FIGURE 6: When cultivated in PBS, cells first enter autophagy and then die when nutrients are lacking. (A) Percentage of autophagosome-containing cells (punctuated ATG8) and TUNEL-positive cells during culture of cells in PBS for 24 h (≥ 1128 cells from a minimum of three independent experiments for the ATG8 assay and from a minimum of five independent experiments for the TUNEL assay). Unpaired t-test: ns not significant, * p<0.05, ** p<0.01. (B) Real time microscopy of a representative GFP-ATG8 cell cultivated in PBS, showing ATG8 punctuated staining disappearance, nucleus and kinetoplast DAPI staining appearance concomitant with cell rounding up, then kinetoplast fluorescence decrease (bar = 5µm). (C) Sequence of events observed in the cell represented in (B), showing the death (characterized by DAPI permeability, cell rounding up and low kinetoplast fluorescence) of an autophagic cell (with GFP-ATG8 puncta). (D) Microscopical images of a cell in control conditions (cell cultivated in medium and not in PBS) representative of all the cells observed in the same conditions as in (B), for 300 min (bar = 5 µm).