Fig. 3. Increased BAF expression promotes KSHV reactivation.

iSLK.219 cells were transfected with either pCMV6 (EV) or pCMV6-BANF1-HA (BAF) expression plasmid for 48 h prior to the addition of 25 ng/mL doxycycline. A Cell lysates were collected at 96 h post-doxycycline treatment and analyzed by 2’3’-cGAMP ELISA. B Cell lysates were collected at 72 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. C Cells were harvested and RNA was isolated at 72 h post-doxycycline treatment and subsequent RT-qPCR was performed to determine ISG mRNA expression levels. D Culture supernatant was harvested at 96 h post-doxycycline treatment and analyzed by IFNβ ELISA. E Fluorescent microscopy imaging of RFP and GFP signal was conducted at 96 h after doxycycline treatment and quantified (F) by plate reader. G Cells were harvested for RNA at 96 h post-doxycycline treatment and subsequent RT-qPCR was performed to quantify the expression of viral mRNA transcripts. H Cell lysates were prepared at 96 h post-doxycycline treatment and analyzed by western blotting with the indicated antibodies. I Culture supernatants were harvested at 96 h post-doxycycline treatment and DNase treated prior to DNA extraction. DNase-resistant KSHV genomes were quantified by real-time qPCR. J At 96 h post-doxycycline treatment, culture supernatants from iSLK.219 cells were used to infect naive HEK293 cells. GFP + infected cells were measured at 48 h post-transfer by fluorescent microscopy and (K) quantified by flow cytometry. L 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.