Figure 5.
Rescue of WT ATG7 restores PV-induced autophagy and control of infection. (A) Immunofluorescence images of primary fibroblasts stained for LC3 (red), and 49-6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). The fibroblasts were transduced with lentiviral vectors encoding eGFP, WT ATG7 or ATG7A388T and infected with PV at an MOI of 0.1 for 24 h. Turquoise arrows indicate cytosolic LC3 (LC3-I), and yellow arrows indicate autophagosome-associated LC3 (LC3-II). (B) Quantification of the data shown in (A). The number of LC3 puncta-positive cells was quantified based on a minimum of three different pictures (from different regions of the slide) and 100 cells per slide. Data are shown with SD, n = 2, Students t-test was used for statistical analysis. (C) Western blotting for ATG7 and GAPDH on lysates from primary fibroblasts from P and the control transduced with lentiviral vectors encoding eGFP, WT ATG7 or ATG7A388T. (D) Primary fibroblasts were transduced with lentiviral vectors encoding eGFP, WT ATG7 or ATG7A388T and infected with PV at an MOI of 0.1. Supernatants were harvested following 72 h for measurement of viral yield. Data are shown with SD, n = 2, nonparametric Mann-Whitney rank sum test was used for statistical analysis. (E) Primary fibroblasts were transduced with lentiviral vectors encoding eGFP, WT ATG7 or ATG7A388T and infected with PV at an MOI of 0.1. Supernatants were harvested following 72 h for measurement of viral yield. Data are shown with SD, n = 2. All experiments were performed twice. Students t-test was used for statistical analysis. WT, wild-type; UT, untreated; P, patient; GFP, green fluorescent protein. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001