Figure 1

Autophagy is reliably detected by HEK cells stably expressing pcDNA3-GFP-LC3-RFP-LC3ΔG. (a) HEK cells in phase contrast, GFP, and RFP channels at 10× after a 24 h treatment with 500 nM Torin1 or 1 µM AlloP. Scale bar: 25 µm. (b) Background-subtracted mean fluorescence ratios relative to their vehicle-treated control. Each point represents a 35 mm dish (250–800 cells analyzed per dish) (n = 8 per group) from 3 independent experiments. Circles represent first experiment, squares second, and triangles third. A one-way ANOVA (F (2, 21) = 74.49, p < 0.0001) showed differences between treatments, and Dunnett’s multiple comparisons revealed a difference between control and Torin1 (****p < 0.0001) and between control and AlloP (**p = 0.0056). (c) Separate GFP and RFP mean fluorescence for each treatment, 3 independent experiments. A repeated measures two-way ANOVA revealed a difference of fluorescent protein (F (1, 21) = 16.25, p = 0.0006) and no difference of treatment (F (2, 21) = 2.077, p = 0.1503), with a significant interaction of treatment x fluorescent protein (F (2, 21) = 7.819, p = 0.0029). Sidak’s multiple comparisons revealed a difference between control and Torin1 GFP (**p = 0.0093).