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. 2021 Oct 27;12:6207. doi: 10.1038/s41467-021-26240-9

Fig. 1. cGAMP activates DNA damage response signaling.

Fig. 1

a Schematic of the proposed hypothesis: the catalysis of cGAMP during genomic instability promotes DNA damage response (DDR). b Immunoblots showing the phosphorylation status of DDR signaling proteins H2AX (γH2AX), ATM (pATM), and CHK2 (pCHK2) in THP1 cells stimulated with cGAMP (+) or vehicle (−) for 16 h. Molecular-weight markers (kDa) are indicated to the left of the blots. Quantification of γH2AX, pCHK2, and 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). c Immunoblots for phosphorylated H2AX (γH2AX), CHK2 (pCHK2), and STAT2 (pSTAT2) in THP1 cells stimulated with vehicle, cGAMP, or signaling incompetent linearized cGAMP (Lin-cGAMP) for 16 h. Bands of interest from representative immunoblots from three independent experiments are shown. d Alkaline comet assay was performed to assess DNA damage in THP1 cells that were mock treated, treated with 2 μM doxorubicin, stimulated with vehicle, or stimulated with cGAMP for 16 h. DNA (green) was visualized by staining with Vista Green DNA Dye. While the comet head is composed of intact DNA, the tail consists of genetic fragments and has a length reflective of the amount of DNA damage the cell has sustained. Representative images are presented. Scale bar = 100 μm. e Comets with n = 28 for Mock, n = 23 for Dox, n = 15 for vehicle, and n = 25 for cGAMP group cells per condition were analyzed using OpenComet; quantification of DNA signal intensity in comet tails as a measure of DNA damage is presented (data presented are mean ± s.d.; two-tailed unpaired t test; *p < 0.05 indicates significance compared to respective groups; ns indicates not significant). f Immunoblots for γH2AX, pCHK2, pSTAT2, and pNF-κB (p-p65) in THP1 stimulated with LPS (500 ng/ml, 6 h) from S. minnesota R595 or cGAMP (16 h). Bands of interest from representative immunoblots from three independent experiments are shown. g Immunoblots show phosphorylated H2AX (γH2AX) and NF-κB (p-p65) in WT primary mouse embryonic fibroblasts stimulated with Pam3CSK4 (500 ng/ml, 6 h) or HT-DNA (4 μg/6 well for 6 h). Bands of interest from representative immunoblots from three independent experiments are shown. h Immunoblots for γH2AX and pSTAT2 in WT primary MEF transfected with 5’ppp-dsRNA (0.5 μg/6 well for 6 h) or cGAMP (16 h). Total H2AX (H2AX), Tubulin, and/or β-actin were used as loading controls for immunoblots, as indicated. Bands of interest from representative immunoblots from three independent experiments are shown.