Short mitochondrial morphology in mouse cortical neurons lacking Bcl-xL. (A) Representative immunoblot of control and bcl-x cKO cortical cultures 4 DIV confirms Bcl-xL deficiency (Boise polyclonal). (B) Immunofluorescence microscopy images show paired cortical cultures (4 DIV) stained with anti–Bcl-xL (Biocarta polyclonal, red) and with DAPI to mark cell nuclei (blue); neurons were identified by costaining with anti-NeuN (not depicted). (C) Proportions of the indicated cell types present in bcl-x cKO cortical neuron cultures (2 DIV) were identified by immunofluorescence microscopy as described in D and quantified; n = 6 samples per genotype in two independent experiments. (D) Immunofluorescence microscopy for Cre recombinase (red) and NeuN (green) in bcl-x cKO cortical cultures (2 DIV); Hoechst 33258 (blue) marks nuclei. Numbered arrows are defined in C. Note, both control and cKO mice have one copy of NEX replaced by Cre recombinase; only control mice contain an unfloxed copy of bcl-x. (E) Viability of Cre-positive neurons in bcl-x cKO (bcl-xflox/flox;cre+/−) and control (bcl-x+/flox;cre+/−) embryonic cortical cultures was determined by nuclear morphology with Hoechst 33258 staining in three independent experiments presented as mean ± SEM (t test, P = 0.01). (F) Representative images of control and bcl-x cKO cortical neurons (8 DIV) transfected with mito-RFP. (G) Mitochondrial length was quantified in three independent experiments as in F and presented as mean ± SEM; n = 1,362 mitochondria from 38 control neurons, and n = 813 mitochondria from 30 bcl-x cKO neurons. t test; *, P < 0.0001
