Journal: Scientific reports

Article Title: Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release.

doi: 10.1038/s41598-023-38210-w

Figure Lengend Snippet: Figure 4. Reversible mitochondrial fragmentation in EtOH treated differentiated PC12 cells, HT22 cells, and Neuro-2a cells. Live cell imaging of differentiated PC12 cells (A), HT22 cells (B), and Neuro-2a cells (C) before EtOH (5%, vol/vol) treatment (Control), after EtOH treatment (EtOH, 2 h), and after removing EtOH and allowing cells to recover in fresh culture medium for 20 h (Recovered). Mitochondria were stained by Mito-tracker (magenta) and nuclei by Hoechst33342 (blue). Scale bar: 10 μm. Confocal microscope images of representative control and EtOH treated differentiated PC12 cells (D), HT22 cells (E), and Neuro-2a cells (F) stained with cytochrome c (Cyto.c, green), TOM20 (red) antibodies and DAPI (blue). Scale bar: 10 μm. (G–I) Quantification results of control, EtOH treated, and recovered differentiated PC12 cells, HT22 cells, and Neuro-2a cells that displayed cell shrinkage, mitochondrial fragmentation, and nuclear condensation. Data are presented as the mean ± SEM based on three independent experiments. ****p < 0.0001, vs. control group; ####p < 0.0001, versus EtOH group. EtOH: ethanol.

Article Snippet: For immunostaining, cells were incubated with primary antibodies against cytochrome c (5 μg/mL; 33–8200, InvitrogenTM) and TOM20 (1:50; 42406S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Live Cell Imaging, Control, Staining, Microscopy

Journal: Scientific reports

Article Title: Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release.

doi: 10.1038/s41598-023-38210-w

Figure Lengend Snippet: Figure 6. EtOH and staurosporine (STS) induced apoptosis in HeLa cells, and cell survival in cell population after removal of EtOH and STS. (A) Confocal images of fixed HeLa cells after treatment with STS (250 nM) for (1, 2, 3, 4, 5, 6, 7, 8) h. Mitochondria were stained by TOM20 antibody (magenta), cytochrome c by cyto.c antibody (green), and nuclei by DAPI (blue). White arrows indicate cells with cytochrome c released into the cytosol, and red arrows indicate cells with cytochrome c contained within the mitochondria. Scale bar: 20 μm. (B) Percentage of cytochrome c released HeLa cells treated with STS (250 nM) for the indicated time. Data are presented as the mean ± SEM based on three independent experiments. At least 300 cells were imaged for each experiment at each time point. (C) Western blot analysis of pro-caspase3, cleaved-caspase3, PARP, and cleaved-PARP in STS (250 nM) treated Hela cells. (D) Cell viability of HeLa cells after STS (250 nM) treatment for the indicated time or after STS treatment, then washing away STS and further culturing cells in fresh culture medium for another 15 h. This was measured by Hoechst33342 and PI double staining. (E) Confocal images of fixed Hela cells expressing Cyto.c-GFP after treatment with EtOH (4.3%, vol/vol) for 2, 4, 6, 8, 10 h. Mitochondria were stained by Mito-tracker (magenta), and nuclei by Hoechst33342 (blue). White arrows indicate cells with cytochrome c released into the cytosol, and red arrows indicate cells with cytochrome c contained within the mitochondria. Scale bar: 20 μm. (F) Percentage of cytochrome c releasing HeLa cells treated with EtOH (4.3%, vol/vol) for the indicated time. Data are presented as the mean ± SEM based on three independent experiments. At least 300 cells were measured for each experiment at each time point. (G) Western blot analysis of pro-caspase3, cleaved-caspase3, PARP, and cleaved-PARP in EtOH (4.3%, vol/vol) treated Hela cells. (H) Cell viability of HeLa cells after EtOH (4.3%, vol/vol) treatment for the indicated time or after EtOH treatment, then washing away EtOH and further culturing cells in fresh culture medium for another 15 h. CON: control. EtOH: ethanol.

Article Snippet: For immunostaining, cells were incubated with primary antibodies against cytochrome c (5 μg/mL; 33–8200, InvitrogenTM) and TOM20 (1:50; 42406S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Staining, Western Blot, Double Staining, Expressing, Control

Journal: bioRxiv

Article Title: Functional characterization of the Met50Val substitution in SLC30A9 as a novel case of adaptive introgression in humans

doi: 10.1101/2022.06.29.498106

Figure Lengend Snippet: (A) Superresolution STED microscopy in cells transfected with ZnT9-50Met and ZnT9-50Val immunoassayed with anti-HA (green) and with the mitochondrial marker anti-TOM20. Scale bar = 10 µm. (B-C) Bar graph representing mean inter-mitochondrial distance (B) and relative mitochondria area (C) in cells transfected with ZnT9-50Met or ZnT9-50Val (n=10). *** p<0.001 using Bonferroni test between conditions. (D) Bar graph measuring FRET between the endoplasmic reticulum and mitochondria transfected with an empty vector (control), ZnT9-50Met or ZnT9-50Val together with the FEMP probe (n=46-65).

Article Snippet: We used the rabbit anti-human TOM20 and the rabbit anti-human Calreticulin antibodies (1:1000) and secondary antibodies Abberior STAR RED or ORANGE (1:350).

Techniques: Microscopy, Transfection, Marker, Plasmid Preparation