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. 2015 Mar 14;34(9):1214–1230. doi: 10.15252/embj.201489920

Figure 5. Che-1 regulates autophagy through Deptor and Redd1.

Figure 5

  • A–C WB analysis with the indicated Abs of TCEs from HCT116 transiently transfected with siRNA GFP (siControl) or siRNA Che-1 (siChe-1) and treated where indicated with brefeldin A (5 μg/ml) (A), chloroquine (100 μM) (CQ) (B) or 2DOG (25 mM for 8 h) (left), hypoxia (1% O2 for 16 h) (center), or I.R. (20 Gy) (right) (C).
  • D HCT116 cells transiently transfected with Stealth siRNA negative controls (siControl) or siRNA Che-1 (siChe-1) and GFP-LC3 expression vector were treated where indicated with 2DOG (25 mM for 8 h) or hypoxia (1% O2 for 16 h). Cells were fixed with 4% formaldehyde and analyzed by fluorescence microscopy. Scale bar, 10 μm.
  • E HCT116 cells were transiently transfected and treated as in (D). Twenty-four hours later, cells were fixed and GFP-LC3 distribution was assessed by fluorescence microscopy and percentages represent punctate-LC3 expressing cells. The data represent the mean ± SD from three independent experiments performed in triplicate (n = 150 for each condition). n.s., not significant, *P ≤ 0.001.
  • F Quantitative RT–PCR (qRT–PCR) for MAP1LC3 mRNA expression was performed after transient transfection of HCT116 cells with siRNA GFP or siRNA Che-1 (siChe-1). Values were normalized to RPL19 expression. Error bars represent the standard error of three different experiments. P = 0.02.
  • G WB analysis with the indicated Abs of TCEs from HCT116 cells transiently transfected with Stealth siRNA negative controls (siControl), siRNA Che-1 (siChe-1), and, where indicated, with Flag-Deptor and Myc-Redd1 expression vectors, exposed to normoxia or hypoxia (1% O2 for 16 h).
  • H Fluorescence microscopy analysis of HCT116 cells transiently transfected with siRNA Che-1 (siChe-1), GFP-LC3, and, where indicated, with Flag-Deptor and Myc-Redd1 expression vectors, treated with hypoxia. Scale bar, 10 μm.
  • I WB analysis with the indicated Abs of TCEs from HCT116 transiently transfected with siRNA GFP (siControl) or siRNA Che-1 (siChe-1), treated with 2DOG (25 mM for 8 h), and, where indicated, with 100 nM CCI-779.
  • J HCT116 cells were transiently transfected with GFP-LC3, and Stealth siRNA negative controls (siControl) or siRNA Che-1 (siChe-1), and treated as in (I). Cells were fixed with 4% formaldehyde and analyzed by fluorescence microscopy. Scale bar, 10 μm.
  • K HCT116 cells were transiently transfected and treated as in (J). Twenty-four hours later cells were fixed, and GFP-LC3 distribution was assessed by fluorescence microscopy and percentages represent punctate-LC3 expressing cells. The data represent the mean ± SD from three independent experiments performed in triplicate (n = 140 for each condition). P ≤ 0.0002.

Source data are available online for this figure.