THP1 and MEF Cell Stimulation

THP1 cells were treated with 500 ng/ml Ultrapure LPS from Escherichia coli 0111: B4 (Invivogen, Cat# tlrl-3pelps) or Salmonella minnesota (Invivogen, Cat# tlrl-smlps) in standard RPMI complete media. Similarly, THP1 cells in RMPI complete media were stimulated with 500 ng/ml Pam3CSK4 (Invivogen, Cat# tlrl-pms). Primary MEFs were transfected with 0.5 μg of 5′ppp-dsRNA or 4 μg HT-DNA per well of a 6-well plate using with Lipofectamine 2000 as described earlier^{117 (link)}. Dox (Sigma, Cat# D1515) was supplemented in the THP1 and MEF culture media at 0.5–2 μM. CPT (Cayman Cat# 11694) was used at a concentration of 1–5 μM for the indicated time periods. Cells were exposed to 5 Gy IR and harvested 1 h post-IR. THP1 cells were pretreated with 2 μM TBK1 inhibitor MRT67307 (EMD Millipore, Cat# 506306) or dimethyl sulfoxide vehicle control as described earlier^{118 (link)} preceding cGAMP treatment. THP1 cells were stimulated with recombinant human IFN-β (0.5–100 ng/ml) (Peprotech, Cat# 300-02BC) in culture media. Cells were pretreated with 25 μM ATM inhibitor KU-55933 (Sigma Cat# SML1109), 10 μM PARP inhibitor olaparib (Cayman Cat# 10621), or 10 μM PARP1 inhibitor rucaparib (Cayman Cat# 15643) for 2 h. Cells were harvested at the indicated time points for analysis. Cellular NAD⁺ levels were boosted by preincubating cells for 24 h with 2–4 mM NAM (Sigma Cat# 47865-U).