Fig. 4. BAF promotes KSHV reactivation through a cGAS-dependent mechanism.

iSLK.219 cells were transfected with NTC, BANF1, cGAS, or BANF1 and cGAS targeting siRNA at a total siRNA concentration of 100 nM for 48 h prior to the addition of 25 ng/mL doxycycline. A Cell lysates were collected at 72 h post-doxycycline treatment and analyzed by 2’3’-cGAMP ELISA. B Cells were harvested for RNA at 48 h post-doxycycline treatment and subsequent RT-qPCR was performed to determine IFNB1 mRNA expression levels. C Culture supernatant was harvested at 72 h post-doxycycline treatment and analyzed by IFNβ ELISA. D Fluorescent microscopy imaging of RFP and GFP signal was conducted at 72 h after doxycycline treatment. E The fluorescence was quantified by a plate reader. F Cell lysates were prepared at 72 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. G Culture supernatants were harvested 72 h post-doxycycline treatment and DNase treated prior to DNA extraction. DNase-resistant KSHV genomes were quantified by real-time qPCR. H At 72 h post-doxycycline treatment, culture supernatants from iSLK.219 cells were used to infect naive HEK293 cells. At 48 h post-transfer, GFP + infected cells were quantified by flow cytometry. I The cells were also analyzed by fluorescent microscopy. J Cell lysates were prepared at 48 h post-siRNA transfection and analyzed by western blotting with the indicated antibody. P values are the result of two-tailed Student’s T tests unless otherwise specified. Error bars indicate the standard error of the mean of three independent biological replicates. Source data are provided as a source data file.