Journal: Frontiers in Physiology

Article Title: Loss of Drosophila Clueless differentially affects the mitochondrial proteome compared to loss of Sod2 and Pink1

doi: 10.3389/fphys.2022.1004099

Figure Lengend Snippet: Mitochondrial respiratory chain proteins are greatly decreased in clu mutants. (A) A pie chart showing clu specific less abundant proteins in different categories as percentage of the whole [total = 92 ]: Mitochondrial Respiratory Chain (MRC) components, Mitochondrial Ribosomal Proteins (Mitochondrial RP) and Others. (B) Bar graph showing the number of subunits, in percentages, from each entire MRC are either decreased (in green) or remain unaltered (in magenta). (C) Heat maps showing the levels of affected individual MRC proteins of different complexes in clu , Sod2 , and Pink1 mutants. The proteins chosen are decreased in clu mutants. Color coded scale bars (green: less abundant and magenta: unaltered) are representing the log2[Fold Change] of individual protein in each mutant compared to wild type. (D) Western blots showing the levels of representative proteins in clu , Sod2 , and Pink1 . Tom20 was used as a loading control. (E) Proteins from isolated mitochondria were run to determine the levels of native MRC complexes on a BN-PAGE (left panel, stained with colloidal blue, right panel, stained with SilverQuest Silver Staining Kit). (F) In-gel activity assays. Dark purple bands indicate CI activity and brown bands indicate CIV activity. (G,H) The activities from CI (G) and CIV (H) were determined by measuring band intensity of the respective complexes using Fiji software. Band intensities from three gels ( n = 3) for CI and two gels ( n = 2) for CIV were used for the measurements. Error bars: S.E.M. calculated in GraphPad-PRISM software. p values were calculated in GraphPad-PRISM software using an unpaired t -test and each mutant was compared to the wildtype control. Statistical significance = p < 0.0001 (****) and p < 0.003 (**).

Article Snippet: For gene expression analysis, quantitative PCR was performed using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, United States) in a 10 μL reaction with 2 μL diluted cDNA and one of the following TaqMan probes: Dm01806850_g1 (Tom20), Dm02136274_g1 (ND-42), Dm01820354_g1 (ND-19), Dm01804649_g1 (Cyp4ac2), Dm02145551_g1 (Mil), Dm01822473_s1 (Hsp23), Dm01816546_s1 (Pepck), Dm01835343_g1 (ND-30), Dm01794109_g1 (COX4), Dm01830822_g1 (Levy) and Dm02151827_g1 (RPL32) (endogenous control) (Thermo Fisher Scientific, Waltham, MA).

Techniques: Mutagenesis, Western Blot, Control, Isolation, Staining, Silver Staining, Activity Assay, Software

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: Phosphoproteomics of CD2 signaling reveals an AMPK-dependent regulation of lytic granule polarization in cytotoxic T cells

doi: 10.1101/795963

Figure Lengend Snippet: (A) Colocalization analysis and (B) immunofluorescence image of LAMP1 and AMPK signals in CTLs. Quantitative measurements were taken for 30 CTLs (n=3 donors). (C) Immunofluorescence images of CTLs electroporated with LAMP1-AIP-mCherry or Tom20-AIP-mCherry constructs and stained with antibodies against perforin (granules). (D) Quantitative analysis of lytic granule polarization in antibody-stimulated mCherry-positive CTLs overexpressing either LAMP1-AIP-mCherry or Tom20-AIP-mCherry (n=3 experiments). (E) Colocalization analysis of immunofluorescence signals in CTLs stained with antibodies against AMPK and perforin (granules) (n=3 experiments). (F) Immunofluorescence image of a CTL stained with antibodies against AMPK, LAMP1 and perforin (granules). (G) CLEM analysis of lytic granules and AMPK-positive compartment in freshly isolated CTLs. N, nucleus; LD, lipid droplet; AMPK, AMPK-positive vesicle; LG, lytic granule. Scale bar, 5 μm. Shown are mean values ± SD; ns, not significant; two-way ANOVA test ****p < 0.0001.

Article Snippet: For overexpression of LAMP1-AIP and Tom20-AIP, CTLs were nucleofected with 2 μg of purified LAMP-mChF-AIP and Tom20-mChF-AIP plasmids (a gift from Takanari Inoue, Addgene plasmids # 61524 and # 61512).

Techniques: Immunofluorescence, Construct, Staining, Isolation

Journal: bioRxiv

Article Title: CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division

doi: 10.1101/723940

Figure Lengend Snippet: (A) Schematic of Mitotrap used in this experiment. In all cases cells expressing endogenously tagged GFP-CLIC4 were co-transfected with Cry2-GFP-VHH and CIB-Tom20 plasmids and pulsed with a 488nm laser to re-target GFP-CLIC4 at the mitochondria. (B) Interphase cell exposed to 488nm to activate Mitotrap. Mitochondria is labeled in red and endogenous GFP-CLIC4 in green. (C-D) Still images from time-lapse microscopy where Mitotrap was activated. Arrows point to blebs or cytokinesis failure induced by GFP-CLIC4 Mitotrap. (E) Quantification of time required for cells to complete mitotic cell division in control and Mitotrapped cells. Data shown are the means and standard deviations.

Article Snippet: For the Mitotrap experiments, cells expressing endogenous GFP-CLIC4 were transfected with Cry2-VHH Addgene #58370) and Tom20-CIB-stop (Addgene #117243).

Techniques: Expressing, Transfection, Labeling, Time-lapse Microscopy