Figure S4.

DUB deficiency does not affect viral DNA release into the cytoplasm. (A) Type I IFN levels in supernatants from PMA-differentiated THP1 (WT), STING^−/−, and cGAS^−/− cells were transfected with dsDNA (4 µg/ml) for 8 h. (B) PMA differentiated THP1 (WT) cells were infected with 5-ethynyl-2′-deoxycytidine (EdC)-labeled HSV1(WT) or HSV1ΔDUB at indicated time points. The cells were fixed and stained for DNA and viral capsid using click chemistry and anti-VP5, respectively. Scale bars, 5 µm. (C) DNA puncta in 6–12 fields were counted using ImageJ. (D and E) HEK293T cells were transfected with cGAS-FLAG, TRAF2m-FLAG for 24 h and infected with HSV1 (WT) or HSV1 ΔDUB for the indicated time intervals. Lysates were immunoprecipitated with anti-FLAG and the eluate was analyzed by (D) immunoblotting for the quality of the precipitation or (E) PCR for viral DNA. Data are shown as HSV1 genome copies per microliter. n = 3. *, P < 0.05; ns, not significant. Error bars represent standard deviation.