Journal: Nature Communications

Article Title: BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects

doi: 10.1038/s41467-020-20679-y

Figure Lengend Snippet: a – e Nontargeting (NT) or GR siRNA was transfected to hippocampal neurons and SH-SY5Y cells for 24 h prior to corticosterone and cortisol for 12 h, respectively. a , b Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) expression was detected in western blot where β-actin was used as a loading control in both cell types. n = 5. c Colocalization of PGC1α (red) and DAPI (blue) in hippocampal neurons was visualized with SRRF imaging system. Scale bars, 20 μm (magnification, ×1000). n = 5. d Colocalization of PGC1α (green) and DAPI (blue) in SH-SY5Y was visualized with SRRF imaging system. Scale bars, 20 μm (magnification, ×1000). n = 5. e PGC1α protein expressions in subcellular fraction samples were detected by western blotting. Lamin A/C and α-tubulin were used as a nuclear and cytosolic loading control, respectively. n = 5. f , g SH-SY5Y cells were transfected with pcDNA3.1/c-eGFP or pcDNA3.1/PPARGC1A-c-eGFP vector for 24 h prior to cortisol treatment for 24 h. f NIX expression was detected in western blot where β-actin was used as a loading control. n = 5. g TOMM20 levels were detected by western blot. Loading control for western blot is β-actin. n = 5. All blots and immunofluorescence images are representative. n = 5 from independent experiments with two technical replicates each. Quantitative data are presented as a mean ± S.E.M. Two-sided two-way ANOVA was conducted. ** indicates p < 0.01 versus control. # , ## indicates p < 0.05, p < 0.01 versus corticosterone in hippocampal neurons and cortisol in SH-SY5Y, respectively. Data are provided as a Source data file.

Article Snippet: The antibodies of LC3 (#NB100-2220, 1:3000 for western blot), PGC1α (#NBP1-04676, 1:3000 for western blot, 1:200 for immunostaining), NIX (#NBP1-88558, 1:3000 for western blot, 1:200 for immunostaining), and PINK1 (#BC100-494, 1:3000 for western blot) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Expressing, Western Blot, Control, Imaging, Plasmid Preparation, Immunofluorescence

Journal: Nature Communications

Article Title: BNIP3L/NIX-mediated mitophagy protects against glucocorticoid-induced synapse defects

doi: 10.1038/s41467-020-20679-y

Figure Lengend Snippet: a – f Mice were exposed to vehicle, corticosterone (10 mg/kg), corticosterone with phorbol 12-myristate 13-acetate (PMA pretreatment, 200 μg/kg), or PMA alone for 7 days. a Slide samples for IHC were immunostained with LAMP1 (green), TOMM20 (red), and DAPI (blue). Scale bars, 100 μm (magnification, ×200). n = 5. b The expressions of NIX, PTEN-induced kinase 1 (PINK1), and BCL2 interacting protein 3 (BNIP3) were detected with western blot where β-actin was used as a loading control. n = 5. c Slide samples for IHC were immunostained with synpatophysin (green), PSD95 (red), and DAPI (blue). Scale bars, 100 μm (magnification, ×200). n = 5. d Synaptophysin and PSD95 were detected by western blot. Loading control is β-actin. n = 5. e The mice were subjected to Y-maze test to evaluate spatial memory function. n = 6. f The mice were subjected to forced swim test to evaluate depression-like behavior. n = 5. g Vehicle or RU 486 (5 mg/kg) injected mice were presented with/without corticosterone (10 mg/kg) for 3 days. The expressions of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and NIX were visualized via western blotting. Loading control is β-actin. n = 5. h The schematic model for mechanisms of inhibition in NIX-dependent mitophagy by glucocorticoid was shown. All blots and immunofluorescence images are representative. n = 5 or 6 from each animal with two technical replicates each in results of IHC and western blot. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging system. Two-sided two-way ANOVA was conducted except Fig. 8b, data of which were analyzed by two-sided unpaired student’s t test. ** indicates p < 0.01 versus control and ## indicates p < 0.01 versus corticosterone, respectively. Data are provided as a Source data file.

Article Snippet: The antibodies of LC3 (#NB100-2220, 1:3000 for western blot), PGC1α (#NBP1-04676, 1:3000 for western blot, 1:200 for immunostaining), NIX (#NBP1-88558, 1:3000 for western blot, 1:200 for immunostaining), and PINK1 (#BC100-494, 1:3000 for western blot) were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Control, Injection, Inhibition, Immunofluorescence, Imaging