Pam3csk4

Mouse anti-TLR2 antibody T2.5 (abcam, ab16894), 1 μg/ml Pam3CSK4 (Tocris, 46331, 1 mg/ml stock in water), 10 μM PP2 (EMD Millipore, 529573, 10 mM stock in DMSO), SuperSignal West Dura chemiluminescent substrate for western blots (ThermoFisher, 34075). Western blots were imaged with a BioRad ChemiDoc Touch Imaging System.

Uric acid, lipopolysaccharide (LPS; from Escherichia coli 0111:B4), Brilliant Blue G, pyrrolidinedithiocarbamate (PTDC), and HEPES were purchased from Sigma-Aldrich (St. Louis, MO, USA). Wortmannin was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Acetyl-YVAD-chloromethylketone and TAK242 were purchased from Calbiochem (Rockland, MA, USA). Pam3CSK4 was purchased from Tocris (Bristol, UK). Antibodies against phosphorylated-Akt (p-Akt), Akt, caspase-1 P10, and caspase-1 P20 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against ABCG2, PDZK1, Na/K ATPase, Lamin A/C, GAPDH, β-actin, TLR2, TLR4, MYD88, P2X7, ASC, and nuclear factor-κB (NF-κB) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Penicillin/streptomycin and TRIzol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).

To quantify cell death, lactate dehydrogenase (LDH) activity in cell supernatants was quantified. BMDMs were seeded in triplicates at 2.5x10⁵ in a 24-well tissue culture plate for 24 h and infected with the indicated L. pneumophila strains at an MOI of 10 in 500 μL of seeding media (RPMP/10% HIFBS/7.5% L929 cell supernatant) and incubated for the indicated times. Supernatants were transferred to a 96-well plate and centrifuged at 200 r.c.f. for 10 min. LDH was quantified using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to manufacturer’s instructions. Absorbance at 490 nm was quantified on a BioTek Epoch2 microplate reader and percent cytotoxicity was calculated by normalizing absorbance values to cells treated with lysis buffer. Where indicated, cells were primed with 1 μM PAM₃CSK₄ (Tocris) for 24 h prior to infection and plasmid expression of legC4 was induced with 1 mM IPTG.

BMDMφs and PMφs were cultured for 24 h with human medium oxLDL (100 μg/ml, Kalen Biomedical, no. 770202) or cholesterol (50 μg/ml, Sigma-Aldrich, no. C3045), followed by ultrapure LPS stimulation (10 ng/ml, InvivoGen, no. tlrl-3pelps) for up to 6 h, or CpG (300 nM) (InvivoGen, no. tlrl-1826), Pam₃CSK₄ (10 ng/ml) (Tocris Bioscience, no. 4633), or bpV (HOpic) (10 µM) (Sigma-Aldrich, no. SML0884) stimulation for up to 3 h. Ethanol (0.5%) was used as a carrier control for cholesterol. For inhibitor experiments, MK-2206 (3 µM) (Cayman Chemical, no. 11593), AKT2i (3 µM) (Sigma-Aldrich, no. 124029), Mito-TEMPO hydrate (25 µM) (Cayman Chemical, no. 16621), and bpV (HOpic) (10 µM) were added 1 h prior to LPS stimulation. For assessing the accumulation of oxLDL, a mixture of DiI-oxLDL and oxLDL (10 and 90 μg/ml, respectively) were added to PMφs for 24 h. In all experiments, Mφs were first cultured for 24 h with or without oxLDL or cholesterol. The groups without oxLDL or cholesterol served as controls. The control group for cholesterol loading was cultured with media containing the same concentration of ethanol as the cholesterol group. Cells were then stimulated with LPS from 0 up to 3 h. At each time point, the groups with versus without oxLDL or cholesterol were compared and changes over time were determined.

Recombinant murine IL-1b (#211-11B) was purchased from PeproTech (Rocky Hill, NJ, USA). ZVAD was purchased from Selleck Chemicals (Houston, TX, USA). A mouse TNF-α ELISA kit was purchased from Bangyi (Shanghai, China). Murine anti-GAPDH antibody (BM3876) and secondary antibodies were purchased from Boster (Wuhan, China). Antibodies against RIPK1 (#3493), p-RIPK1 (83613), p-JNK (#9255), p-IkBa (#2859), p-P65 (#3033), TRAF2 (#4712), cleaved PARP (#9544), and cleaved caspase-3 (#9964) were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit antibodies against JNK (24164-1-AP), IkBa (10268-1-AP), p65 (10745-1-AP), matrix metalloproteinase (MMP) 1 (10371-2-AP), and Myd88 (23230-1-AP) were purchased from Proteintech Group (Wuhan, Hubei, China). Antibodies against p-MLKL (ab196436), TRIF (180619), MMP3 (ab53015), and MMP13 (ab39012) were obtained from Abcam (Cambridge, UK). Pam3CSK4 and Poly (I:C) were obtained from Tocris Bioscience (Bristol, UK). A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) apoptosis detection kit was obtained from Beyotime (Shanghai, China).

The A549 cell line (ATCC: CCL-185) was used in this study as a model of type II alveolar epithelial cells. Cells were cultured in Dulbecco Modified Eagle Medium (DMEM), low glucose (1g/L) (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS) and antibiotics (10,000 U/ml penicillin and 10 mg/ml Streptomycin). Cells were seeded in 24-well plates at a final density of 4×10⁵ cells/ml and were stimulated with different concentrations of SARS-CoV-2 Spike-Membrane Recombinant Fusion Protein (10, 20, 50, 100 ng/ml; TP701119, OriGene, Rockville, USA) in the presence or absence of the TLR ligand PAM3csk4 (1μg/ml; Tocris, Bristol, UK) for 6 and 12 hours. In another set of experiments, A549 cells were pre-treated with the selective Akt 1/2/3 inhibitor MK-2206 2HCl (5μM; cat# S1078, SelleckChem, Berlin, Germany) for 24 hours and then stimulated with 50ng/ml SARS-CoV-2 Spike-Membrane Recombinant Fusion Protein for 18 hours to collect cell culture supernatants.