Amlexanox

Primary antibodies used were: mouse anti-GFAP (Thermo Fisher Scientific), mouse anti-nestin (Rat-401), mouse anti-p62 (Abcam), rat anti-phosphorylated p62 (MBL: D343-3), mouse anti-ubiquitin (Santa Cruz Biotechnology), rabbit anti-TBK1 (Cell Signaling), rabbit anti- phosphorylated TBK1 (Novus Biologicals), rabbit anti-GFAP (Dako), rabbit anti-Ki67 (Spring Bioscience), rabbit anti-p62 (Enzo), rabbit anti-Sox2 (Millipore), rat anti-Ki67 (BioLegend), and guinea pig anti-doublecortin (anti-DCX; EMD Millipore). Secondary antibodies were goat anti–rabbit IgG-FITC, goat anti–rabbit IgG–Texas red, goat anti–mouse IgG-FITC, goat anti–mouse IgG–Texas red, goat anti–mouse IgG-HRP, and goat anti–rabbit IgG-HRP (Jackson Immunology). Dihydroethidium (DHE) was purchased from Sigma-Aldrich and Amlexanox was purchased from MedChemExpress. Transfections were carried out using Lipofectamine 3000 reagent (Invitrogen).

Human primary RA synovial cells were treated for 3 h with 100 μM amlexanox (MedChem Express), previously identified to be a GRK5 inhibitor^{24 (link)}. After amlexanox treatment for 3 h, cells were stimulated with LPS (1 μg/mL) for 6 h.

Stocks of aminoglycoside antibiotics (All purchased from Sigma-Aldrich, St. Louis, MO, USA) were dissolved in sterile H₂O at a 100 mg/mL concentration, and stored at 4 °C. PTC124 (3-[5-(2-Fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid, MedChemExpress, Cat. #HY-14832), escin (Sigma-Aldrich, Cat. #E1378), VX-661 (MedChemExpress, Monmouth Junction, NJ, USA Cat. #HY-15448), NMDI-14 (Ethyl 2-(((6,7-dimethyl-3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetyl)amino)-4,5-dimethyl-3-thiophenecarboxylate, EMD Millipore, Burlington, MA, USA, Cat. #530838), and amlexanox (MedChemExpress Cat. #HY-B0713) were dissolved in dimethylsulphoxide (DMSO). SMG1i (2-chloro-N,N-diethyl-5-((4-(2-(4-(3-methylureido)phenyl)pyridin-4-yl)pyrimidin-2-yl)amino)benzenesulfonamide) was received from Dr. Robert Bridges from Rosalind Franklin University of Medicine and Science through the CFFT compound distribution program, and was dissolved in DMSO. Working solutions were all ≤0.1% DMSO, with the exception of NMDI-14, which was 1% DMSO. The increased DMSO did not prevent G418-facilitated FIS (Supplementary Figure S9). Stocks of forskolin (Sigma-Aldrich, Cat. #F6886) were dissolved in 100% EtOH and stored at −20 °C.

Products from the following vendors were used as reagents: MSA-2 (MedChemExpress, catalog no. HY-136927); phorbol-12-myristate-13-acetate (PMA) (Beyotime, catalog no. S1819); fostamatinib (MedChemExpress, catalog no. HY-13038A); and amlexanox (MedChemExpress, catalog no. HY-B0713). In addition, antibodies from the following manufacturers were used: TBK1 (Beyotime, catalog no. AF8103); p-TBK1 (Cell Signaling Technology, catalog no. 5483 T); p-p65 (Cell Signaling Technology, catalog no. 3303 T); p-IRF3 (Cell Signaling Technology, catalog no. 29047S); TDP-43 (HUABIO, catalog no. ET1703-74); SOD1 (HUABIO, catalog no. ET1702-36); β-actin (Cell Signaling Technology, catalog no. 3700S); STING (Proteintech, catalog no. 19851–1-AP); cGAS (HUABIO, catalog no. HA500023); p-SYK (Cell Signaling Technology, catalog no. 2710S); Flag (Beyotime, catalog no. AF2852); IKK epsilon (Zenbio, catalog no. R382375); IRF3 (HUABIO, catalog no. ET1612-14); and NF-κB p65 (HUABIO, catalog no. ET1603-12).

GFP-AZI2 -4OHT cells were seeded in 24-well plates and left overnight before treatment with respective inhibitors. Upon treatment, images of cells were acquired every 2 h with an Incucyte live-cell imaging system (Essen Bioscience) to monitor GFP-AZI2 puncta formation. Inhibitor concentrations used in this screen were; SB203580, 10 µM (MedChemExpress, HY-10256); Tozasertib, 5 µM (MedChemExpress, HY-10161); Chloroquine, 100 µM (Sigma Aldrich, C6628); GDC0994, 5 µM (Cayman Chemical, 21107); Prexasertib, 5 µM (MedChemExpress, HY-18174); LTURM34, 10 µM (MedChemExpress, HY-101667); GSK650394, 5 uM (MedChemExpress, HY-15192); Amlexanox, 100 µM (MedChemExpress, HY-B0713); U0126, 10 µM (Cayman Chemical, 70970); IRAK 1–4 Inhibitor I, 10 µM (MedChemExpress, HY-13329); SP600125, 25 µM (MedChemExpress, HY-12041); SBI0206965, 10 µM (Cayman Chemical, 18477); BMS345541, 10 µM (MedChemExpress, HY-10519); Triciribine, 10 µM (MedChemExpress, HY-15457); Silmitasertib, 10 µM (MedChemExpress, HY-50855); PP242, 5 µM (MedChemExpress, HY-10474); Lys05, 20 µM (provided by Dr. Ravi Amaravadi); Palbociclib, 5 µM (MedChemExpress, HY-50767); and D4476, 50 µM (MedChemExpress, HY-10324). The puncta/image at 24 h were quantified and plotted.