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. 2022 May 18;21(7):100247. doi: 10.1016/j.mcpro.2022.100247

Fig. 2.

Fig. 2

Oncogenes disproportionately downregulate dsRNA sensing compared to dsDNA sensing pathways at proteomic and functional level.A, volcano plot depicts dsRNA sensor, dsDNA sensor, and adaptor protein fold changes and p values for oncogene versus nononcogene cells. Each data point is labeled by the protein and the associated tumor model. dsRNA sensors are labeled in blue text. B–E, cells with or without oncogenes were treated with transfection agent alone (B) or complexed with polyI:C (C), salmon dsDNA (D), or cGAMP (E). Phosphorylation at Ser172 of TBK1 was immunoblotted. Densities were normalized to the value for respective EV cells for each treatment. Bar graphs represent mean and standard deviation for five biological replicates. Statistics were calculated using Student’s t test between EV and oncogene. F, cells with or without oncogenes were treated with transfection agent alone or complexed with polyI:C. Transcript level of IFNβ relative to GUSβ reference gene was quantified by qPCR. Extent of IFNβ induction was calculated as the fold change in IFNβ mRNA between polyI:C treatment and transfection agent alone. Data are normalized to induction value of EV. Bar graphs represent mean and standard deviation of at least three biological replicates. Statistics were calculated using Student’s t test between EV and oncogene. G–H, cells with or without oncogenes were treated with transfection agent alone or complexed with polyI:C and activation of RNASEL was quantified by capillary electrophoresis. G, representative capillary electrophoresis experiment for LHS EV and LHS MYC cells. Cleavage of RNA was quantified by RINe values. H, the reported ΔRINe in bar graphs is the difference between RINe(Oncogene)—RINe(EV). Bar graph reports mean and standard deviation of at least two biological replicates. RINe values are tabulated in supplemental Fig. S3G. ∗p ≤ 0.05, ∗∗p ≤ 0.005, ∗∗∗p ≤ 0.0005. dsRNA, double-stranded RNA; dsDNA, double-stranded DNA; ISG, interferon-stimulated gene; IFN, interferon; T1IFN, type 1 interferon.