Fig 4. Depletion of the m⁶A writer and readers does not impact ORF50 nascent transcription in iSLK.219 cells.

(A) Schematic of the experimental setup for measuring nascent RNA synthesis. Cells were transfected with the indicated siRNAs for 48 hr then reactivated for 24 hr with dox. 4sU was added for 30 minutes, whereupon 4sU-labeled RNA was isolated using biotin/streptavidin affinity purification, reverse transcribed, and analyzed by RT-qPCR using primers specific to ORF50 or ORF37. (B-D) Levels of 4sU-labeled total ORF50 (B), ORF37 (C), and ORF50 transcribed from the viral genome (virus-derived) (D) determined as described above. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values <0.05 (*), ≤ 0.01 (**), and ≤ 0.001 (***).