A. Cell death was assessed with trypan blue exclusion in cells treated with DMSO or with BTA, in the presence or absence of 5mM methyl-pyruvate or 2 mM NAC. ATP (B) and Oxygen consumption levels (C) in cells cultured in 5 mM glucose and mock treated (DMSO, Control) or treated with BTA, or in cells expressing CIC or CIC-DN, 16 hours after treatments. D. Cell cycle profiles of control H1299 cells, or of cells treated with 1 mM BTA, or expressing the indicated CIC proteins, cultured in either 25 mM (upper panels) or in 2 mM Glucose (bottom panels). The percentage of cells distributed in the G1-, S-, or G2- phases of the cells cycle is shown. Apoptotic cells are identified as the Sub-G1 (SG) population. E. Immuno-fluorescence experiments similar to those described in Figure , were performed in cells expressing epitope tagged Flag-CIC (panels 1 to 3) or Flag-CIC-DN (panels 3 to 6) proteins. Cells were stained with anti-Flag (green, panels 1 and 4) and anti-LC3 (red, panels 2 and 5) antibodies, and counterstained with DAPI. Merged images are shown in panels 3 and 6. Note the intense LC3 staining in the mitochondria of panel 5. The results of these immunofluorescence experiments where confirmed with cellular sub-fractionation experiments shown in F. Cytoplasmic (c) and mitochondrial (m) fractions derived from naïve 293T cells (lanes 1,2) or 293T cells expressing CIC (lanes 3,4) or CIC-DN (lanes 5,6) or treated with BTA (lanes 7,8), were probed with anti-CIC, anti-LC3, or anti-mitofilin antibodies, as indicated. The two arrows in the LC3 blot indicate cytosolic (-I) or activated (-II) LC3 forms. Note the enrichment of LC3-II in the mitochondria of cells expressing CIC-DN or BTA (lanes 6 and 8, respectively). All images shown are derived from the same autoradiogram at identical times of exposures, but lanes in between samples of interest were eliminated. G. Time course (in hours, indicated at the top of each panel) of BTA treated- (lanes 1 to 3) or CIC-DN induced cells (lanes 4 to 6). The anti-mitofilin, anti-p62 (employed as a general autophagic marker), anti-CIC, or anti LC3 specific immuno-blots are shown.