Fig. 2.

Mitochondrial mass and distribution in HeLa cells cultured under AL and CR serum conditions. (A) Mitochondrial mass in cells cultured after 48 h with AL or CR sera was determined by flow cytometry by using MTG as indicated in Methods. (Left) Data indicate the mean of the MFI from three different experiments performed in duplicate. *, Significant differences vs. AL treatment, P < 0.01. (Right) MTG signal in AL and CR cells visualized by confocal microscopy. Images were acquired by using a ×40 objective. Settings for detectors were maintained along the study. (B) Mitochondrial staining in HeLa cells cultured under AL and CR serum conditions. Representative images of mitochondrial staining in AL and CR cells by using 20 nM MTG, 10 μM NAO, and immunostaining with anticytochrome c (Cyt c) or anticytochrome c oxidase I (Cox). Images were acquired by using a ×63 objective. (C) NAO staining of FAO and primary rat hepatocytes cultured in AL and CR conditions. (Left) Data indicate the mean of the MFI from three different experiments performed in duplicate. *, Significant differences vs. AL treatment, P < 0.01. (Right) NAO signal in AL and CR cells visualized by confocal microscopy. Images were acquired by using the ×40 objective. (D) Citrate synthase activity in HeLa cells incubated with rat serum grown under AL or CR conditions and from rat liver from animals fed under these conditions for 8 months. *, Significant differences vs. AL treatment, P < 0.01. (E) Quantification of the mitochondrial numbers from micrographs of 24-month-old AL and CR rat hepatocytes as described in Methods. *, Significant differences vs. AL treatment, P < 0.01. (F) EM of AL (Left) and CR (Right) rat hepatocytes prepared and imaged as described in Methods.