Fig. 3. Mitotic hyperphosphorylation suppresses cGAS activity.

. (A) HeLa cells stably expressing cGAS-Flag were synchronized with thymidine and nocodazole or center untreated (async). cGAS-Flag proteins isolated from these cells were analyzed using PhosTag electrophoresis (left) or an in vitro enzymatic assay (right). (B) THP-1 cGAS^KO cells stably expressing cGAS^WT, cGAS^20A, or cGAS^20DE were transfected with HT-DNA. The level of cGAMP was measured at 4 hours after transfection. (C) 5 μM recombinant cGAS^WT, cGAS^20A, or cGAS^20DE was incubated with 5 μM 100 bp dsDNA in a physiological buffer at 37°C for the indicated time. Phase separation was visualized using bright field microscopy. Size bars = 10 μm. (D) STING-deficient BJ-5ta cells stably expressing Flag tagged cGAS^WT, cGAS^20A, cGAS^R236E or cGAS^{R236E, 20A} were synchronized with thymidine and nocodazole or center untreated (async). Hypotonic lysates of resulting cells were incubated with ATP, ¹⁵N₅-GTP, and with or without HT-DNA at 37°C for 1 hr to measure the cGAS product ¹⁵N₅-cGAMP with quantitative mass spectrometry (upper). PhosTag electrophoresis shows the level of cGAS phosphorylation and immunoblotting shows the level of pH3S10 (lower). (E) The levels of ifnβ and cxcl10 RNA in HeLa cGAS^KO cells stably expressing C-terminally 3xFlag tagged cGAS, cGAS^20A, or cGAS^20DE were quantified by RT-qPCR. (F) The growth curves of the cells shown in (E). (A, D-F) Data represent mean ± SD (N=3). Unpaired t-test: ns, not significant; *, p≤0.05; **, p≤0.01; *** p≤0.001.