Journal: The EMBO Journal

Article Title: Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

doi: 10.15252/embj.2018100715

Figure Lengend Snippet: A–E (A) Representative images (and details from boxed regions) of WT MEF cells transfected with YFP Parkin (green) and treated with FCCP (10 µM) to induce mitophagy. Cells were immunostained with anti‐Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time points for the different conditions tested (the specific time point of each example is indicated in the image). From top to bottom: (i) “Diffuse Parkin” defines cells where Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). (ii) Soon after mitochondrial damage, Parkin appears enriched in small “puncta” throughout the cytoplasm localising within isolated mitochondria (quantified in C). (iii) Later in the process, these puncta aggregate into bigger structures accumulating in perinuclear regions (quantified in D). (iv) The process of Parkin translocation eventually affects all the mitochondrial population which usually collapses into several large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments ( n = 3; one‐way ANOVA with Dunnett post‐test). F, G (F) Representative images (and details from boxed regions) of WT MEF cells transfected with GFP PINK1 (green) and treated with FCCP (10 µM) to induce mitophagy. Cells were immunostained with anti‐Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 stabilisation on mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 stabilisation on mitochondria in WT and Miro DKO cells at different time points shortly after mitochondrial insult (G). Data collected from three independent experiments ( n = 3). Data information: Error bars represent SEM. Significance: * P < 0.05 and ** P < 0.01.

Article Snippet: GFP PINK1 construct was obtained from Addgene [plasmid #13316, (Beilina et al , )].

Techniques: Transfection, Expressing, Isolation, Translocation Assay

Journal: The EMBO Journal

Article Title: Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

doi: 10.15252/embj.2018100715

Figure Lengend Snippet: Representative confocal images of the soma of WT and Miro1 KO cortical neurons expressing YFP Parkin and MtDsRed after valinomycin treatment at the indicated time points (scale bars = 5 µm). Airyscan confocal images of Parkin recruitment after 5 h of valinomycin treatment in WT and Miro1 KO neuronal processes (scale bars = 2.5 µm). Fluorescent linescans of YFP Parkin and MtDsRed signal in WT and Miro1 KO neurons after 5 h of valinomycin treatment (lines shown in A). Quantification of Parkin recruitment to mitochondria ( YFP Parkin signal overlapping MtDsRed signal. Intensity adjusted and normalised to t = 0) in WT and Miro1 KO neurons following valinomycin treatment ( n = 15 cells for all conditions per genotype over 3 neuronal preparations; two‐way ANOVA). Quantification of mitochondrial occupancy (area of MtDsRed signal in soma/entire area of soma) in the somas of WT and Miro1 KO neurons following valinomycin treatment ( n = 15 cells for all conditions per genotype over 3 neuronal preparations; two‐way ANOVA). Quantification of mitochondrial clearance in somas of WT and Miro1 KO neurons between 2 and 5 h of valinomycin treatment ( n = 15 cells for all conditions per genotype over 3 neuronal preparations; unpaired t ‐test). Representative confocal images of WT, Miro1 KO and PINK1 KO cortical neurons immunostained with MAP2 (cyan) and pS65‐Ub (green) after valinomycin treatment (scale bars = 20 µm). Quantification of pS65‐Ub signal intensity within MAP2 signal (normalised to MAP2 area and t = 0) in Miro1 WT , Miro1 KO and PINK1 KO neurons following valinomycin treatment ( n = 3, 4 and 3 embryos from WT, Miro1 KO and PINK1 KO embryos, respectively, 6 ROIs per condition; two‐way ANOVA). Representative confocal images of the soma of Miro1 KO cortical neurons expressing Miro1 WT and mutant forms of Miro1, YFP Parkin and MtDsRed without valinomycin treatment (scale bars = 10 µm). Quantification of Parkin co‐localisation with MtDsRed ( YFP Parkin signal overlapping MtDsRed signal. Intensity adjusted) in the soma of Miro1 KO and PINK1 KO cortical neurons ( n = 12 cells for all conditions per genotype over 3 neuronal preparations; one‐way ANOVA). Representative confocal images of the soma of Miro1 KO cortical neurons expressing WT and mutant forms of Miro1, YFP Parkin and MtDsRed after 5 h of valinomycin treatment (scale bars = 10 µm). Quantification of Parkin recruitment to mitochondria (normalised to t = 0) in Miro1 KO and PINK1 KO cortical neurons following valinomycin treatment ( n = 12 cells for all conditions per genotype over 3 neuronal preparations, two‐way ANOVA). Data information: Error bars represent SEM. Significance: * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: GFP PINK1 construct was obtained from Addgene [plasmid #13316, (Beilina et al , )].

Techniques: Expressing, Mutagenesis

Journal: Carcinogenesis

Article Title: BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

doi: 10.1093/carcin/bgt242

Figure Lengend Snippet: Fig. 1. BH4 domain is required for bcl-2 protein-induced in vivo angiogenesis under hypoxic conditions. (A) HIF-1α protein expression by western blot analysis and (B) VEGF protein secretion by ELISA in melanoma M14 control clone (puro), clones overexpressing bcl-2 protein wild-type (Bcl-2/37) or deleted of BH4 domain (BH4del/8, BH4del/42) exposed to hypoxia for 24 h. (C and D) In vivo vessel formation assessed after injection of C57BL/6 mice with Matrigel plugs containing CM from M14 derivatives exposed to hypoxia for 24 h. (C) Macroscopic analysis (D) and Hb content of Matrigel plugs from one representative experiment out of three with superimposable results are shown. (D) The values were expressed as optical density (OD)/g Matrigel plug. (E) HUVEC proliferation evaluated after exposure for 48 h to CM from M14 derivatives exposed to hypoxia for 24 h in presence or absence of VEGF-neutralizing antibody (anti-VEGF). The results represent the mean ± SD of three independent experiments, performed in sextuplicate. The values were expressed as mean of OD ± SD. (F) HUVEC morphogenesis on Matrigel after exposure for 6 h to CM from M14 derivatives grown under hypoxia for 24 h. (G) HUVEC migration in response to CM from M14 derivatives exposed to hypoxia for 24 h, in presence or absence of anti-VEGF. The values are expressed as percentage of migrated cells compared with exposure to CM from control cells. (A) Western blots representative of two independent experiments with similar results are shown. HSP 72/73 is shown as loading and transferring control. (B, E and G) P values were calculated between (B) control cells and cells overexpressing bcl-2 wild-type or deleted of BH4 domain, (E and G) CM from control cells and cells overexpressing bcl-2 wild-type or deleted of BH4 domain (*P < 0.05) or (G) CM from untreated cells and cells exposed to VEGF-neutralizing antibody (§P < 0.05).

Article Snippet: M14 and A375SM-SC1 cells were stably transfected using Lipofectamine (Invitrogen, Carlsbad, CA) with expression vectors encoding the human bcl-2 protein wild-type or deleted of BH4 domain (aa 1–36), or mutated in BH1 (G145E) or BH2 (W188A) domains (13), with Flag-tagged vector encoding the only BH4 domain of bcl-2 (aa 1–30) or with green fluorescent protein (GFP)-tagged vector encoding bcl-2 protein wild-type (Addgene plasmid 17999) or targeted to ER (Addgene plasmid 18000).

Techniques: In Vivo, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Clone Assay, Injection, Migration, Transferring

Journal: Carcinogenesis

Article Title: BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

doi: 10.1093/carcin/bgt242

Figure Lengend Snippet: Fig. 2. The bcl-2 protein lacking BH4 domain decreases in vivo vascularization and metastatization. (A) Representative images and (B) quantification of microvessel density, (C) VEGF mRNA and (D) VEGF protein expression in xenografted tumors from M14 control clone (puro) and clones overexpressing bcl-2 wild-type (Bcl-2/37) or deleted of BH4 domain (BH4del/8, BH4del/42). (A) Original magnification ×200. (E) Representative images and (F) quantification of hematoxylin and eosin staining of lung micrometastases. (E) Micrometastases are indicated by arrow heads. Original magnification ×100. (B, C and F) The results represent the mean ± SD of three independent experiments. P values were calculated between control and bcl-2 wild-type or deleted of BH4 domain overexpressing cells (*P < 0.05).

Article Snippet: M14 and A375SM-SC1 cells were stably transfected using Lipofectamine (Invitrogen, Carlsbad, CA) with expression vectors encoding the human bcl-2 protein wild-type or deleted of BH4 domain (aa 1–36), or mutated in BH1 (G145E) or BH2 (W188A) domains (13), with Flag-tagged vector encoding the only BH4 domain of bcl-2 (aa 1–30) or with green fluorescent protein (GFP)-tagged vector encoding bcl-2 protein wild-type (Addgene plasmid 17999) or targeted to ER (Addgene plasmid 18000).

Techniques: In Vivo, Expressing, Control, Clone Assay, Staining

Journal: Carcinogenesis

Article Title: BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

doi: 10.1093/carcin/bgt242

Figure Lengend Snippet: Fig. 3. BH4 domain is required for progression-related features enhanced by bcl-2 protein. (A) Cell migration evaluated by wound-healing assay of M14 control clone (puro) and its clones overexpressing bcl-2 wild-type (Bcl-2/37) or deleted of BH4 domain (BH4del/8, BH4del/42), in presence or absence of VEGF- neutralizing antibody (anti-VEGF). (B) Metalloproteinases MMP-2 and MMP-9 enzymatic activity evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis gelatin zymography of CM from M14 transfectants exposed to hypoxia for 24 h. Zymographyes representative of two independent experiments with similar results are shown.

Article Snippet: M14 and A375SM-SC1 cells were stably transfected using Lipofectamine (Invitrogen, Carlsbad, CA) with expression vectors encoding the human bcl-2 protein wild-type or deleted of BH4 domain (aa 1–36), or mutated in BH1 (G145E) or BH2 (W188A) domains (13), with Flag-tagged vector encoding the only BH4 domain of bcl-2 (aa 1–30) or with green fluorescent protein (GFP)-tagged vector encoding bcl-2 protein wild-type (Addgene plasmid 17999) or targeted to ER (Addgene plasmid 18000).

Techniques: Migration, Wound Healing Assay, Control, Clone Assay, Activity Assay, Polyacrylamide Gel Electrophoresis, Zymography

Journal: Carcinogenesis

Article Title: BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

doi: 10.1093/carcin/bgt242

Figure Lengend Snippet: Fig. 4. Stable expression of BH4 domain is sufficient to induce proangiogenic HIF-1/VEGF signaling. (A) Apoptosis evaluated by Annexin V-fluorescein isothiocyanate versus propidium iodide (PI) flow cytometry analysis in M14 control cells (Flag) and cells expressing Flag-BH4 protein (Flag-BH4/1, Flag-BH4/4) untreated or treated with 0.5 μM CPT for 48 h. The percentages of the Annexin V+/PI− (early apoptotic cells) and Annexin V+/PI+ (late apoptotic cells) are shown. Panels representative of one out of three independent experiments with comparable results are shown. (B) HIF-1α protein expression by western blot analysis. (C) HIF-1 transcriptional activity by reporter assay and (D) VEGF protein secretion by ELISA in M14 derivatives exposed to normoxia or hypoxia for 24 h. (B) Western blots representative of two independent experiments with similar results are shown. HSP72/73 is shown as loading and transferring control. (E) Quantitative evaluation of capillary-like structures of HUVEC incubated with CM from M14 transfectants exposed to hypoxia for 24 h. HUVEC exposed to serum-free medium and complete medium were used as negative (NEG) and positive (POS) controls, respectively. (C, D and E) The results are reported as the mean ± SD of three independent experiments. P values were calculated between control (Flag) and Flag-BH4-expressing cells (*P < 0.05).

Article Snippet: M14 and A375SM-SC1 cells were stably transfected using Lipofectamine (Invitrogen, Carlsbad, CA) with expression vectors encoding the human bcl-2 protein wild-type or deleted of BH4 domain (aa 1–36), or mutated in BH1 (G145E) or BH2 (W188A) domains (13), with Flag-tagged vector encoding the only BH4 domain of bcl-2 (aa 1–30) or with green fluorescent protein (GFP)-tagged vector encoding bcl-2 protein wild-type (Addgene plasmid 17999) or targeted to ER (Addgene plasmid 18000).

Techniques: Expressing, Flow Cytometry, Control, Western Blot, Activity Assay, Reporter Assay, Enzyme-linked Immunosorbent Assay, Transferring, Incubation

Journal: Carcinogenesis

Article Title: BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

doi: 10.1093/carcin/bgt242

Figure Lengend Snippet: Fig. 5. The bcl-2 protein lacking BH4 domain fails to localize into the nucleus and to bind HIF-1α protein. (A) Coimmunoprecipitation of HIF-1α/ bcl-2 proteins in M14 control clone (puro) and clones overexpressing bcl-2 wild-type (Bcl-2/37) or deleted of BH4 domain (BH4del/8) exposed to hypoxia. Total protein extracts were immunoprecipitated (IP) with anti-HIF- 1α antibody and then western blot analysis was performed using anti-bcl-2 antibody. (B) Coimmunoprecipitation of HIF-1α/bcl-2 proteins in M14 derivatives exposed to hypoxia mimetic CoCl2 (50 μM, 16 h). Nuclear (Nu) and cytoplasmic (Ct) protein extracts were immunoprecipitated with anti-HIF-1α or anti-bcl-2, respectively, and then western blot analysis was performed using anti-bcl-2 and anti-HIF-1α antibodies. (C) The bcl-2 protein expression in nuclear and cytoplasmic protein extracts of M14 derivatives. Lamin A/C (Lam A/C) protein was used as marker for nuclear fraction. (A and C) HSP72/73 or β-actin protein amounts were used to check equal loading and transfer of proteins. (A–C) Western blots representative of two independent experiments with similar results are shown.

Article Snippet: M14 and A375SM-SC1 cells were stably transfected using Lipofectamine (Invitrogen, Carlsbad, CA) with expression vectors encoding the human bcl-2 protein wild-type or deleted of BH4 domain (aa 1–36), or mutated in BH1 (G145E) or BH2 (W188A) domains (13), with Flag-tagged vector encoding the only BH4 domain of bcl-2 (aa 1–30) or with green fluorescent protein (GFP)-tagged vector encoding bcl-2 protein wild-type (Addgene plasmid 17999) or targeted to ER (Addgene plasmid 18000).

Techniques: Control, Clone Assay, Immunoprecipitation, Western Blot, Expressing, Marker