FIG 2.

5′pppRNA induces STING expression through secreted proteins. (A, B) A549 cells were transfected with increasing amount of 5′pppRNA, and STING mRNA and protein levels were analyzed by qPCR and Western blotting, respectively. (C, D) A549 cells were transfected with 5′pppRNA (10 ng/ml), and whole-cell extracts were prepared at different times after transfection (time zero to day 4) and then subjected to SDS-PAGE and probed for STING, RIG-I, ISG56, and actin (C) or qPCR analysis (D). (E) Schematic outline of the timeline used for experiments whose results are shown in panel F. (F) A549 cells were transfected with 5′pppRNA for 24 h. Fresh A549 cells were transfected with control siRNA or RIG-I siRNA and then incubated with supernatants from 5′pppRNA-transfected cells for 24 h or 48 h. Cells were collected, and whole-cell extracts were prepared and subjected to immunoblot analyses. Statistical analysis was performed by Student's test. NT, not treated; SN, supernatants.