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: Journal of Nanobiotechnology

Article Title: Liver-specific delivery of spherical DNA frameworks for alleviation of hepatic ischemic reperfusion injury

doi: 10.1186/s12951-024-02661-8

Figure Lengend Snippet: Detection of cytokines in liver tissues. A-H ) Cytokines of IFN-γ ( A ), TNF-α ( B ), IL-1 ( C ), IL-6 ( D ), IL-12 ( E ), and NOS2 ( F ) from activated monocyte/macrophages and Kupffer cells and MPO ( G ) from activated neutrophil were measured in liver homogenates and SOD( H ) form each group. Data represent means ± s.d. from five independent replicates, and P values were calculated by one-way ANOVA with Tukey’s honest significant difference post-hoc test (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, non-significant)

Article Snippet: The following measurements of secreted cytokines by ELISA were conducted according to the manufacturer’s instructions (Mouse IL-1 ELISA development Kit, PromoKine; Mouse TNF-α ELISA Kit, Cayman; Mouse IL-12, IFN-γ and IL-6 ELISA Kit, Bioleagend; Mouse Myeloperoxidase ELISA Kit, R&D system; Mouse NOS2 ELISA Kit, G-Bioscience).

Techniques: