Journal: Journal of Translational Medicine

Article Title: Pro-apoptotic activity of α-bisabolol in preclinical models of primary human acute leukemia cells

doi: 10.1186/1479-5876-9-45

Figure Lengend Snippet: BID and NADH-supported state 3 respiration in normal PBMCs and leukemic blasts treated with α-bisabolol . (A) 24-hour α-bisabolol did not induced the cleavage of BID (full length 22 kDa, cleaved 15 kDa) at any concentration. Etoposide-treated Jurkat cells were used as a positive control for tBID. (B) No BID translocation was detected in mitochondrial fraction at different times and solubilized doses of α-bisabolol. α-tubulin and Hsp60 were used as markers for the cytosol and mitochondria fractions, respectively. A representative case is shown. (C) Permeabilized leukemic cells and healthy lymphocytes were incubated for 10 minutes in respiration buffer at 30°C in the presence or in the absence of 3 μM α-bisabolol. In treated leukemic cells, the G/M oxygen consumption was clearly lower than in untreated leukemic controls ( p < 0.05). The S/G3P oxygen consumption was not modified by treatment, and the mitochondrial respiration was not stimulated by FCCP addition. This is in line with a direct effect of α-bisabolol on mitochondrial integrity. Healthy lymphocyte respiration was not affected by treatment. G/M: glutamate plus malate; S/G3P: succinate plus glycerol-3-phosphate; FCCP: carbonylcyanide-4-(trifluoromethoxy)-phenyl-hydrazone. Means ± SD of 6 leukemias and 6 normal donors are depicted.

Article Snippet: Then, the membrane with the cytosolic and mitochondrial fractions were probed with a rabbit polyclonal IgG antibody to α-tubulin (Cell Signaling Technology) and with a mouse monoclonal IgG antibody to Hsp60 (Abcam, Cambridge, UK), respectively.

Techniques: Concentration Assay, Positive Control, Translocation Assay, Incubation, Modification

Journal: Cell metabolism

Article Title: Succinyl-CoA Ligase Deficiency in Pro-inflammatory and Tissue-Invasive T Cells

doi: 10.1016/j.cmet.2020.10.025

Figure Lengend Snippet: CD4+CD45RA+ T cells from RA patients and controls were activated for 72 h and stained for the mitochondrial marker HSP60. Nuclei were marked with DAPI.

Article Snippet: The following primary antibodies were used: Acetyl-alpha Tubulin (Lys40) mouse mAb (1:100, Thermo Fisher Scientific), α-tubulin rabbit mAb (1:100, Cell signaling technology), GM-130 rabbit mAb (1:100, Cell signaling technology), Pericentrin rabbit pAb (1:100, Abcam), Hsp60 rabbit mAb (1:100, Cell signaling technology).

Techniques: Staining, Marker

Journal: Cell metabolism

Article Title: Succinyl-CoA Ligase Deficiency in Pro-inflammatory and Tissue-Invasive T Cells

doi: 10.1016/j.cmet.2020.10.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: Acetyl-alpha Tubulin (Lys40) mouse mAb (1:100, Thermo Fisher Scientific), α-tubulin rabbit mAb (1:100, Cell signaling technology), GM-130 rabbit mAb (1:100, Cell signaling technology), Pericentrin rabbit pAb (1:100, Abcam), Hsp60 rabbit mAb (1:100, Cell signaling technology).

Techniques: Recombinant, Activation Assay, SYBR Green Assay, Cell Isolation, Citrate Assay, Activity Assay, Staining, Plasmid Preparation, Software

Journal: Life Science Alliance

Article Title: High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo

doi: 10.26508/lsa.202101034

Figure Lengend Snippet: (A) Western blot analysis of TFAM protein levels in liver whole cell lysates of CAG-TFAM (+/CAG) mice. Litter mates were used as controls (Con). Actin was used as a loading control. A representative image is shown (n = 2 independent experiments). (B) TFAM protein levels in control and CAG-TFAM animals were quantified by densitometry and are expressed as folds of control (means ± SEM, n = 4–5 biological replicates; P < 0.05: *, two-way ANOVA with Sidak’s test for multiple comparisons). (C) Quantification of steady-state mtDNA levels in liver tissue of CAG-TFAM (+/CAG) animals and control litter mates (Con). mtDNA levels were quantified by qPCR using specific probes against COX1 and 18S. Data are expressed as means ± SEM (n = 6–7 biological replicates). (D) Southern blot analysis of PstI-digested mtDNA derived from heart and skeletal muscle of CAG-TFAM (+/CAG) animals and control litter mates (Con). mtDNA was quantified by radiolabeling with a specific probe against COX1, nuclear DNA was probed with 18S. A representative image is shown (n = 3 independent experiments). (E) Analysis of steady-state mitochondrial transcript levels in liver tissue of CAG-TFAM (+/CAG) animals and control litter mates (Con) by qRT-PCR. Data are expressed as means ± SEM (n = 5 biological replicates). (F) De novo RNA synthesis in skeletal muscle and liver mitochondria isolated from CAG-TFAM (+/CAG) mice and control litter mates. Mitochondria were pulse labelled for 1 h. Mitochondrial HSP60 was used as a loading control. A representative image is shown (n = 2–3 independent experiments). (G) Representative images of fixed liver tissue from control (Con) and CAG-TFAM animals at 16 d of age analysed by transmission electron microscopy. For each genotype, six biological replicates were analysed. Scale bars, 20 (upper panels), 5 (middle), 2 μm (lower panels). (H) Measurement of triglyceride content. Approximately 100 mg of liver tissue was homogenized and triglycerides were quantified using the triglycerides quantification kit (Sigma-Aldrich). Data are expressed as means ± SEM (n = 5 biological replicates; P < 0.001:***, unpaired t test). Source data are available for this figure.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-TFAM (Abcam), mouse monoclonal anti-actin (Abcam), Total OXPHOS Rodent Western Blotting antibody cocktail (Abcam), mouse monoclonal anti-tubulin (Sigma-Aldrich), rabbit anti-Tubulin (Cell Signaling), rabbit anti-vinculin (Abcam), mouse monoclonal anti-UQCRFS1/RISP (Abcam), mouse monoclonal anti-COX5a (Invitrogen/ThermoFisher Scientific), mouse monoclonal anti-VDAC (Millipore), rabbit polyclonal anti-ACOT2 (Proteintech), mouse monoclonal anti-cytochrome C (Abcam), rabbit anti-HSP60 (Cell Signaling), sheep anti-mouse IgG (GE Healthcare), and donkey anti-rabbit (GE Healthcare).

Techniques: Western Blot, Southern Blot, Derivative Assay, Radioactivity, Quantitative RT-PCR, Isolation, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: A subcellular map of the human kinome

doi: 10.7554/eLife.64943

Figure Lengend Snippet: ( A ) Sub-mitochondrial localizations of new mitochondrial kinases. Transfected HeLa cells were stained and imaged by 3D structured illumination microscopy (3D-SIM). ( B ) MOK2 is imported into mitochondria. Transfected HeLa cells were fixed and permeabilized with digitonin or Triton X-100 before staining. ( C ) MOK is not a mitochondrial membrane protein. Mitochondria purified from transfected HeLa cells were incubated in indicated buffers, and then centrifuged. Supernatants (S) and pellets (P) were analyzed by western blotting. Equal loading was achieved by normalizing to the same number of cells. ( D ) MOK is an IMS protein. COS-7 cells transfected with MOK2-GFP were imaged by Hessian SIM. MitoTracker Red marked inner membrane (IMM) and co-transfected HSP60-mCherry marked matrix. ( E ) MOK2 has better mitochondrial localization than MOK1. HeLa cells were transfected with full-length (FL) or kinase domain (KD) of MOK1 and MOK2. Cells with mitochondrial MOK were quantified. Data is represented as mean ± SD; n = 3 independent experiments, two-tailed Student’s t test; n = 100 cells were analyzed in each experiment. ( F ) Mitochondrial localization of MOK mutants. Transfected HeLa cells were stained with anti-Myc and anti-TOM20 antibodies. ( G ) TOM20-TIM23 complex is required for mitochondrial importing of MOK. HeLa cells expressing specific shRNAs were transfected with MOK2-Myc and stained . Figure 6—source data 1. Uncropped western blot for . Figure 6—source data 2. Raw data for and .

Article Snippet: Antibody , Rabbit monoclonal anti-HSP60 (D6F1) antibody , Cell Signaling Technology , Cat# 46611, RRID: AB_2799305 , WB (1:1000), IF (1:500).

Techniques: Transfection, Staining, Microscopy, Purification, Incubation, Western Blot, Two Tailed Test, Expressing

Journal: eLife

Article Title: A subcellular map of the human kinome

doi: 10.7554/eLife.64943

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-HSP60 (D6F1) antibody , Cell Signaling Technology , Cat# 46611, RRID: AB_2799305 , WB (1:1000), IF (1:500).

Techniques: Transfection, Construct, Sequencing, shRNA, Protease Inhibitor, Software