The image contains four panels (A, B, C, and D) of Western blot and R N A profiling data. Panel A shows R N A profiling results with visible 28 S and 18 S r R N A bands under W T, D B R 1 K O, and D B R 1 K O plus D B R 1 conditions, each treated with poly I:C either negative or positive. The 28 S and 18 S bands appear intact across all lanes, indicating no obvious rRNA degradation after four hours of poly I:C treatment. Panel B presents western blot analysis showing decreased phosphorylation levels of P K R and e I F 2 alpha in D B R 1 knockout cells compared to wild-type cells after poly I:C treatment. In W T cells treated with poly I:C, P-P K R is 48 percent, and P-e I F 2 alpha is 100 percent, whereas in D B R 1 K O cells treated with poly I:C, P-P K R is 14 percent and P-e I F 2 alpha is 54 percent. Total P K R, total e I F 2 alpha, and beta-Actin serve as loading controls and remain relatively constant. Panel C displays western blot results in D B R 1 K O cells with and without D B R 1 add-back following poly I:C treatment. Phosphorylation levels increase upon D B R 1 re-expression, with P-P K R values of 0 percent, 20 percent, and 42 percent, and P-e I F 2 alpha values of 10 percent, 26 percent, and 51 percent, indicating restoration of signaling activity. Panel D shows western blot results demonstrating that phosphorylation of P K R and e I F 2 alpha occurs specifically in response to A l u d s R N A. P-P K R reaches 81 percent, and P-e I F 2 alpha reaches 19 percent with A l u d s R N A, while minimal phosphorylation (0 to 1 percent) is observed with No R N A, A l u plus s s R N A, or A l u s s R N A. Total P K R, total e I F 2 alpha, and beta-Actin are shown as controls. All values are approximated.
DBR1 loss suppresses, while Alu dsRNA activates, innate dsRNA-sensing pathways. (A) Total RNA profiling using an Agilent Bioanalyzer. The absence of rRNA degradation was observed in DBR1 KO cells following 4 h of poly I:C treatment, whereas rRNA degradation was restored upon add-back of DBR1 (DBR1 KO +DBR1) in the presence of poly I:C. Data are representative of three independent experiments. (B) In DBR1 KO cells, the phosphorylation levels of PKR and eIF2α were significantly decreased compared with those in WT cells (HEK293T) following 2 h of poly I:C treatment, as determined by western blot analysis. Data are representative of three independent experiments. (C) The phosphorylation levels of PKR and eIF2α significantly increased upon add-back of DBR1 following a 1-h treatment with poly I:C, as determined by western blot analysis. Quantification of phosphorylated PKR and eIF2α was performed using ImageJ. Data are representative of three independent experiments. (D) Western blot results showed that the phosphorylation of PKR and eIF2α occur specifically in response to Alu dsRNA. Data are representative of three independent experiments. Source data are available for this figure: SourceData F4.