Figure 7. Bcl-xL promotes Drp1-independent mitochondrial growth. (A) The number density of mitochondrial organelles was determined as the number of mitoRFP-expressing mitochondria per 100 microns of neuron process in four independent experiments per condition and presented as mean ± SEM. **, P < 0.003 compared with vector or Bcl-xL, except vector compared to both; *, P < 0.03. See “Computational methods” and Table S2 (available at http://www.jcb.org/cgi/content/full/jcb.200809060/DC1). (B) Mitochondrial mass was approximated using the formula: [(mean mitochondrial length from Fig. 2 B) × (mitochondrial number density/micrometer from panel A)]; SEM < 0.03 for all conditions. Bootstrap significance tests were used for effective mitochondrial mass; *, P < 0.02; **, P < 0.002; ***, P = 0.0002 compared with vector control. (C) Diagram of findings from B. (D) Bcl-xL increases the mitochondrial content of hippocampal neuronal processes determined by electron microscopy. The total area of neuronal processes that was occupied by mitochondria was calculated for at least 50 mitochondria per condition in 19 images for GFP–Bcl-xL and in 29 images of GFP, and presented as mean ± SEM. t test; *, P < 0.002. (E) The area within control and bcl-x cKO mouse cortical neurons (6–8 DIV) that is occupied by mitochondria was determined by immunofluorescence microscopy for cytochrome c and analyzed using ImageJ. Neurons were identified by staining with anti-Cre (not depicted). Data are presented as mean ± SEM for 28–34 healthy neurons in 9–10 independent cultures per condition in two experiments. t test; *, P < 2 × 10−7. (F) Immunoblots of lysates and indicated subcellular fractions prepared from transiently transfected HeLa cells. Some lanes from the same exposures of the same blots were rearranged for presentation. Bcl-xL was estimated by densitometry to increase VDAC levels in mitochondrial fractions over loading control by 2.7-fold (as shown), and in two additional independent experiments by 13.0-fold and 2.3-fold. These values do not take into account <100% transfection efficiency. Molecular mass markers are shown (kD).