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. 2015 Dec 10;12(2):327–342. doi: 10.1080/15548627.2015.1125071

Figure 1.

Figure 1.

Inhibition of starvation-induced autophagy by ectopic expression of TRS1 and IRS1. (A) Representative images of GFP-LC3 HeLa cells transfected with an empty vector (vector), IRS1, or TRS1 plasmids and then fixed after starvation. (B) The number of GFP-LC3-dots in TRS1 or IRS1-expressing cells in the transfected HeLa cells was quantified. The results are the mean of 6 independent experiments; 50 to 100 cells were analyzed per assay. (C) (Microscopy panels). (Left) Cellomics ArrayScan images of GFP-LC3 HeLa cells transfected with empty vector, IRS1 or TRS1. (Right) Image analysis software detected DAPI-labeled nuclei (blue), cell outline (red line), GFP-LC3 dots (green dots) and nontransfected cell nuclei (yellow line). The number of GFP-LC3 dots per cell, total intensity, total area and average area were quantified using Cellomics spot detector software. (D) (Top panel) SQSTM1 staining in HeLa cells under starvation or after 3-methyladenine (3MA) treatment. (Lower panel) SQSTM1 staining in HeLa cells expressing IRS1 or TRS1 and after starvation. (E) Autophagy was quantified by counting the number of SQSTM1 dots per transfected cell. The results are the mean of 3 independent experiments; 20 cells were analyzed per assay. (F) Immunoblot analysis of SQSTM1 and LC3 proteins in HeLa cells transfected with IRS1, TRS1 or ICP34.5 plasmids. CM, complete medium. ACTB was used as a loading control. **, P<0.01; ***, P<0.001 (One-way ANOVA).