Journal: Bone Research

Article Title: Osteoclasts control endochondral ossification via regulating acetyl-CoA availability

doi: 10.1038/s41413-024-00360-6

Figure Lengend Snippet: Rheb1 deletion mainly impairs osteoclast mitochondrial respiration. a Mitochondrial OCR in the routine, noncoupled (oligomycin), and maximal (FCCP) steady state in Rheb1-deficient osteoclasts or control cells were determined by the real-time Seahorse assay. b Extracellular acidification rate (ECAR) in Rheb1-deficient osteoclasts or their respective control cells was determined by the real-time Seahorse assay. c The basal respiration and FCCP-induced maximal respiration in the OCR shown in a were compared between Rheb1-deficient osteoclasts and their controls ( n = 26). Unpaired t test. **** P < 0.000 1. d The basal and maximal glycolysis levels in the ECAR shown in b were compared between Rheb1-deficient osteoclasts and their controls ( n = 26). Unpaired t test. * P < 0.05; n.s., no significant difference. e The mitochondrial and glycolytic ATP production rates were compared between Rheb1-deficient osteoclasts and their respective control cells ( n = 26). Unpaired t test. **** P < 0.000 1; n.s., no significant difference. f Schematic of the type I collagen - based ex vivo model. g OCR in Rheb1-deficient osteoclasts adherent on type I collagen-coated substrates was determined by the Seahorse assay in real time, which was compared to control cells. h The basal and maximal respiration in the OCR shown in g ( n = 4). i Schematic of the type II collagen-based ex vivo model. Unpaired t test. ** P < 0.01, *** P < 0.001. j OCR in Rheb1-deficient osteoclast adherent on type II collagen-coated substrates was determined by the Seahorse assay in real time, which was compared to control cells. k The basal and maximal respiration in the OCR shown in j ( n = 5 for control, n = 8 for Rheb1 OC ). Unpaired t test. ** P < 0.01, **** P < 0.000 1. All data are presented as mean ± SEM

Article Snippet: Inhibitors and substrates used were as follows: rotenone (HY-B1756, MedChemExpress, NJ, USA), oligomycin A (HY-16589, MedChemExpress), FCCP (HY-100410, MedChemExpress), 2-DG (HY-13966, MedChemExpress), 3BrPA (HY-19992, MedChemExpress), bafilomycin A1 (HY-100558, MedChemExpress), pyruvate (103578-100, Agilent Technologies, Santa Clara, CA, USA), glutamine (103579-100, Agilent Technologies), citrate (C0759, Sigma Aldrich, St. Louis, USA), acetate (AM9740, Invitrogen, Waltham, Massachusetts, USA), DMKG (349631, Sigma Aldrich, St. Louis, USA), ACSS2 inhibitor (HY-148104, MedChemExpress), ACLY inhibitor (HY-16450, MedChemExpress) and integrin α v β 3 inhibitor (HY-P0023, MedChemExpress).

Techniques: Control, Ex Vivo

Journal: Bone Research

Article Title: Osteoclasts control endochondral ossification via regulating acetyl-CoA availability

doi: 10.1038/s41413-024-00360-6

Figure Lengend Snippet: Mitochondrial respiration is critical to osteoclast procathepsin K production. a Labeling of mitochondria with Mito Tracker Dyes in osteoclasts adherent on type I collagen-coated or type II collagen-coated interfaces, bone slices or plastic interfaces without collagen-coating, respectively (scale bar, 10 µm). b Statistical analysis of immunofluorescence signal density in a ( n = 5 for type I collagen-coating, n = 4 for type II collagen-coating, n = 5 for bone slice, n = 4 for plastic). One-way ANOVA. * P < 0.05, ** P < 0.01; n.s., no significant difference. c OCR in osteoclasts adherent on type I or type II collagen-coated substrates compared to cells culture on plastic substrates were determined by the Seahorse assay in real-time. d The basal respiration and FCCP-induced maximal respiration in the OCR shown in c were compared between osteoclasts adherent on collagen-coated substrates and plastic substrates ( n = 3 for collagen-coating, n = 6 for plastic). One-way ANOVA. * P < 0.05, ** P < 0.01, **** P < 0.000 1. e Immunofluorescence staining analysis to show CTSK protein abundance in osteoclasts adherent on different substrates (scale bar, 100 Pixel, n = 3 for plastic, n = 4 for type I collagen-coating, n = 3 for type II collagen-coating). One-way ANOVA. * P < 0.05. f Immunofluorescence staining to show the CTSK expression level in osteoclasts that were treated with oligomycin A (10 nmol/L), rotenone (100 nmol/L), FCCP (1 μmol/L), 2DG (1 μmol/L) or 3BrPA (1 μmol/L) for 6 h (scale bar, 100 µm). g Immunoblots of the pre-proenzyme and active CTSK levels in osteoclasts treated with 2DG (1 μmol/L), FCCP (1 μmol/L), rotenone (100 nmol/L) or 3-BrPA (1 μmol/L) for 10 h. The MMP-9 and CTSB expression levels were also detected. DMSO-treated osteoclasts were used as controls. h, i Immunoblots of the levels of the CTSK pre-proenzyme and active forms in osteoclasts treated with 10 nmol/L oligomycin A or 100 nmol/L rotenone, with or without lysosome inhibitor bafilomycin A1 (10 ng/mL) treatment for 4 h. Bafilomycin A1 administration was performed followed 2 h later by treatment with OxPhos inhibitors. All data are presented as mean ± SEM

Article Snippet: Inhibitors and substrates used were as follows: rotenone (HY-B1756, MedChemExpress, NJ, USA), oligomycin A (HY-16589, MedChemExpress), FCCP (HY-100410, MedChemExpress), 2-DG (HY-13966, MedChemExpress), 3BrPA (HY-19992, MedChemExpress), bafilomycin A1 (HY-100558, MedChemExpress), pyruvate (103578-100, Agilent Technologies, Santa Clara, CA, USA), glutamine (103579-100, Agilent Technologies), citrate (C0759, Sigma Aldrich, St. Louis, USA), acetate (AM9740, Invitrogen, Waltham, Massachusetts, USA), DMKG (349631, Sigma Aldrich, St. Louis, USA), ACSS2 inhibitor (HY-148104, MedChemExpress), ACLY inhibitor (HY-16450, MedChemExpress) and integrin α v β 3 inhibitor (HY-P0023, MedChemExpress).

Techniques: Labeling, Immunofluorescence, Staining, Quantitative Proteomics, Expressing, Western Blot

Journal: bioRxiv

Article Title: Genetically encoded tool for manipulation of ΔΨm identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction

doi: 10.1101/2023.12.27.573435

Figure Lengend Snippet: a , Effect of various concentrations of FCCP (left) or Bam15 (right) on the ΔΨm using TMRM. The intensity of TMRM was normalized by the intensity of Hoechst. Data is normalized to control condition (Ctrl, lane 1). Means ± SEM, n=3 biological replicates. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad ( n.s. , not significant). b , Effect of UCP1 on the ΔΨm in the presence of 50 mM oleic acid (Methyl-b-cyclodextrin conjugated) using TMRM. The intensity of TMRM was normalized by the intensity of Hoechst. Data is normalized to oleic acid-treated condition (lane 1). Means ± SEM, n=3 independent experiments. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad ( n.s. , not significant). c , Effect of oleic acid (OA, 300 mM) or doxycycline (Dox, 300 ng/ml) on proliferation of wild type parental C2C12 (left) or UCP1-expressing (right) C2C12 cells in the presence of 1 mM pyruvate. Means ± SEM, n=3 biological replicates. d , Effect of various concentrations of FCCP (left) or Bam15 (right) on the proliferation of C2C12 cells in the presence of 1 mM pyruvate. Means ± SEM, n=4 independent experiments. e , Effect of various concentrations of FCCP (left) or Bam15 (right) on basal and oligomycin (1 mM)-treated oxygen consumption. Antimycin A (1 mM) was injected at the last as a negative control. Means ± SEM, n=3 independent experiments.

Article Snippet: Cells were incubated at 37 °C with the following mitochondrial toxins for 16 hr: 1 μM oligomycin (Signa-Aldrich, 75351), 5 μM FCCP (TOCRIS, 45310), 1 μM antimycin A (Sigma-Aldrich, A8674), 1 μM rotenone (Sigma-Aldrich, R8875), 50 μgml -1 doxycycline (Sigma-Aldrich, D9891).

Techniques: Control, Expressing, Injection, Negative Control

Journal: bioRxiv

Article Title: Genetically encoded tool for manipulation of ΔΨm identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction

doi: 10.1101/2023.12.27.573435

Figure Lengend Snippet: a , A simple schematic diagram showing the molecular mechanism of how oligomycin induces mitochondrial stress signaling. b , Effect of various chemical drugs on inducing ATF4 in C2C12 cells. Immunoblotting analyses were performed with the indicated antibodies. Representative gel from one of three experiments. C, control; OLG, 1 mM oligomycin; FCCP, 5 mM FCCP; Ant, 1 mM Antimycin A; Rot, 1 mM Rotenone; Dox, 50 mg/ml doxycycline. Bar graphs show the quantification of the intensity of western blot data, relative amount of ATF4 compared to control condition. Means ± SEM, n=3independent experiments. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad (n.s., not significant). c,d,e , Effect of UCP1 (c), LbNOX (d), or mitoLbNOX (e) on mitochondrial stress signaling. Representative gel from one of four (UCP1 and LbNOX) or five (mitoLbNOX) experiments Bar graphs show the quantification of the intensity of western blot data, relative amount of ATF4 (left) compared to control condition. Means ± SEM, n=4 or 5 independent experiments. P values were determined with two-way ANOVA followed by Tukey’s multiple comparisons test in Graphpad (n.s., not significant).

Article Snippet: Cells were incubated at 37 °C with the following mitochondrial toxins for 16 hr: 1 μM oligomycin (Signa-Aldrich, 75351), 5 μM FCCP (TOCRIS, 45310), 1 μM antimycin A (Sigma-Aldrich, A8674), 1 μM rotenone (Sigma-Aldrich, R8875), 50 μgml -1 doxycycline (Sigma-Aldrich, D9891).

Techniques: Western Blot, Control