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. 2016 Jun 22;35(15):1656–1676. doi: 10.15252/embj.201694401

Figure 4. C9orf72 regulates translocation of the ULK1 complex.

Figure 4

  1. HEK293 cells were transfected with non‐targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho‐ULK1 (Ser757), total ULK1, and GAPDH Abs (loading control).
  2. HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry‐FIP200. Twenty‐four hours post‐transfection, cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ns: not significant, ****P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT–qPCR (Appendix Fig S2). Scale bar = 10 μm.
  3. Primary cortical neurons (DIV5/6) were transfected with EmGFP non‐targeting (Ctrl) or C9orf72 miRNA (green) and mCherry‐FIP200 (red); for rescue experiments, the cells were additionally transfected with mCerulean‐tagged C9orf72s and C9orf72L (cyan). Three days post‐transfection, neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per soma from 2 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ns: not significant, ***P ≤ 0.001, ****P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 μm.
  4. HeLa cells were co‐transfected with mCherry‐FIP200 (red) and empty vector (EV), FLAG‐C9orf72L, or FLAG‐C9orf72S (green). As positive control, EV‐transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry‐FIP200‐positive puncta per cell from 3 independent experiments (mean ± SEM; one‐way ANOVA with Fisher's LSD test, ***P ≤ 0.001, ****P ≤ 0.0001; N (cells) = EV: 47, EV+Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 μm.

Source data are available online for this figure.