1. Fig. 1 Cellular PFAS negatively regulates RTA-dependent transcriptional activation.
Cellular PFAS negatively regulates RTA-dependent transcriptional activation. (A) 293T/RTA cells were infected with lentivirus carrying control (CTL) shRNA or shRNA targeting indicated GAT. At 48 hours after infection, cells were infected with rKSHV.219 and induced with doxycycline for RTA expression. Representative RFP+ and GFP+ cells of the screen were recorded at 24 hours after KSHV infection. Scale bars, 5 μm. (B) RFP+ cells from (A) were quantified at 48 hours after infection by counting five randomly selected areas. The results are shown as the mean ± SD of three independent experiments in duplicate. n = 3. (C) 293T cells were transfected with plasmids containing RTA or the indicated GAT, and a reporter plasmid cocktail. RTA-mediated transcriptional activation of the PAN promoter was determined by luciferase activity at 30 hours after transfection. (D) 293T cells were infected with lentivirus carrying control shRNA (CTL) or shRNA against PFAS and selected with puromycin. Stable 293T cells were transfected with a plasmid containing RTA and a reporter plasmid cocktail. RTA-mediated transcriptional activation of the PAN promoter was determined by luciferase assay at 24 hours after transfection. For (C) and (D), the data are shown as the median ± SD of three independent experiments in duplicate (n = 3). **P < 0.01 and ***P < 0.001, unpaired two-tailed Student’s t test. (E to H) iSLK/rKSHV.219 cells were infected with lentivirus containing control (CTL) shRNA or shRNA against PFAS. Cells were induced with doxycycline (1.0 μg/ml) for the indicated times. When cells were harvested, total RNA was extracted for reverse transcription and RT-PCR analysis with primers specific for TK (or ORF21) and vGAT (or ORF75) (E), whole-cell lysates (WCLs) were analyzed by immunoblotting with antibodies against indicated viral and cellular proteins (F), viral genome copies were quantified by RT-PCR (G), and viral titer in the medium was determined by flow cytometry analysis of a KSHV-infected 293T monolayer (H). For (E) to (H), the data represent three independent experiments (n = 3). For (E), (G), and (H), the results are shown as the median ± SD of three independent experiments. PFU, plaque-forming units.
Junhua Li et al. Sci Adv 2019;5:eaaw7373
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