Fig. 4

ZCCHC3 acts at the level of cGAS. a Effects of ZCCHC3-deficiency on cGAMP synthesis induced by transfected HSV120. Zcchc3^+/+ and Zcchc3^−/− MLFs were left untreated or treated with HSV120 (3 μg/ml) for 4 h, and then cell extracts containing cGAMP were separated by chromatography using a C18 column, the abundance of cGAMP was quantitated by mass spectrometry using SRM. The concentration of cGAMP (ng/ml) in Zcchc3^+/+ and Zcchc3^−/− MLFs was shown in the right histograph (mean ± SD, n = 3 technical repeats, **P < 0.01. b Effects of ZCCHC3-deficiency on transcription of downstream genes induced by cGAMP. Zcchc3^+/+ and Zcchc3^−/− MLFs were left un-treated or treated with 2′3′-cGAMP (0.2 mg/ml) for 4 h before qPCR analysis. c ZCCHC3 is associated with cGAS. HEK293 cells were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting analysis. d Colocalization of ZCCHC3 with cGAS. HeLa cells were transfected with GFP-ZCCHC3 and RFP-cGAS for 20 h before confocal microscopy. Scale bars, 10 μm. e PLA analysis of ZCCHC3 and cGAS interaction. Zcchc3^−/− MEFs reconstituted with an empty vector or Flag-ZCCHC3 were infected with HSV-1 or transfected with HSV120 (3 μg/ml) for 4 h before PLA analysis. Scale bars, 20 μm. f Endogenous ZCCHC3 is associated with cGAS in THP1 cells. THP1 cells were left un-infected or infected with HSV-1or transfected with HSV120 (3 μg/ml) for the indicated times before co-immunoprecipitation and immunoblotting analysis. Data are representative of at least two experiments with similar results