Carbocyanines

Carbocyanine dye DiI (Invitrogen) was used to label neurons66 (link). DiI crystals were applied using a thin needle by delicately touching region of interest on both sides of 2 mm coronal sections prepared from previously cardiacally fixed with 1.5% PFA in 0.1 M phosphate buffer (PB). DiI was left to diffuse for 1 day in the dark at room temperature, then sections were fixed again with 4% PFA in PB 0.1 M for 45 min at 4 °C. 150 μm coronal sections were then obtained using a vibratome, the first section was discarded. Sections were mounted on glass slides with Fluoromount mounting medium (Sigma-Aldrich) for confocal imaging. DiI crystals were also used on primary hippocampal neurons previously fixed with 1.5% PFA-4% sucrose in 0.1 M PB, where they were applied to the coverslip with a thin needle and let to diffuse for 30 min in 0.1 M PB and then fixed with 4% PFA-4% sucrose in PB for 5 min at 4 °C. After washing, coverslips were mounted on glass slides with Fluoromount mounting medium (Sigma-Aldrich) for confocal imaging.

HeLa cells (untransfected or transfected with plasmids or siRNA, as indicated), were washed with PBS and incubated for 25 min with MitoTracker Red CMXRos (100 nM; Invitrogen). Incubation of cells with 20 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone; Sigma–Aldrich) confirmed the mitochondrial membrane potential sensitivity of the MitoTracker Red localization. For co-staining studies, cells were then fixed with 4% (w/v) paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and blocked in 10% (v/v) FBS in PBS [at room temperature (25°C), 20 min]. Primary antibodies were added to sterile-filtered 1% (w/v) BSA in PBS and incubated with the cells (at room temperature, 1 h). The primary antibodies used and their dilutions were as follows: mouse monoclonal anti-(p32 N_m-terminus) antibody (1:100 dilution; Abcam); mouse monoclonal anti-c-Myc antibody (1:100 dilution, sc-40; Santa Cruz Biotechnology); rabbit anti-calnexin antibody (1:100 dilution, C4731; Sigma–Aldrich); and mouse monoclonal anti-α-tubulin (1:100 dilution, sc-5286, Santa Cruz Biotechnology). Following three rounds of 5-min washes with PBS, Cy2 (carbocyanine)-conjugated anti-mouse or anti-rabbit secondary antibody (1:400 dilution) were added to sterile filtered 1% (w/v) BSA in PBS and incubated for 1 h at room temperature. Nuclei were stained by DAPI (Sigma–Aldrich; 1:15000 dilution in PBS) for 5 min at room temperature before final washes in PBS and mounting on to glass slides with Biomedia Gel Mount (ProSciTech).
Stained cells were examined by confocal laser scanning microscopy using the Leica TCS SP2 imaging system (100×objective). Normal/elongated, fragmented/punctate or fibrillar mitochondrial morphologies were defined by width/length parameters of 1:1, 1:3 and 1:10 respectively. Quantification of each type of mitochondrial morphology upon p32 knockdown or p32 overexpression was assessed by counting 60–80 cells per condition on three independent occasions.

AS‐IV was obtained from Nanjing Jingzhu Biotechnology Company (purity > 98% measured by HPLC, Nanjing, China). Human pulmonary artery endothelial cells were purchased from BLUEFBIO (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma Aldrich (St. Louis, MO, United States). CCN1‐small interfering lentiviruses were purchased from Shanghai Just Science. (Shanghai, China). Recombinant CCN1 protein was purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS, LOT 0001644044) was purchased from Sigma. EGF was purchased from purchased Sigma (St. Louis, MO, USA). The A5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐lbenzimidazol‐carbocyanine iodide (JC‐1) kit and Fluo‐4 were obtained from the Beyotime Institute of Biotechnology (Nanjing, China). Antibodies against Bcl‐2 (A0208), Bax (A0207), Caspase‐3 (A2156), CCN1 (A7632), ERK (A16686) and p‐ERK (AP0886), and β‐actin (AC038) were purchased from ABclonal (ABclonal Technology Co.,Ltd.). A terminal deoxynucleotidyl transferase‐mediated dUTP Nick‐End Labeling (TUNEL) kit was purchased from Roche (Darmstadt, Germany).