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. 2018 Jan 30;14(1):e1006877. doi: 10.1371/journal.ppat.1006877

Fig 6. TDRD7 inhibits autophagy-inducing kinase AMPK, whose activity is required for SeV replication.

Fig 6

(A) L929 cells, transfected with Flag.VPS34 and V5.Tdrd7, were infected with SeV for the indicated time, when VPS34 was immunoprecipitated from the cell lysates and PI3K III activity was analyzed as described in Materials and Methods. (B) L929 cells stably expressing V5.Tdrd7 were transfected with GFP.p40.phox and then serum-starved (SS) for 8 h, when the cells were fixed and analyzed by confocal microscopy. The GFP.p40.phox puncta were counted from at least 100 cells and the results are presented on the right panel. (C) L929 cells stably expressing V5.Tdrd7 were serum-starved for 8 h, when pULK1 (Ser317) was analyzed by immunoblot. (D) L929 cells stably expressing V5.Tdrd7 were serum-starved for 8h, when pULK1 (Ser757) was analyzed by immunoblot. (E) HeLa cells expressing AMPK shRNA were infected with SeV and viral protein (SeV C) expression was analyzed by immunoblot at 16 hpi. Lower panel indicates the AMPK levels in these cells. (F) L929 cells ectopically expressing HA-AMPK (lower panel) were infected with SeV and viral protein expression was analyzed at 16 hpi by immunoblot. (G) L929 cells were pre-treated with various concentrations of Compound C for 1h, and then infected with SeV. Viral protein (SeV C) expression and pAMPK (on Thr172) and AMPK levels were analyzed at 16 hpi by immunoblot. (H) HeLa cells were pre-treated with various concentrations of Compound C for 1h, and then infected with SeV. Viral protein (SeV C) expression and pAMPK (on Thr172) and AMPK levels were analyzed at 16 hpi by immunoblot. (I) L929 cells stably expressing V5.Tdrd7 were serum-starved (SS) for the indicated time, when pAMPK (Thr172) levels were analyzed by immunoblot. (J) L929 cells stably expressing V5.Tdrd7 were infected with SeV for the indicated time, when pAMPK (Thr172) levels were analyzed by immunoblot. EV, empty vector, NT, no treatment, * indicates p<0.05. The results presented here are representatives of at least three biological repeats.