Journal: Molecular Cancer Therapeutics

Article Title: BH3-only proteins Mcl-1 and Bim as well as endonuclease G are targeted in spongistatin 1–induced apoptosis in breast cancer cells

doi: 10.1158/1535-7163.mct-08-1179

Figure Lengend Snippet: Figure 4. Spongistatin 1 induces the translocation of AIF and EndoG to the nucleus. A, cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Nonnucleic and nuclear protein fractions were prepared and AIF and EndoG were detected by specific antibodies using Western blot analysis. Cytochrome c oxidase served as control for the quality of the extraction procedure. B, cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Translocation of AIF and EndoG from mitochondria to the nucleus was analyzed by confocal microscopy. Blue, nuclei; green, AIF and EndoG. All experiments were done three times with consistent results. C, AIF and EndoG assume a functional role in spongistatin 1–induced apo- ptosis. MCF-7 cells were transfected or cotransfected with oligonucleotides encoding for either AIF siRNA and EndoG siRNA or a nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 48 h. Apoptotic cells were quantified by flow cytometry. Results are percent specific apoptosis. Downregulation of AIF and EndoG protein levels was verified by Western blot. Equal protein loading was controlled by staining membranes with β-actin. Columns, mean of three independent experiments done in triplicate; bars, SE. ***, P < 0.001 (ANOVA/Bonferroni).

Article Snippet: Cells were blocked with 0.2% bovine serum albumin and incubated with specific antibodies against AIF (rabbit polyclonal IgG; Upstate) and EndoG (rabbit polyclonal antibody; Prosci).

Techniques: Translocation Assay, Western Blot, Control, Extraction, Confocal Microscopy, Functional Assay, Transfection, Sequencing, Flow Cytometry, Staining

Journal: Molecular Cancer Therapeutics

Article Title: BH3-only proteins Mcl-1 and Bim as well as endonuclease G are targeted in spongistatin 1–induced apoptosis in breast cancer cells

doi: 10.1158/1535-7163.mct-08-1179

Figure Lengend Snippet: Figure 6. Bim functions as a proapoptotic regulator upstream of mitochondria. A, MCF-7 cells were transfected with oligonucleotides encoding for either Bim siRNA or nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 24 h. Nonnucleic and nuclear protein fractions were prepared and AIF and EndoG were detected by specific antibodies using Western blot analysis. Cytochrome c oxidase served as control for the quality of the extraction procedure. As a control, Bim protein level in cell lysates from nonsense and Bim siRNA cells was analyzed by Western blot. B, Bim and EndoG are the major regulators of spongistatin 1–induced cell death. MCF-7 cells were cotransfected with oligonucleotides encoding for either Bim siRNA or EndoG siRNA or nonsense sequence. Cells were left untreated or treated with spongistatin 1 (500 pmol/L) for 48 h. Apoptotic cells were quantified by flow cytometry. Results are percent specific apoptosis. Downregulation of Bim and EndoG protein levels was verified by Western blot. Equal protein loading was controlled by staining membranes with β-actin. All experiments were done three times with consistent results. Columns, mean of three independent experiments done in triplicate; bars, SE. ***, P < 0.001 (ANOVA/Bonferroni). C, proposed mechanism of spongistatin 1–induced apoptosis. Thick arrows, main signaling pathway of spongistatin 1. The tubulin depolymerizing agent spongistatin 1 frees Bim from its sequestration both by the microtubule network and by the antiapoptotic protein Mcl-1. Bim triggers the translocation of AIF and EndoG from mitochondria to the nucleus leading to caspase-independent apoptosis. Thin arrows, inferior role of the caspase-dependent cell death induced by spongistatin 1.

Article Snippet: Cells were blocked with 0.2% bovine serum albumin and incubated with specific antibodies against AIF (rabbit polyclonal IgG; Upstate) and EndoG (rabbit polyclonal antibody; Prosci).

Techniques: Transfection, Sequencing, Western Blot, Control, Extraction, Flow Cytometry, Staining, Translocation Assay

Journal: Frontiers in Pediatrics

Article Title: Case Report: A Novel Intronic Mutation in AIFM1 Associated With Fatal Encephalomyopathy and Mitochondrial Disease in Infant

doi: 10.3389/fped.2022.889089

Figure Lengend Snippet: (A) Family pedigree. Black symbols represent affected persons and symbols with a dot, carriers. The proband is indicated by an arrow. (B) Chromatograms for the mutations confirmed by Sanger sequencing. (C) Agarose gel electrophoresis of the transcripts generated by cDNA amplification of the patient and control, with adjacent schematic representation of the resulting splicing events. The PCR product of patient was marked by red arrow. (D) Agarose gel electrophoresis of cloning PCR. S1–S9 represent the different clones picked. (E) Sanger sequencing electropherogram of gel-purified fragments for the upper band and lower band from the patient cDNA samples. (F) AIFM1 Sashimi plot of RNA extracted from patient’s and control’s fibroblasts.

Article Snippet: A mouse monoclonal anti-GAPDH (4A Biotech Co., Ltd., Shanghai, China, Cat. No. 4ab030004) and mouse monoclonal anti-AIFM1 antibody binding to the C-terminus of human AIF (Boster Biological Technology, Wuhan, China, Cat. No. M01571-1) were used.

Techniques: Sequencing, Agarose Gel Electrophoresis, Generated, Amplification, Control, Cloning, Clone Assay, Purification

Journal: Frontiers in Pediatrics

Article Title: Case Report: A Novel Intronic Mutation in AIFM1 Associated With Fatal Encephalomyopathy and Mitochondrial Disease in Infant

doi: 10.3389/fped.2022.889089

Figure Lengend Snippet: (A) Relative quantification of mRNA levels for AIFM1 in controls and patient’s fibroblasts using different primers. The results of mRNA are the average of the values assessed after three reaction tests. (B) Western blot analysis in controls and patient’s fibroblasts using AIF antibody. GAPDH was used as loading control. The patient’s sample shows a clear reduction in the AIF amount compared with the control lines. (C) Western blot analysis in controls and patient’s muscles using AIF antibody. GAPDH was used as loading control. (D) Electron microscopy of quadriceps muscle demonstrating large, irregularly shaped mitochondria, including one with concentric cisternae. (E) TOMM20 staining of the patient’s and control fibroblast. DAPI was used to mark the nucleus.

Article Snippet: A mouse monoclonal anti-GAPDH (4A Biotech Co., Ltd., Shanghai, China, Cat. No. 4ab030004) and mouse monoclonal anti-AIFM1 antibody binding to the C-terminus of human AIF (Boster Biological Technology, Wuhan, China, Cat. No. M01571-1) were used.

Techniques: Quantitative Proteomics, Western Blot, Control, Muscles, Electron Microscopy, Staining

Journal: Frontiers in Pediatrics

Article Title: Case Report: A Novel Intronic Mutation in AIFM1 Associated With Fatal Encephalomyopathy and Mitochondrial Disease in Infant

doi: 10.3389/fped.2022.889089

Figure Lengend Snippet: (A) Schematic model representing the AIF protein. AIF is a flavoprotein (with an oxidoreductase enzymatic activity) containing a FAD-bipartite domain (amino-acids 128–262 and 401–480), a NADH-binding motif (amino-acids 263–400), and a C-terminal domain (amino-acids 481–608) where the proapoptotic activity of the protein resides. In addition, it has a Mitochondria Localization Sequence (MLS, amino-acids 1–41) placed in its N-terminal region. AIF also possesses two DNA-binding sites, which are located in amino-acids 255–265 and 510–518, respectively. (B) The mutant causes a frameshift resulted in the change of amino acid coding after R358, and the early termination codon was generated at the position of amino acid 362. (C) Protein structure modeling of wild-type and mutated AIFM1. A part of the amino acid sequence has been eliminated in the mutated protein compared to the wild type protein. (D) The NAD + /NADH ratios in fibroblasts of patient and control. (E) The human COX of cell culture supernatant in patient and control. *Statistically significant difference at p < 0.05.

Article Snippet: A mouse monoclonal anti-GAPDH (4A Biotech Co., Ltd., Shanghai, China, Cat. No. 4ab030004) and mouse monoclonal anti-AIFM1 antibody binding to the C-terminus of human AIF (Boster Biological Technology, Wuhan, China, Cat. No. M01571-1) were used.

Techniques: Activity Assay, Binding Assay, Sequencing, Mutagenesis, Generated, Control, Cell Culture

Journal: Cell death and differentiation

Article Title: Apoptosome inactivation rescues proneural and neural cells from neurodegeneration.

doi: 10.1038/sj.cdd.4401476

Figure Lengend Snippet: Figure 1 Apaf1-deficiency leads to accumulation of NPCs expressing neural markers in vivo and in vitro. (a) BrdU staining on coronal sections from e12.5 wt and Apaf1/ (/) littermates embryos. nt: neural tube (hindbrain), fb: forebrain. (b) Analysis of differentiation markers from e14 wt and Apaf1/ littermate embryos. nestin, delta1, notch1 mRNA levels were revealed by in situ mRNA hybridization as previously described (Stoykova and Gruss, 1994) on embryonic coronal sections. Class III b- tubulin was revealed by immunohistochemistry on embryonic e14.5 coronal sections with a mouse monoclonal anti-class III b-tubulin antibody. The corresponding histological structures are indicated in the sketch and in the right panel as follows: I: intermediate zone, M: marginal zone, V: ventricular germinal zone, ve: brain ventricle. Scale bar: 0.4 mm. (c) ETNA þ / þ ( þ / þ ) and ETNA/ (/) cell lines were isolated from e14 wt and mutant embryos, respectively, and immortalized with a retrovirus transducing the tsA58/U19 large T antigen. Total extracts from both cell lines were immunoprecipitated with an anti-Apaf1 rabbit polyclonal antibody and analysed with a rat monoclonal antibody anti-Apaf1. Ctrl: human neuroblastoma cells (SH-SY5Y) stably overexpressing Apaf1. (d) Total cell lysates from ETNA þ / þ and ETNA/ cells, either naı¨ve (N) or incubated for 48 h with a differentiation medium (D), were analysed by Western blotting for the expression of NeuN, class III b-tubulin, ChAT, PSD95, tau; b-actin was also assayed as a control of equal protein loading. (e) ETNA þ / þ and ETNA/ cells, either naı¨ve or differentiated, were immunostained with an antibody recognizing the 160 kDa form of neurofilament (NF-M). Scale bar: 40 mm

Article Snippet: We used an anti-cytochrome c mouse monoclonal antibody (clone 6H2.B4, BD PharMingen), an anti-cytochrome c rabbit polyclonal antibody (Santa Cruz), an anti-AIF rabbit polyclonal antibody (a generous gift of Guido Kroemer), an anti-EndoG rabbit polyclonal antibody (ProSci Incorporated), an anti-Neurofilament 160 mouse monoclonal antibody (clone NN18, SIGMA), anti-Apaf1 polyclonal antibody (AB16941, Chemicon Int.) and an anti- Large T Antigen monoclonal antibody (clone PAb419, Oncogene).

Techniques: Expressing, In Vivo, In Vitro, BrdU Staining, In Situ, Hybridization, Immunohistochemistry, Isolation, Mutagenesis, Immunoprecipitation, Stable Transfection, Incubation, Western Blot, Control