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. 2018 Sep 10;15(1):58–77. doi: 10.1080/15548627.2018.1507439

Figure 9.

Figure 9.

Rescue of ATG7 normalized NFE2L2 and DDIT3 levels and restores GH-IGF1 functions in atg7Δ nSCs. WT and atg7Δ nSCs were infected with control lentivirus (LV ctr) or lentivirus expressing ATG7 (LV Atg7). (a) Representative immunoblot analysis of ATG7, MAP1LC3, SQSTM1 and GAPDH, as a loading control. (b) Representative SQSTM1 immunostaining (red). DAPI is used for nuclear counterstaining (blue); phalloidin (ThermoFisher Scientific, A22287) is used for cell outline (gray) (scale bar: 10 µm). (c) Percentage of MKI67+ cells of total DAPI nuclear counterstaining (n = 3 experiments). (d) Mean number of myonuclei/myotube (left panel) and percentage of myotubes with 5 or more nuclei (right panel) (n = 3 experiments). (e) RT-qPCR analysis of myogenic markers Myog, Myod1 and Myh2 (n ≥ 3 experiments). (f) Representative immunoblotting analysis of ATG7, MAP1LC3, NFE2L2 and GAPDH as a loading control (n = 3 experiments). (g) Representative DDIT3 nuclear immunostaining (red) and DAPI as nuclear counterstaining (blue) (scale bar: 25 µm). (h) RT-qPCR analysis of Ddit3, Ghr and Igf1 (n ≥ 3 per treatment). * vs WT nSCs infected with LV ctr (* P < 0.05, ** P < 0.01, *** P < 0.001); + vs atg7Δ nSCs infected with LV ctr (+ P < 0.05, ++ P < 0.01). Values are expressed as mean ± SEM.