Journal: Scientific reports

Article Title: Disordered metabolism in mice lacking irisin.

doi: 10.1038/s41598-020-74588-7

Figure Lengend Snippet: Figure 6. Increased bone resorption in irisin lacking mice. (A, B) Sections of the distal shaft of the femur were stained with osteoprotegerin (OPG) (1:60) and receptor activator of nuclear factor-kB ligand (RANKL) (1:80) antibodies. Positive (black arrows) osteoblasts are shown, 200×. (C) Representative images of the distal metaphyseal region of the femur, together with cell counts (osteoclasts) per bone perimeter (Bpm). Red arrows, tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, 100×, (NIS-Elements Viewer, v4.2.0; https:// www.downza.cn/soft/2751-21.html); (D) serum levels of osteocalcin TRAP (with three replicates). (Graphpad Prism, v7.0, https://www.xue51.com/soft/3932.html). Data are presented as the mean ± SEM (n = 15 per group). *P < 0.05, **P < 0.01 compared to the WT group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit for osteocalcin (OCN) (E06917m), Alkaline Phosphatase (ALP) (E0200m), and Tartrate-resistant acid phosphatase (TRAP) (E08492m) were purchased from Elabscience (Wuhan, China).

Techniques: Staining

Journal: JBMR Plus

Article Title: Geranylgeranyl diphosphate synthase inhibition impairs osteoclast differentiation, morphology, and resorptive activity

doi: 10.1093/jbmrpl/ziae133

Figure Lengend Snippet: RAM2061 does not significantly impact normal bone remodeling in CD-1 mice. (A) Schematic representation of drug administration/dosing and downstream readouts conducted. Abbreviations: procollagen type 1 N-propeptide (P1NP); C-terminal telopeptide (CTX), tartrate resistant acid phosphatase 5b (TRAP5b). (B) Immunoblot analysis of unmodified Rap1a and GAPDH (loading control) using 30 μL of bone homogenate from long bones of mice; positive control = cell lysate from RAW264.7 osteoclasts (D5) treated with 400 nM RAM2061 for 48 h). (C) Quantification of RAM2061 accumulation in long bones and mandibles of CD-1 mice ( n = 5 per group, data are represented as mean ± SD). (D) MicroCT analysis portraying morphometric analysis of the proximal tibia specimens. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 5; zoledronic acid (ZA) n = 4; * denotes p <.05, ** denotes p <.01 per t-test). (E) The growth plate in the distal femur of tartrate resistant acid phosphatase (TRAP)-stained sections were identified and 1000 μm from the most distal area was used to encapsulate the area of interest. Quantification of osteoclast numbers in the defined area per femur was performed. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 4; ZA n = 4, * denotes p<.05, *** denotes p <.001 per t-test). (F) Representative images of TRAP-stained sections of distal femurs (scale bar = 50 μm). The arrows indicate TRAP+ osteoclasts (Tb = trabecular bone, BM = bone marrow).

Article Snippet: Commercial ELISA kits (Novus Biologics) were utilized for measuring plasma levels of TRAP5b (NBP2-76463), c-terminal telopeptide (CTX) (NBP2-82400), and procollagen type I N-propeptide (P1NP) (NBP2-76466).

Techniques: Western Blot, Control, Positive Control, Staining

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a Relative quantification of Kif5a, Kif5b and Kif5c transcripts by real-time PCR in CD8α + and CD11b + DCs and BMDCs from WT or cKO Kif5b mice. Transcript levels for each sample are expressed relative to Kif5b. Graph shows mean ± S.E.M. ( n = 3). b Contour plots of DCs from the spleen of either WT or cKO Kif5b mice pregated on CD19 − , Gr − , F4/80 − CD11c + , and IA/IE + . The data are representative of three independent experiments. c Absolute numbers of CD11b + DCs and CD8α + DCs in the spleen of WT (blue circles) and cKO Kif5b (red squares) mice. The data are representative of five independent experiments. d – g The efficiency of cross-presentation of different concentration of sOVA and the presentation of OVA peptide SIINFEKL in vitro by CD8α + DCs ( d , g ), CD11b + DCs ( e , g ) and BMDCs ( f , g ) from WT (blue histogram or line) or cKO Kif5b (red histogram or line) mice was measured as IL-2 secretion by OT-I T cells after 16 h of co-culture. Graph shows mean ± S.E.M. ( n = 4). Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. * P < 0.05; ** P < 0.005; *** P < 0.0001.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Quantitative Proteomics, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro, Co-Culture Assay, Comparison

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a Tumour growth curves for WT (blue line) or cKO Kif5b (orange line) mice injected subcutaneously with B16-OVA cells and adoptively transferred with OT-I T cells (WT purple line, cKO Kif5b yellow line). Tumour growth was monitored daily, and non-survival was defined as ulceration of the tumour or a mean tumour diameter of 15 mm. The data are quoted as the mean ± SEM from two independent experiments (WT, WT OT1, cKO Kif5b n = 10 mice per group, cKO Kif5b OT1 n = 9 mice). Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. * P < 0.05; ** P < 0.005; *** P < 0.0001. b Survival curves for WT or cKO Kif5b mice, treated as in a . Statistical significance was determined by the log-rank test and by the Gehan–Breslow–Wilcoxon test. * P < 0.05; ** P < 0.005. c Mice were injected with Violet-labelled transgenic OT-I T cells and primed 1 day later with CD11c/P3UOVA (WT blue line or circles, cKO Kif5b red line or circles; WT mice n = 7, cKO Kif5b mice n = 7). The left panel shows representative profiles gated on CD8 + , TCR Vα2 + cells. Statistical analysis of the division index is shown in the right panel: ** P < 0.005 in a two-tailed unpaired Student’s t test. Graph shows mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Injection, Comparison, Transgenic Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a CD8α + , CD11b + DCs and BMDCs from WT (blue histogram) or cKO Kif5b (red histogram) mice were incubated for 10, 30 or 60 min at 37 °C or for 10 min at 4 °C with Alexa-Flour-647-OVA prior to flow cytometry analysis. The figure shows a histogram that was representative of three independent experiments. b CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice were incubated for 30 min at 37 °C with DQ-OVA. Next, the cells were washed. DQ-OVA degradation was analysed by flow cytometry after different chase periods. Graphs are representative of four independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. *** P < 0.0001. c Degradation of DQ-OVA in BMDCs from WT (DMSO blue line, Conc-A or Cath I purple line) or cKO Kif5b (DMSO orange line, Conc-A or Cath I yellow line) mice pretreated or not with concanamycin A (Conc-A) or a cathepsin inhibitor-I (Cath I). Graphs are representative of four independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. ** P < 0.005; *** P < 0.0001. d The pH values in CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice were determined by FACS after a pulse and different chase periods. Graphs are representative of four independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. * P < 0.05; ** P < 0.005; *** P < 0.0001. e Protease activity of CatB/L in early (20 min) or late (120 min) using total cell lysate from BMDCs from WT (blue histogram) or cKO Kif5b (red histogram) mice was measured with a specific fluorescent substrate. Graphs are representative of three independent experiments. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. *** P < 0.0001. a – e Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Incubation, Flow Cytometry, Comparison, Activity Assay

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: BMDCs from WT or cKO Kif5b mice were incubated for 15, 30 or 60 min with Alexa-Flour-647-OVA and then plated on glass coverslips. The cells were fixed, permeabilized and stained with anti-EEA1, anti-Lamp1 or anti-Rab11 antibodies. The left panel shown representative images for the 15 min time point observed in three independent experiments. Bars: 5 μm. n = 32 cells per condition. Statistical significance (shown in the right panel; WT blue circle, cKO Kif5b red square) was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. ** P < 0.005; *** P < 0.0001. Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Incubation, Staining, Comparison

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice were labelled for H-2K b and analysed using FACS. The figure shows a FACS histogram that was representative of three independent experiments. b MHC-I recycling ability in CD8α + , CD11b + DCs and BMDCs from WT (blue line) or cKO Kif5b (red line) mice was measured using FACS at the indicated time points. Statistical significance was determined by the two-way ANOVA and Sidak test’s correction for multiple comparison. ** P < 0.005; *** P < 0.0001. c TrfR (CD71) recycling ability was measured using FACS at the indicated time periods in CD8α + , CD11b + DCs and BMDCs from WT or cKO kif5b mice. Statistical analysis: * P < 0.05; ** P < 0.005; *** P < 0.0001 in the two-way ANOVA and Sidak test’s correction for multiple comparison. d MHC-I recycling in the presence of primaquine in BMDCs from WT or cKO Kif5b mice was measured using FACS at the indicated time points. Statistical analysis: ** P < 0.005; in the two-way ANOVA and Sidak test’s correction for multiple comparison. b – d Graphs are representative of four independent experiments. e BMDCs from WT or cKO kif5b mice were plated on glass coverslips and were fixed, permeabilized and stained with anti-EEA1, anti-Rab11 or anti-MHC-I antibodies. Representative images are shown in the left panel observed in three independent experiments. ( n = 33 cells per condition upper panel, n = 29 cells per condition lower panel) Bars: 5 μm. Statistical analysis (shown in the right panel; WT blue circle, cKO Kif5b red square): ** P < 0.005; *** P < 0.0001 in an two-tailed unpaired Student’s t test. b – e Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Comparison, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

doi: 10.1038/s41467-020-15692-0

Figure Lengend Snippet: a Confocal microscopy of BMDCs from WT or cKO Kif5b mice stained with anti-tubulin and anti-Kif5b. Bars: 5 μm. The indicated box is shown at a higher magnification in the inset. Bars: 2 μm. b Confocal microscopy of WT BMDCs stained with anti-EEA1 and anti-Kif5b. The intersection between the two stainings is shown in white. The indicated boxes are shown at higher magnification in the insets. Bars: 2 μm. c A schematic representation of early endosome WGA labelling. d WT and cKO Kif5b BMDCs were labelled with WGA-488 and stained with anti-EEA1. Bars: 2 μm (left panel). Quantification of the colocalization between WGA-488 and EEA1-555 (right panel; WT blue histogram, cKO Kif5b red histogram; n = 7 cells per condition). All images of single cells are representative of >100 cells observed in three independent experiments ( a – d ). e Spinning disk video microscopy was performed on WT and cKO Kif5b BMDCs labelled with WGA-488. Representative series of images are shown every 25 s. Bars: 2 μm. Images are representative of four independent experiments. See also Supplementary Movies and . f The number of tubulations unable to detach per cell during the 5 min acquisition. (WT blue circles, cKO Kif5b red squares; n = 20 cells per condition). Statistical analysis: *** P < 0.0001 in a two-tailed unpaired Student’s t test. g The number of WGA-488-positive vesicular structures >1 μm in size was measured in WT (blue line) and cKO Kif5b (red line) BMDCs during the 5 min acquisition ( n = 13 cells per condition). Statistical analysis: *** P < 0.0001 in a two-way ANOVA and Sidak test’s correction for multiple comparison. h Schematic representation of the WGA labelling protocol (upper panel). Spinning disk video microscopy was performed on WT and cKO Kif5b BMDCs labelled with WGA-555 and WGA-488. Representative series of images observed in three independent experiments are shown at the indicated time periods (lower panel). Bars: 2 μm. i n = 10 cells per condition. Statistical analysis of the experiment in (WT blue histogram, cKO Kif5b red histogram) ( h ); *** P < 0.0001 in a two-way ANOVA and Sidak test’s correction for multiple comparison. d – i Graphs show mean ± S.E.M.

Article Snippet: Anti-CD11c (BV711 (BioLegend), APC (BD Biosciences) PB (Sony)), anti-IA/IE FITC (BD Biosciences), anti-CD8α BV450 (Sony), anti-CD11b (PE (BD Biosciences), APC-CY7 (Sony)), anti-F4/80 PE/Cy7 (Sony), anti-GR1 APC (BD Biosciences), anti-CD19 (PerCP (BD Biosciences), PerCP/Cy5.5 (Sony)), anti-B220 BV650 (BioLegend), anti-CD86 BV650 (Sony), anti-CD40 PE/Cy7 (Sony), mouse monoclonal Kif5b antibody (Biolegend), purified rabbit anti-α-tubulin (Rockland), goat anti-EEA1 antibody (Santa Cruz Biotechnology), rat anti-Lamp1 antibody (Invitrogen), H-2Kb PE antibody (Biolegend), AF6-88.5 clone (Biolegend), rabbit Rab11 antibody (Life Technologies), anti-CD71 PE antibody (Sony), Alexa Fluor-594 donkey anti-rat IgG (H+L) (Life Technologies), Alexa Fluor-555 donkey anti-goat IgG (H+L) (Life Technologies), Alexa Fluor-488 donkey anti-mouse IgG (H+L) (Life Technologies), and Alexa Fluor-555 donkey anti-rabbit IgG (H+L) (Life Technologies) were used.

Techniques: Confocal Microscopy, Staining, Microscopy, Two Tailed Test, Comparison

Journal: Molecular Medicine

Article Title: Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

doi: 10.1186/s10020-024-00856-1

Figure Lengend Snippet: Inhibition of ACP5 suppresses CF proliferation, migration, and transition into myofibroblasts. ( A ) Relative mRNA expression levels after ACP5 silencing ( n = 3/group). ( B - C ) Western blot analysis of the silencing efficiency of ACP5 ( n = 3/group). ( D - E ) EdU assay was used to detect the cell proliferation rate of CFs ( n = 4/group); scale bar = 100 μm. ( F ) OD values of CFs in different groups ( n = 4/group). ( G - H ) Cell migration was assessed by a wound-healing assay ( n = 4/group); scale bar = 100 μm. ( I - J ) Cell migration was assessed by Transwell assays ( n = 4/group); scale bar = 100 μm. ( K - N ) Relative mRNA expression levels of ACP5, α-SMA, COL1, and COL3 in CFs in different groups ( n ≥ 3/group). ( O - Q ) Western blot analysis of α-SMA and COL1 ( n = 3/group). * P < 0.05, ** P < 0.01, *** P < 0.001, and ns: P > 0.05

Article Snippet: The Mouse ACP5 ELISA Kit was purchased from Signalway Antibody (USA, #EK12408), and the Human ACP5 ELISA Kit was purchased from BOSTER (China, #EK2138).

Techniques: Inhibition, Migration, Expressing, Western Blot, EdU Assay, Wound Healing Assay

Journal: Molecular Medicine

Article Title: Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

doi: 10.1186/s10020-024-00856-1

Figure Lengend Snippet: Overexpression of ACP5 promotes CF proliferation, migration, and transition into myofibroblasts. ( A ) Relative mRNA expression levels of ACP5 and the overexpression efficiency ( n = 4/group). ( B - C ) Western blot analysis of the overexpression efficiency of ACP5 ( n = 3/group). ( D - E ) The EdU assay was used to detect the cell proliferation rate of CFs ( n = 4/group); scale bar = 100 μm. ( F ) OD values of CFs in different groups ( n = 3/group). ( G - H ) Cell migration was assessed by a wound-healing assay ( n = 4/group); scale bar = 100 μm. ( I - J ) Cell migration was assessed by Transwell assays ( n = 4/group); scale bar = 100 μm. ( K - N ) Relative mRNA expression levels of ACP5, α-SMA, COL1, and COL3 in CFs in different groups ( n = 4/group). ( O - Q ) Western blot analysis of α-SMA and COL1 ( n = 3/group). * P < 0.05, ** P < 0.01, *** P < 0.001, and ns: P > 0.05

Article Snippet: The Mouse ACP5 ELISA Kit was purchased from Signalway Antibody (USA, #EK12408), and the Human ACP5 ELISA Kit was purchased from BOSTER (China, #EK2138).

Techniques: Over Expression, Migration, Expressing, Western Blot, EdU Assay, Wound Healing Assay

Journal: Molecular Medicine

Article Title: Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

doi: 10.1186/s10020-024-00856-1

Figure Lengend Snippet: ACP5 inhibition suppresses fibrosis in MI mice and enhances cardiac function. ( A ) Masson staining and Sirius red staining in each group ( n = 6/group; upper layer, scale bar = 1000 μm; middle layer, scale bar = 100 μm; lower layer, scale bar = 100 μm). ( B ) Masson staining of fibrosis. ( C ) Sirius red staining of the collagen area. ( D ) Representative images of echocardiography. ( E - F ) Echocardiographic measurements of LVEF (E) and LVFS (F) ( n = 6/group). ( G - I ) Western blot analysis of α-SMA and COL1 in the hearts of mice in different groups ( n = 3/group). ( J - K ) Immunofluorescence staining of α-SMA in the hearts of mice in different groups ( n = 4/group); scale bar = 50 μm. (The bottom-layer image in Fig. 4J is the locally enlarged image circled in the Merge graph, bar = 20 μm). * P < 0.05, ** P < 0.01, *** P < 0.001, and ns: P > 0.05

Article Snippet: The Mouse ACP5 ELISA Kit was purchased from Signalway Antibody (USA, #EK12408), and the Human ACP5 ELISA Kit was purchased from BOSTER (China, #EK2138).

Techniques: Inhibition, Staining, Western Blot, Immunofluorescence

Journal: Molecular Medicine

Article Title: Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

doi: 10.1186/s10020-024-00856-1

Figure Lengend Snippet: ACP5 affects the GSK3β/β-catenin signal transduction pathway. ( A - D ) Western blot analysis of the expression levels of ACP5, p-GSK3β, GSK3β, and β-catenin in ACP5-deficient CFs ( n = 3/group). ( E - H ) Western blot analysis of the expression levels of ACP5, p-GSK3β, GSK3β, and β-catenin in CFs overexpressing ACP5 ( n = 3/group). ( I - L ) Western blot analysis of the expression levels of ACP5, p-GSK3β, GSK3β, and β-catenin in the hearts of mice in different groups ( n ≥ 3/group). * P < 0.05, ** P < 0.01, *** P < 0.001, and ns: P > 0.05

Article Snippet: The Mouse ACP5 ELISA Kit was purchased from Signalway Antibody (USA, #EK12408), and the Human ACP5 ELISA Kit was purchased from BOSTER (China, #EK2138).

Techniques: Transduction, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

doi: 10.1186/s10020-024-00856-1

Figure Lengend Snippet: ACP5 affects CF activation by regulating ERK. ( A - B ) Western blot analysis of the expression levels of p-ERK and ERK in ACP5-deficient CFs ( n = 3/group). ( C - D ) Western blot analysis of the expression levels of p-ERK and ERK in CFs overexpressing ACP5 ( n = 3/group). ( E - F ) Western blot analysis of the expression levels of p-ERK and ERK in the hearts of mice in different groups ( n = 3/group). ( G - K ) Western blot analysis of the expression levels of ACP5, p-ERK, ERK, p-GSK3β, GSK3β, and β-catenin in CFs pretreated with Ro 67-7476 (an ERK agonist) ( n = 3/group). ( L - M ) The EdU assay was used to detect the proliferation rate of CFs pretreated with Ro 67-7476 (an ERK agonist) ( n = 4/group); scale bar = 100 μm. ( N - O ) Cell migration was assessed by Transwell assays ( n = 4/group); scale bar = 100 μm. ( P - R ) Western blot analysis of α-SMA and COL1 in the hearts of mice in different groups ( n = 3/group).* P < 0.05, ** P < 0.01, *** P < 0.001, and ns: P > 0.05

Article Snippet: The Mouse ACP5 ELISA Kit was purchased from Signalway Antibody (USA, #EK12408), and the Human ACP5 ELISA Kit was purchased from BOSTER (China, #EK2138).

Techniques: Activation Assay, Western Blot, Expressing, EdU Assay, Migration

Journal: Molecular Medicine

Article Title: Inhibition of tartrate-resistant acid phosphatase 5 can prevent cardiac fibrosis after myocardial infarction

doi: 10.1186/s10020-024-00856-1

Figure Lengend Snippet: Schematic diagram of the mechanism of ACP5 in myocardial fibrosis after MI. Under the stimulation of MI or Ang II, the increased expression of ACP5 activates the ERK/GSK3β/β-catenin signaling pathway, which promotes the transformation of CFs into myofibroblasts with more active proliferation, migration and fibrosis, leading to the onset of myocardial fibrosis. (Generated by Figdraw)

Article Snippet: The Mouse ACP5 ELISA Kit was purchased from Signalway Antibody (USA, #EK12408), and the Human ACP5 ELISA Kit was purchased from BOSTER (China, #EK2138).

Techniques: Expressing, Transformation Assay, Migration, Generated

Journal: Journal of Cellular Physiology

Article Title: Excessive osteoclast activation by osteoblast paracrine factor RANKL is a major cause of the abnormal long bone phenotype in Apert syndrome model mice

doi: 10.1002/jcp.30682

Figure Lengend Snippet: Abnormally enhanced osteoclast formation and activity in EIIA‐Fgfr2 S252W/+ mice are attributed to abnormal osteoblast differentiation. (a) Osteoclast identification by tartrate‐resistant acid phosphatase (TRAP) staining. Bone marrow‐derived macrophages (BMMs) isolated from wild‐type (WT) and EIIA‐Fgfr2 S252W/+ mice were differentiated into osteoclasts in the presence of CSF1 (20 ng/ml) and receptor activator of nuclear factor‐κB ligand (RANKL) (80 ng/ml) for 5 days (scale bar: 100 μm). (b) Number of TRAP‐positive osteoclasts with more than three nuclei ( n = 5 in each group). (c, d) Messenger RNA (mRNA) levels of Fgfr1 and Fgfr2 measured by quantitative real‐time PCR (qPCR) analysis of WT BMMs isolated from the spleen (c) and bone marrow (d). BMMs from the spleen were cultured in osteoclast differentiation medium for each indicated day and BMMs from bone marrow were cultured for 6 days. Relative mRNA expression was normalized to Gapdh expression. (e) ERK phosphorylation of WT and EIIA‐Fgfr2S252W/+ mice BMMs from bone marrow. BMMs were differentiated into osteoclasts in osteoclast differentiation medium for 5 days. (f, g) TRAP staining of BMMs from WT and EIIA‐Fgfr2 S252W/+ mice cocultured with primary calvaria osteoblasts (OBs) from WT and EIIA‐Fgfr2 S252W/+ mice, respectively, for 7 days with vitamin D3 (10 nM) and prostaglandin E2 (PGE2) (1 µM) in osteogenic medium for osteoclast differentiation (scale bar: 100 μm). (h) Number of TRAP‐positive osteoclasts. (i–l) Primary calvarial OBs of WT and EIIA‐Fgfr2 S252W/+ were cocultured with WT BMMs and mRNA levels of osteoclast marker genes were determined by reverse‐transcription (RT) quantitative PCR (qPCR). Data are expressed as the mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001. NS, not significant

Article Snippet: To measure TRAP activity, tibial tissue sections and differentiated osteoclasts were stained using a TRAP‐staining kit (PMC‐AK04F, Cosmo Bio) in accordance with the manufacturer's protocol.

Techniques: Activity Assay, Staining, Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Marker

Journal: Journal of Cellular Physiology

Article Title: Excessive osteoclast activation by osteoblast paracrine factor RANKL is a major cause of the abnormal long bone phenotype in Apert syndrome model mice

doi: 10.1002/jcp.30682

Figure Lengend Snippet: Increased osteoblast‐mediated osteoclast activation and reduced bone formation in tibia trabecular bones of Col1a1‐Fgfr2 S252W/+ mice. (a) Representative micro‐computed tomography (CT) images of the tibial growth plate at postnatal Day 21 (P21) in midsagittal and coronal views are shown for each genotype. ( n ≥ 8, scale bar: 1 mm). (b–e) Histomorphometric analyses of three‐dimensional (3D) micro‐CT data. (f) Tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts as determined by TRAP staining of trabecular bone and secondary ossification center (SOC) of proximal tibiae in P21 wild‐type (WT) and Col1a1‐Fgfr2 S252W/+ mice ( n = 3, scale bars: top, 200 μm; bottom, 100 μm). (g) Graph of the number of osteoclasts per bone surface. Data are expressed as the mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001. (h) Mechanisms of reduced long bone growth in Fgfr2 S252W/+ mice

Article Snippet: To measure TRAP activity, tibial tissue sections and differentiated osteoclasts were stained using a TRAP‐staining kit (PMC‐AK04F, Cosmo Bio) in accordance with the manufacturer's protocol.

Techniques: Activation Assay, Micro-CT, Staining

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: iSN40 inhibits the RANKL-induced osteoclastogenesis of RAW264.7 cells. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.1–3.0 μM iSN40 for 7 days. Scale bar, 100 μm. ( B ) The number of nuclei in the TRAP + osteoclasts was quantified. No TRAP + cells were observed in any of the RANKL(−) groups. ** p < 0.01 vs. RANKL(+)/control. n = 4 fields. ( C ) Representative images of the pits generated in the bone matrix resorption assay of RAW264.7 cells treated with 100 ng/mL RANKL and 0.3 μM iSN40 for 6 days. Scale bar, 100 μm. ( D ) The pit area was quantified. ** p < 0.01 vs. RANKL(−)/control; †† p < 0.01 vs. RANKL(+)/control. n = 3 fields.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control, Generated

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: iSN40 inhibits osteoclastogenesis in a TLR9-dependent manner. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODN, and 1 μM E6446 for 6 days. Scale bar, 100 μm. ( B ) The number of nuclei in TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40/E6446(−) and CpG-2006/E6446(−) groups. NS, no significant difference, ** p < 0.01 vs. each E6446(−) group. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODNs, and 1 μM E6446 for 24 h. ** p < 0.01 vs. control/E6446(−), †† p < 0.01 vs. iSN40/E6446(−), ‡‡ p < 0.01 vs. CpG-2006/E6446(−). n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: The CpG motif is essential for the anti-osteoclastogenic effect of iSN40. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.3 μM ODN for 7 days. Scale bar, 100 μm. ( B ) The number of nuclei in the TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40 group. NS, no significant difference vs. control. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL, 0.3 μM ODNs for 24 h ( Il1b ) or 5 days ( Nfatc1 , Ctsk , and Dcstamp ). * p < 0.05, ** p < 0.01 vs. RANKL(−)/control; † p < 0.05, †† p < 0.01 vs. RANKL(+)/control; ‡ p < 0.05, ‡‡ p < 0.01 vs. RANKL(+)/iSN40. n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: Anti-osteoclastogenic effects of the iSN40 variants. ( A ) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.3 μM ODN for 7 days. Scale bar, 100 μm. ( B ) The number of nuclei in the TRAP + osteoclasts was quantified. No TRAP + cells were observed in the iSN40 group. * p < 0.05, ** p < 0.01 vs. control. n = 4 fields. ( C ) qPCR results of RAW264.7 cells treated with 30 ng/mL RANKL and 0.3 μM ODN for 24 h. * p < 0.05, ** p < 0.01 vs. control. n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: Anti-osteoclastogenic effect of iSN40 in the coculture of RAW264.7 and MC3T3-E1 cells. ( A ) Representative images of the TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL for 4 days. RW, undifferentiated RAW264.7 cells (dense nuclei in black); OC, differentiated TRAP + multinucleated osteoclasts; OB, fibroblast-like MC3T3-E1 osteoblasts. Scale bar, 25 μm. ( B ) qPCR results of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL, 50 μg/mL ascorbic acid (AA), and 0.3–10 μM iSN40 for 4 days. ** p < 0.01 vs. RANKL(+)/AA(+)/iSN40(−). n = 3. ( C ) Representative images of the TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL and 0.3 μM ODN for 4 days (top panels). Representative images of the pits generated in the bone matrix resorption assay of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL and 0.3 μM ODN for 6 days (bottom panels). Scale bars, 100 μm. ( D ) The number of nuclei in TRAP + osteoclasts in panel C was quantified. ** p < 0.01 vs. RANKL(−)/control, †† p < 0.01 vs. RANKL(+)/control. n = 3 fields. ( E ) Scale bar, 100 μm. ( E ) The pit area in panel C was quantified. ** p < 0.01 vs. RANKL(−)/control, †† p < 0.01 vs. RANKL(+)/control. n = 3 fields. ( F ) qPCR results of the ratio of RANKL ( Tnfsf11 ) to OPG ( Tnfrsf11b ) of the same samples in panel B. * p < 0.05 vs. RANKL(−)/AA(−)/iSN40(−), †† p < 0.01 vs. RANKL(+)/AA(−)/iSN40(−). n = 3.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Generated, Control

Journal: Life

Article Title: Osteogenic CpG Oligodeoxynucleotide, iSN40, Inhibits Osteoclastogenesis in a TLR9-Dependent Manner

doi: 10.3390/life14121572

Figure Lengend Snippet: Time course of anti-osteoclastogenic effect of iSN40. ( A ) Representative images of TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL for 4 days and 0.3 μM iSN40 for defined periods. d, differentiation days with iSN40 treatment. Scale bar, 100 μm. ( B ) The number of nuclei in TRAP + osteoclasts was quantified. ** p < 0.01 vs. control/RANKL(−); †† p < 0.01 vs. control/RANKL(+); ‡‡ p < 0.01 vs. d0–3, d0–4, d1–3, and d1–4; §§ p < 0.01 vs. d2–3 and d2–4. n = 3 fields.

Article Snippet: The TRAP enzymatic activity of the differentiated osteoclasts was visualized using a TRAP Staining Kit (Fujifilm Wako Chemicals) according to the manufacturer’s instructions.

Techniques: Staining, Control