Thp1 dual cells

THP1-Dual cells (Cat. code: thpd-nfis), THP1-Dual KO-STING cells (Cat. code: thpd-kostg), 293T-Dual hSTING-H232 cells (Cat. code: 293d-h232), 293T-Dual hSTING-R232 cells (Cat. code: 293d-r232), Raw-Lucia (Cat. code: rawl-isg) and Raw-Lucia-KO-STING (Cat. code: rawl-kostg) were purchased from InvivoGen (San Diego, USA). All the cells were incubated following manufacturer’s protocols at 37 ^oC in a humidified atmosphere of 5% CO₂. QUANTI-Luc solution (Cat. code: rep-qlc2) and QUANTI-Blue solution (Cat. code: rep-qbs2) were purchased from InvivoGen (San Diego, USA). Antibodies against phospho-TBK1 (Ser172) (Cat. code: 5483S), TBK1 (Cat. code: 3504S), phospho-IRF3 (Ser396) (Cat. code: 4947S), IRF3 (Cat. code: 4302S), and GAPDH (Cat. code: 5174S) were purchased from Cell Signaling Technology (Beverly, MA). diABZI-compound 3 (Cat. code: S8796) and MSA-2 (Cat. code: S9681) were purchased from Selleck (Houston, USA).

THP1-Dual cells were purchased from InvivoGen and cultured according to the manufacturer’s manual. BMDMs were isolated from mice and differentiated for 5–6 days, following standard procedures^{31 (link)}. Adherent BMDMs were cultured overnight at a density of 1 × 10⁶ cells per ml before being treated with 5, 2.5, 0.5, or 0.25% WE (equivalent to 12.5, 6.25, 1.25, or 0.625 mg MDP residue per ml, respectively) or with the indicated chemicals for 6 h. The cells were then stimulated with TLR ligands at the indicated concentrations^{32 (link)}.

All of the cell lines used in this work were maintained according to supplier specifications. RAW-Dual cells (InvivoGen) were cultured in DMEM (Gibco) supplemented with 4.5 g/L glucose, 2 mmol/L l-glutamine, 10% heat-inactivated FBS (Gibco), 100 U mL⁻¹ penicillin/100 µg mL⁻¹ streptomycin (Gibco), and 100 µg/mL Normocin. To maintain selection pressure of the RAW-Dual cells, 200 µg/mL Zeocin was added every other passage. THP1-Dual cells (InvivoGen) were cultured in RPMI1640 Medium (Gibco) supplemented with 25 mmol/L HEPES, 2 mmol/L l-glutamine, 10% heat-inactivated FBS (Gibco), 100 U mL⁻¹ penicillin/100 µg mL⁻¹ streptomycin (Gibco), and 100 µg/mL Normocin. To maintain selection pressure of the THP1-Dual cells, 10 µg/mL Blasticidin and 100 µg/mL Zeocin were added every other passage. B16.F10 cells (ATCC) and B16.F10 IFN-LUC cells were cultured in DMEM (Gibco) supplemented with 4.5 g/L glucose, 2 mmol/L l-glutamine, 10% heat-inactivated FBS (Gibco), and 100 U mL⁻¹ penicillin/100 µg mL⁻¹ streptomycin (Gibco). To maintain selection pressure of the B16.F10 IFN-LUC cells, 10 µg/mL puromycin was added every passage. All of the cell lines used in this work were cultured in a humidified environment (37°C; 5% CO₂) and routinely tested for Mycoplasma contamination.

Human monocyte THP-1 Dual cells (InvivoGen), THP1-Dual™ KO-STING Cells (InvivoGen) and THP1-Dual KI-hSTING-R232 cells (InvivoGen) were cultured in RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/ml Normocin, Pen-Strep, and 10 µg/ml of blasticidin, 100 μg/ml of Zeocin. Mouse RAW 264.7 cells (ATCC) were cultured in DMEM with 10% heat-inactivated fetal bovine serum and Pen-Strep. MV4-11 cells (ATCC) were cultured in ATCC-formulated Iscove's Modified Dulbecco's Medium with 10% heat-inactivated fetal bovine serum and Pen-Strep. MOLM-16 cells (DSMZ) were cultured in RPMI 1640 with 20% heat-inactivated fetal bovine serum. Frozen human peripheral blood mononuclear cells (PBMCs) and Cynomolgous monkey PBMCs were purchased from Stem Cell Technologies and iQ Biosciences respectively.