cells using Lipo3000 (Invitrogen) following the manufacturer's instructions. Then, the cells were exposed to lipopolysaccharides (LPS, 1 µg/ml for 8 h) and adenosine triphosphate (ATP, 5 mM for 2 h) obtained from Sigma–Aldrich (St. Louis, MO, USA) to induce pyroptosis as previously described,²³, ²⁴ with or without the treatment of pacritinib (SB1518; Selleck Chemicals; 10 µM for 48 h) 24 h after siRNA or plasmid transfection. After 8 h, the mRNA and protein were harvested for subsequent experiments.