Fig 2. TRIM32-mediated alphavirus inhibition is independent of the TRIM32-STING-IFN axis.

A. HeLa-sgSTING or HeLa-sgNT with or without TRIM32 expression were infected VEEV-TC83-GFP at MOI of .01 or 1 for 24h, and viral infectivity was quantified by flow cytometry. The asterisks denote the monoubiquitinated TRIM32. B. Western blot analysis of STING protein expression from the indicated cells. C. Huh7 cells expressing TRIM32-3xF were infected with VEEV-TC83-GFP at 0.1 MOI for 12h, virus infectivity was quantified by flow cytometry. D. HeLa-sgTBK1 or HeLa-sgNT with or without TRIM32 expression were infected VEEV-TC83-GFP at MOI of .01 1 for 24h, and viral infectivity was quantified by flow cytometry. The asterisks denote the monoubiquitinated TRIM32. E. Western blot analysis of IRF9 and STAT1 from the indicated cells. The asterisks denote the monoubiquitinated TRIM32. F. The indicated cells were pretreated with IFNβ at a concentration of 100U for 16h, then infected with VEEV-TC83-GFP at 0.1 MOI for 24h, and viral infectivity was determined by flow cytometry. Data represent averages of independent biological replicates and are presented as means ± SD (n = 2–4). In Statistical significance was determined by unpaired students’ t-test (****P<0.0001). In E, the statistical significance was compared between sgNT/Empty and sgNT/TRIM32-3xF, sgIRF9/TRIM32-3xF, and sgSTAT1/TRIM32-3xF in the presence or absence of INFβ treatment, respectively.