Journal: Bone & Joint Research

Article Title: Down-expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenesis and accelerates age-related bone loss via PTEN/PI3K/AKT pathway

doi: 10.1302/2046-3758.132.BJR-2023-0146.R2

Figure Lengend Snippet: Effects of senescent MLO-Y4 cell-derived exosomes (abbreviated tert-Butyl hydroperoxide (TBHP)-exos in the figure) on osteogenic differentiation of MC3T3-E1 cells. a) Cell Counting Kit 8 (CCK-8) assay of MC3T3-E1 cells treated with different concentrations (20, 100, or 300 μg/ml) of senescent MLO-Y4 cell-derived exosomes for different lengths of time (0, 24, 48, or 72 hours). b) Alkaline phosphatase (ALP) and Alizarin Red S staining of MC3T3-E1 cells on days 7, 14, and 21 after treatment with the indicated concentrations of exosomes obtained from senescent MLO-Y4 cells. c) and d) Western blotting and grayscale analysis of ALP, Runt-related transcription factor 2 (Runx2), and collagen type I α1 (Col-I) expression in MC3T3-E1 cells in different treatment groups. Data are shown as column charts and the p-values were calculated by one-way analysis of variance with Sidak’s multiple comparison tests. The p-values were specified only when p < 0.05 (statistically significant). GD, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The primary antibodies used were P16 (1:1000, ab51243; Abcam, USA), Runt-related transcription factor 2 (Runx2, 1:1,000, 12,556 S; Cell Signaling Technology), collagen type I α1 (Col-I, 1:1,000, 72,026 T; Cell Signaling Technology), ALP antibody (1:1,000, AF2910; R&D Systems, USA), AKT (1:1,000, 60203-2-Ig; Proteintech, USA), phosphorylated (p)-AKT (1:1,000, 66444-1-Ig; Proteintech), PI3K (1:1,000, 20584-1-AP; Proteintech), PTEN (1:1,000, 22034-1-AP; Proteintech), CD9 (1:1,000, 60232-1-Ig; Proteintech), and CD63 (1:1,000, 67605-1-Ig; Proteintech), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000, 60004-1-Ig; Proteintech) was used as a loading control.

Techniques: Derivative Assay, Cell Counting, CCK-8 Assay, Staining, Western Blot, Expressing, Comparison

Journal: Bone & Joint Research

Article Title: Down-expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenesis and accelerates age-related bone loss via PTEN/PI3K/AKT pathway

doi: 10.1302/2046-3758.132.BJR-2023-0146.R2

Figure Lengend Snippet: miR-494-3p in MLO-Y4 cell-derived exosomes regulated osteogenic differentiation of MC3T3-E1 cells via the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/AKT pathway. a) Alkaline phosphatase (ALP) and Alizarin Red S staining of MC3T3-E1 cells on day 21 in different treatment groups. The magnification is 10× (lower part). b) and c) Western blotting and grayscale analysis of ALP, Runt-related transcription factor 2 (Runx2), collagen type I α1 (Col-I), p-AKT, AKT, PI3K, and PTEN expression in MC3T3-E1 cells in different treatment groups. Data are shown as column charts and the p-values were calculated by one-way analysis of variance with Sidak’s multiple comparison tests. The p-values were specified only when p < 0.05 (statistically significant). GD, glyceraldehyde 3-phosphate dehydrogenase; miR, microRNA; TBHP, tert-Butyl hydroperoxide.

Article Snippet: The primary antibodies used were P16 (1:1000, ab51243; Abcam, USA), Runt-related transcription factor 2 (Runx2, 1:1,000, 12,556 S; Cell Signaling Technology), collagen type I α1 (Col-I, 1:1,000, 72,026 T; Cell Signaling Technology), ALP antibody (1:1,000, AF2910; R&D Systems, USA), AKT (1:1,000, 60203-2-Ig; Proteintech, USA), phosphorylated (p)-AKT (1:1,000, 66444-1-Ig; Proteintech), PI3K (1:1,000, 20584-1-AP; Proteintech), PTEN (1:1,000, 22034-1-AP; Proteintech), CD9 (1:1,000, 60232-1-Ig; Proteintech), and CD63 (1:1,000, 67605-1-Ig; Proteintech), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000, 60004-1-Ig; Proteintech) was used as a loading control.

Techniques: Derivative Assay, Staining, Western Blot, Expressing, Comparison

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: (a) PCr’ase activities of total mitochondrial protein extracts from different tissues of cold-acclimated mice. BAT, interscapular brown adipose tissue; and iWAT, inguinal white adipose tissue. Mitochondrial protein extract was prepared from tissues excised from 10 mice for BAT or 20 mice for iWAT. Each reaction contains 10 mM of PCr and 0.4 mg/mL of mitochondrial protein extract, except the buffer control. Data are presented as the estimated parameters ± uncertainties. Uncertainties are represented by the standard errors of non-linear regression that fits a straight-line model to the initial linear phase of PCr hydrolysis kinetics measured by 31P NMR over 11 time points for BAT and iWAT and 6 time points for the buffer control (shown in Source Data).

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Control

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: (a) Confocal fluorescence images showing subcellular localization of endogenous TNAP in brown adipocytes (upper and middle panels) and hepatocytes (lower panels). Primary brown preadipocytes were prepared from Alpl fl/fl mice, transduced with either AdGFP (WT) or AdCRE (ALPL KO) on day 4 of differentiation, and fixed for imaging on day 8. Arrows denote selected peri-nuclear areas of TNAP signal that colocalize with mitochondria signal. Antibodies for TNAP (Red) and HSP60 (Green) were used to visualize TNAP and mitochondria. Scale bar: 5 μm.

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Fluorescence, Transduction, Imaging

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: (a) Confocal fluorescence images showing subcellular localization of ectopically expressed TNAP in immortalized brown adipocytes and primary hepatocytes. The insets show an enlarged region of the image outlined by the dotted box. Arrows denote selected signals of TNAP that colocalize with mitochondria signal. Anti-TNAP (Red) and anti-HSP60 (Green) were used to visualize TNAP and mitochondria, respectively. Scale bar: 10 μm.

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Fluorescence

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: Confocal fluorescence microscopic images showing subcellular localization of ectopically expressed TNAP in different cell types. PTEC stands for kidney proximal tubule epithelial cells. The insets show a zoomed-in region of the image outlined by a dotted box. Anti-TNAP and anti-HSP60 were used to visualize TNAP and mitochondria, respectively. Scale bar: 10 μm.

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Fluorescence

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: (a) Confocal fluorescence microscopic images of immortalized brown adipocytes showing colocalization of the GFP signal from 3XHA-EGFP-OMP25 construct (mGFP, channel: 488 nm) with different mitochondria markers. Endogenous antibodies, OxPhos (Upper red, channel: 561 nm) and HSP60 (Lower red, channel: 640 nm), were used to visualize mitochondria. The insets show a zoomed-in region of the image outlined by the dotted box. Scale bar: 5 μm.

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Labeling, Fluorescence, Construct

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: (a) Model of inhibition of FCC by the TNAP-selective inhibitor, SBI-425.

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Inhibition

Journal: Nature

Article Title: Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine

doi: 10.1038/s41586-021-03533-z

Figure Lengend Snippet: (a) Effect of SBI-425 treatment (10 μM) on oxygen consumption rate (OCR) of beige fat-derived mitochondria from WT vs Adipo-Alpl KO mice in the presence of 0.01 mM creatine and 0.1 mM ADP (limiting ADP) or 1 mM ADP (saturating ADP), as measured by a Seahorse XF24 Extracellular Flux Analyzer; n=7 independent measurements per group.

Article Snippet: For immunofluorescence staining, anti-TNAP mouse monoclonal antibody (R&D Systems, MAB29092) at 1:100 and anti-HSP60 rabbit monoclonal antibody (Cell Signaling, 12165) at 1:500 in blocking solution were used for primary labeling of TNAP and mitochondria, respectively; and anti-mouse Alexa Fluor 568 and anti-rabbit Alexa Fluor 647 at 1:1000 (Invitrogen) were used for secondary labeling.

Techniques: Inhibition, Derivative Assay

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Monoclonal antibodies used in this study

Article Snippet: MSCA-1 , PE , Mouse IgG1κ , Miltenyi Biotec , 130-099-198.

Techniques: Bioprocessing