Fig 2. Upregulation of dsRNA sensors in 293FT TMEM41B KO and VMP1 KO cells upon DENV infection.

(A) Western blotting analysis to detect RIG-I, MDA5, TBK1, and p-TBK1 protein levels in 293FT WT cells, TMEM41B KO clone #1, and VMP1 KO clone #1, upon DENV infection. Cells were infected with DENV1 at MOI of 1 and lysates were harvested at 48 hours post-infection. (B) Western blotting analysis to detect RIG-I and MDA5 protein levels in 293FT WT, TMEM41B KO clone #1, and VMP1 KO clone #1 cells, upon HCoV-OC43 infection. Cells were infected with HCoV-OC43 at MOI of 0.5 and lysates were harvested at 48 hours post-infection. (C,D) IFN-β mRNA (C) and DENV RNA (D) abundance in uninfected and DENV1 infected (MOI of 1, 48 hours post-infection) WT and KO cells. Values demonstrated are relative to the expression levels in uninfected WT cells (C), or DENV infected WT cells (D). (E,F) DENV viral protein accumulation and infectious viral particles produced in 293FT WT, TMEM41B KO clone #1, and VMP1 KO clone #1 cells, upon further knocking out of RIG-I and MDA5. Infection assays were carried out at MOI of 1 and (E) the amount of infectious virus in the supernatant was determined at 48 hours post-infection by plaque assay, and (F) protein levels of the indicated proteins were determined by western blotting analysis. GAPDH was used as a loading control for all blots. 18S rRNA was used as the control for the RT-qPCR experiments. All data shown represent results from at least two independent experiments. Single-dashed lines divide blots from two gels loaded with the same set of lysates. Double-dashed lines divide blots from gels belonging to separate experiments using the same experimental conditions. Error bars represent mean +/- SEM, n = 3. * indicates p-value < 0.05 as determined by one-way ANOVA. LOD indicates the limit of detection.