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: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: A , B MDA-MB-231 and MCF-7 cells were treated with or without flubendazole (0.5 μM) for 24 h. A fluorescence microscope evaluated the intensity of Calcein AM. Representative images and quantification of Calcein AM were shown. Scale bar, 5 µm. C , D Flow cytometric analysis and quantification of mitochondrial membrane potential changes in MDA-MB-231 and MCF-7 cells treated with or without flubendazole (0.5 μM) for 24 h. E Mitochondria were stained with MitoTracker TM Deep Red FM probes for 30 min and observed with a confocal microscope. Representative images of mitochondrial morphology were shown. Scale bar, 5 µm. F RT-qPCR analysis of mitochondrial DNA copies in MDA-MB-231 and MCF-7 cells. G , H Mitochondria were stained with MitoSOX TM Red FM for 30 min, and mitochondrial ROS accumulation was analyzed by flow cytometry. I ATP content measurement in MDA-MB-231 and MCF-7 cells treated with or without flubendazole (0.5 μM) for 24 h. J Immunoblotting analysis of VDAC1, SOD2, COX IV, TOM20 expression in MDA-MB-231 and MCF-7 cells treated with the indicated concentration of flubendazole for 24 h. β-actin was used as the loading control. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Fluorescence, Microscopy, Membrane, Staining, Quantitative RT-PCR, Flow Cytometry, Western Blot, Expressing, Concentration Assay, Control

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: A MDA-MB-231 and MCF-7 cells were treated with or without flubendazole (0.5 μM) for 24 h. The images were captured with a transmission electron microscope. Scale bar, 500 nm. B , C The autophagosomes are labeled by LC3 (green fluorescence) protein and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 5 µm. D Immunoblotting of PINK1, Parkin, p-Parkin ser65 and LC3 in MDA-MB-231 and MCF-7 cells treated with the indicated concentrations of flubendazole for 24 h. β-actin was used as the loading control. E Immunoblotting of Parkin in the cytosolic and mitochondrial fractions of MDA-MB-231 and MCF-7 cells treated with or without Flubendazole (0.5 μM) for 24 h. β-actin (cytoplasmic fraction) and VDAC1 (mitochondrial fraction) were used as the loading controls. F , G Colocalization of PINK1 (green fluorescence) protein and Parkin (red fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized PINK1 and Parkin was quantified. Scale bar, 10 µm. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Transmission Assay, Microscopy, Labeling, Fluorescence, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: A Immunoblotting of DRP1, p-DRP1 ser616 and Mitofusin-2 in MDA-MB-231 and MCF-7 cells treated with the indicated concentrations of flubendazole for 24 h. β-actin was used as the loading control. B DRP1 mRNA expression in MDA-MB-231 and MCF-7 cells was analyzed by RT-qPCR. C , D MDA-MB-231 and MCF-7 cells were transfected with negative-control or DRP1 shRNA for 24 h, respectively. After treatment with or without flubendazole (0.5 μM) for 24 h. The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 5 µm. E , F Colocalization of PINK1 (green fluorescence) protein and Parkin (red fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized PINK1 and Parkin was quantified. Scale bar, 10 µm. G Immunoblotting of Parkin, p-Parkin ser65 and PINK1 expression. β-actin was measured as the loading control. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Transfection, Negative Control, shRNA, Labeling, Fluorescence

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: MDA-MB-231 and MCF-7 cells were transfected with EVA1A siRNA or negative-control for 24 h, respectively, followed by treatment with or without flubendazole (0.5 μM). A Immunoblotting of EVA1A, DRP1, p-DRP1 ser616 , Parkin, p-Parkin ser65 and PINK1 expression. β-actin was measured as the loading control. B , C The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 10 µm. D , E Colocalization of Parkin (red fluorescence) protein and TOM20 (green fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized Parkin and TOM20 was quantified. Scale bar, 10 µm. F ATP content measurement. G , H Flow cytometric analysis and quantification of mitochondrial membrane potential changes. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Transfection, Negative Control, Western Blot, Expressing, Control, Labeling, Fluorescence, Membrane

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: MDA-MB-231 and MCF-7 cells were co-transfected with DRP1 shRNA and Flag-EVA1A or vehicle control respectively for 48 h. A Immunoblotting of DRP1, EVA1A, p62, Parkin, p-Parkin ser65 , PINK1, and LC3 expression. β-actin was measured as the loading control. B , C The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 10 µm. D , E Colocalization of Parkin (red fluorescence) protein and TOM20 (green fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized Parkin and TOM20 was quantified. Scale bar, 10 µm. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Transfection, shRNA, Control, Western Blot, Expressing, Labeling, Fluorescence

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