Jsh 23

For METTL3 knockdown or overexpression, lentivirus containing specific short hairpin RNA (#1 sh-METTL3 or #2 sh-METTL3, GenePharma, Shanghai, China) or METTL3-overexpressing fragment was transfected into MODE-K cells using polybrene (Invitrogen). 48 h later, the cells were harvested for further investigation. The scramble shRNA was used as a negative control (sh-NC). The sequences were listed in Table S1.
For LPS stimulation, MODE-K cells were incubated with 200 ng/ml LPS (Sigma-Aldrich, USA) for 48 h.
For NF-κB inhibition, MODE-K cells were incubated with 10 μM JSH-23 (Sigma-Aldrich) for 48 h.

R848 (TLR7 ligand) and CpG-B ODN 1826 (murine TLR9 ligand) were purchased from Invivogen (San Diego, CA, USA). Lipopolysaccharide (LPS, TLR4 ligand from Salmonella Minnesota) was from Sigma-Aldrich(St. Louis, MO). Recombinant murine BAFF was from Biolegend (San Diego, CA). ODN2088 and ODN20958 were from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). JSH23 (4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine, Sigma-Aldrich), SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole) (Sigma-Aldrich). Amino-actinomycin D (7-AAD) was obtained from BD Bioscience (San Jose, CA, USA). The non-secreting mouse myeloma cell line SP2/0 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The murine anti-U1-70 K (RNP) hybridoma cell line 2.73 was a gift of Dr. Sally Hoch [17 (link)]. Additional hybridomas included 172.4 (murine anti-CD100, ATCC), 11B11 (rat IgG1 anti-mouse IL-4, ATCC), Pab101 (murine IgG2a anti-SV40 large T antigen, ATCC), 111 (murine IgG1 anti-Ku), and 162 (murine IgG2a anti-Ku).

Recombinant IL-1α/β or cancer cell CM were used to stimulate MRC-5 cells or primary mouse lung fibroblasts from mice (NSG, Il1r1^tm1Roml C57BL/6 or WT C57BL/6). Primary lung fibroblasts were cultured in fibroblast-specific MEMα medium. When treatments included IL-1R blockade or NF-κB inhibition, MRC-5 were seeded in the presence of 20 µg/ml anti-IL-1R1 antibody or Normal Goat IgG control (R&D), or 5 µM JSH-23 (Sigma-Aldrich) or 0.1% DMSO as a vehicle. The next day, medium was aspirated, fibroblasts were washed with 1 ml PBS per well, and 1 ml CM or serum-free MEMα were added. Overall, 1 ng/ml human recombinant IL-1α/β (Peprotech) or 0.1% BSA in PBS as carrier control as well as 20 µg/ml anti-IL-1R1/IgG control or 5 µM JSH-23/ 0.1% DMSO vehicle were added to the respective wells. After 48 h incubation at 37 °C, fibroblasts were washed with PBS and lysed in RLT buffer (Qiagen) for RNA extraction.

Bacterial lipopolysaccharide (LPS) from E. coli serotype 0111:B4, trans-cinnamaldehyde (TCA), and JSH-23 were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS was dissolved in sterile saline. TCA and JSH-23 were reconstituted in dimethyl sulfoxide (DMSO). Anti-CD11b monoclonal antibody (OX-42, microglial marker) was from Abcam (Cambridge, UK). Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute medium (RPMI), and fetal bovine serum (FBS) were purchased from Gibco (Rockville, MD, USA). All other reagents were purchased from Sigma-Aldrich unless otherwise described.

Rat primary embryonic metanephric mesenchymal precursor cells (MM), KSHV-transformed MM cells (KMM)[8 (link)], MM cells infected by KSHV mutants with a cluster of 10 precursor miRNAs deleted (ΔmiRs)[10 (link)], vFLIP deleted (ΔvFLIP)[18 (link)], vCyclin deleted (ΔvCyclin)[12 (link)] and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; with 25 mM glucose, 4 mM L-glutamine and 2 mM sodium pyruvate) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Mo) and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin). Only MM and KMM cells at early passage (<15) were used for the experiments. For glucose starvation, cells were cultured in DMEM without glucose (with 4 mM L-glutamine and 2 mM sodium pyruvate), supplemented with 10% FBS (Sigma-Aldrich). PEL cell lines BCBL1, BC3 and BCP1, and EBV-negative Burkitt’s lymphoma cell line BJAB and KSHV-infected BJAB (BJAB-KSHV) were cultured in RPMI-1640 medium with 10% FBS. JSH-23 (inhibitor of NF-κB nuclear translocation), BAY 11–7082 (an inhibitor of IκBα phosphorylation) and LY294002 (PI3K inhibitor) were purchased from Sigma-Aldrich.