Figure 6: Cordycepin activates interferon through RIG-I-MAVS signaling pathway.(A) and (B) HT29 cells infected with SA11 (2 M.O.I.) were treated with cordycepin and were harvested at 3 hpi and 12 hpi for real time PCR and western blotting. (A) Immunoblotting confirmed induction of RIG-I protein in presence of cordycepin (64μM). GAPDH was used as an internal loading control. (B) Results show induction of rig-I transcripts in presence of cordycepin in both uninfected and rotavirus infected cells. Simultaneously, HT29 cells were incubated with Poly-IC (10 μg/ml) as a positive control. Fold changes were obtained by normalizing relative gene expression to gapdh using formula 2-ΔΔC T (ΔΔCT=ΔCT Sample-ΔCT Untreated control). Results shown are mean of three independent experiments (P < 0.05). (C) HT29 cells infected with SA11 (2 M.O.I.) were treated with cordycepin (64μM) for 3 hpi and 8 hpi. Whole-cell extracts were immunoprecipitated with anti-MAVS antibody followed by immunoblotting with anti-RIG-I antibody. CO-IP revealed positive interaction between RIG-I and MAVS in cordycepin treated cells. For analyzing expression of proteins in input, cell lysates were immunoblotted with anti-MAVS and anti-RIG-I antibody. (D) 293T cells were transfected with pEF-BOS-RIG-I followed by cordycepin or Poly-IC treatment (10 h). Whole cell lysates were immunoprecipitated with anti-FLAG antibody (for RIG-I). Western blotting with anti- MAVS antibody indicates interaction of FLAG tagged RIG I and MAVS in cordycepin and Poly-IC treated cells. Whole cell lysates were immunobloted with anti- MAVS and anti-FLAG antibody to assess expression of MAVS and RIG-I in input. (E) 293T cells were transfected with siRIG-I and control siRNA (scrambled siRNA) followed by SA11 infection and treatment with either cordycepin or Poly-IC. Total RNA was isolated by TRIZOL to quantitate ifn-β mRNA levels over control cells. Reduction of ifn-β transcripts was observed in RIG-I siRNA treated cordycepin or Poly-IC treated cells compared to control siRNA transfected cells. Results shown are mean of three independent experiments (P < 0.05). Reduced expression of RIG-I in siRIG-I transfected cells was confirmed by immunoblot. (F) Total RNA was isolated from 293T and 293T MAVS-/-cells, infected with SA11 (2 M.O.I.) followed by treatment with cordycepin (64 μM) and expression of ifn-β was measured by real time PCR. Results are representations of mean of three independent experiments (P < 0.05) Reduced MAVS expression in 293T MAVS-/- cells was confirmed by immunoblotting with anti-MAVS antibody. GAPDH was used as an internal loading control.