Figure 6

Starvation differentially activates autophagy in cardiac cells. (A) NMCFs (n = 7–14 wells) were transfected with Ad-Cre or Ad-GFP for 5 days. The cells were incubated overnight with serum-free medium followed by 4 h of incubation with EBSS (amino acid-free, AA⁻) medium with or without CQ (10 µM, CQ⁺). WB was performed for LC3II, p62, and GAPDH (upper panel) with quantification (lower panel). The increase in the LC3II/LC3I flux was calculated as the delta after both treatments (AA⁻ CQ⁺) to untreated cells (p = 0.033). B and C. NMCMs (n = 4–8 wells) were transfected with Ad-Cre or Ad-GFP for 5 days. Cells were incubated overnight with serum-free medium and subjected to EBSS (amino acid-free, AA⁻) medium (B) for 4 h or RPMI (glucose-free, Gluc⁻) medium (C) for 24 h with or without CQ (10 µM, CQ⁺) before being harvested. All results were obtained from 2–5 different cell isolations and were normalized to control (Ad-GFP). The results are displayed as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 by Kruskal–Wallis test with Dunn’s multiple comparison post-test or Mann–Whitney nonparametric test as described in the Methods.