Fig. 5. cGAS-cGAMP-induced TBK1 kinase activity stimulates ATM autophosphorylation.

a Immunoblots for γH2AX, pCHK2, and pATM in THP1 cells pretreated with vehicle or 2 μM TBK1 inhibitor (MRT67307) and then stimulated with cGAMP (+) or vehicle (−) for 16 h. Bands of interest from representative immunoblots from three independent experiments are shown. b Schematic of TBK1 kinase assay. c Endogenous ATM in WT-THP1 cells that were pulled down using immunoprecipitation and used as substrates in kinase assays performed with a recombinant TBK1 protein and radiolabeled γ-ATP. Upper panel shows immunoblot of immunoprecipitated ATM. Lower panel shows autoradiogram of ³²P incorporated into beads bound to endogenous ATM. Bands of interest from representative immunoblots from three independent experiments are shown. d Immunoblot showing phosphorylated ATM from the kinase assay reaction using Phospho-ATM (Ser1981) (IP: ATM beads) antibody. Bands of interest from representative immunoblots from three independent experiments are shown. e Immunoblot showing phosphorylated ATM from the kinase assay reaction with recombinant ATM and TBK1 proteins. The immunoblotting was carried out using Phospho-ATM (Ser1981) antibody. Quantification of pATM bands is presented in the bar graph (n = 4 independent experiments; data presented are mean ± s.d.; two-tailed, paired t test; *p < 0.05 indicates significance compared to respective groups; ns indicates not significant). f Immunoblot showing phosphorylated ATM from the kinase assay reaction using recombinant ATM and catalytically active TBK1, kinase-dead TBK1 (kd-TBK1), or heat-killed TBK1 (HK TBK1) as indicated. Bands of interest from representative immunoblots from three independent experiments are shown. g Immunoblot showing phosphorylated ATM from the kinase assay reaction using recombinant ATM and TBK1 in the presence of inhibitors of ATM or TBK1, as indicated. Bands of interest from representative immunoblots from three independent experiments are shown. h Immunoblot showing phosphorylated ATM from the kinase assay reaction entailing incubation of wild-type (wt) or catalytically dead (kd) ATM with recombinant TBK1. Bands of interest from representative immunoblots from three independent experiments are shown. i Immunoblot showing TBK1 enrichment in ATM immunoprecipitate in cells mock treated or treated with CPT 5 μM, etoposide (ETO) 10 μM, or 2 μg cGAMP for 16 h each. WCE is the whole-cell extract. Quantification of pATM bands is presented in the bar graph (n = 3 independent experiments; data presented are mean ± s.d.; *p < 0.0.016, two-tailed paired t test; ns = not significant; adjustments are made for multiple comparisons). j Interaction of ATM and TBK1 shown by Co-IP analysis. ATM and GFP were immunoprecipitated from GFP-positive HEK293 cells using target-specific or isotype antibodies. The resulting bead-bound ATM and GFP complexes were incubated with recombinant TBK1. The beads with immune complexes were washed and immunoblotted to examine for the presence of TBK1. TBK1 was found in complex with ATM but not with GFP. Bands of interest from representative immunoblots from three independent experiments are shown. k Immunofluorescence imaging of γH2AX and TBK1 in U2OS-STING cells mock treated or treated with etoposide 10 μM for 16 h. Representative images from three independent biological replicates are shown.