Journal: Cell stem cell

Article Title: Distinct Bone Marrow Sources of Pleiotrophin Control Hematopoietic Stem Cell Maintenance and Regeneration

doi: 10.1016/j.stem.2018.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-Leptin receptor Polyclonal Antibody, FITC , Bioss Antibodies , Cat. bs-0961R-FITC; RRID:AB_11074292.

Techniques: Staining, Recombinant, Expressing, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Imaging

Journal: PLoS ONE

Article Title: Dietary protein restriction increases hepatic leptin receptor mRNA and plasma soluble leptin receptor in male rodents

doi: 10.1371/journal.pone.0219603

Figure Lengend Snippet: Primers for realtime PCR.

Article Snippet: Anti-leptin receptor polyclonal antibody (Novus Biologicals, NB120-5593, 1:2000 dilution) and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, sc-69879, 1:500 dilution) were used as 1 st antibodies and goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004, 1:50000 dilution) and goat anti-mouse IgG-HRP (Santa Cruz, sc-2005, 1:50000 dilution) were used as 2 nd antibodies, respectively.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Dietary protein restriction increases hepatic leptin receptor mRNA and plasma soluble leptin receptor in male rodents

doi: 10.1371/journal.pone.0219603

Figure Lengend Snippet: (A) Rats were fed a control diet (20C) or a low-protein diet (5C) for 7 d and sacrificed after 12-h fasting (20CF, 5CF) or 12-h fasting followed by 12-h re-feeding (20CR, 5CR). Leptin receptor mRNA was measured by real-time PCR and results were expressed relative to that of 20CF, means ± standard errors (n = 5). Results of two-way ANOVA are given below the graph (NS, not significant; **, p < 0.01). (B) Rats were fed a control diet ad libitum (20C), fed a low-protein diet ad libitum (5C), or pair-fed a control diet with 5C (20R) for 16 d. Ob-R mRNA was measured by real-time PCR and expressed as means ± standard errors (n = 5). Results with different alphabet are statistically different ( P < 0.05).

Article Snippet: Anti-leptin receptor polyclonal antibody (Novus Biologicals, NB120-5593, 1:2000 dilution) and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, sc-69879, 1:500 dilution) were used as 1 st antibodies and goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004, 1:50000 dilution) and goat anti-mouse IgG-HRP (Santa Cruz, sc-2005, 1:50000 dilution) were used as 2 nd antibodies, respectively.

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Dietary protein restriction increases hepatic leptin receptor mRNA and plasma soluble leptin receptor in male rodents

doi: 10.1371/journal.pone.0219603

Figure Lengend Snippet: C57BL/6 mice were fed 20C or 5C ad libitum for 7 d. Plasma sOb-R (A) and plasma leptin (B) concentrations were measured with ELISA. Free leptin index was calculated from plasma leptin and sOb-R (C). Results are expressed as means ± standard errors (n = 5). Statistical difference between the groups are shown as **, P < 0.01.

Article Snippet: Anti-leptin receptor polyclonal antibody (Novus Biologicals, NB120-5593, 1:2000 dilution) and anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, sc-69879, 1:500 dilution) were used as 1 st antibodies and goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004, 1:50000 dilution) and goat anti-mouse IgG-HRP (Santa Cruz, sc-2005, 1:50000 dilution) were used as 2 nd antibodies, respectively.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: Representative confocal images of VE-cadherin + (magenta) and CD31 + (green) BM vessels, and nuclei (blue) in femurs from a non-IRR mice and at b day +3 and day +7 following 500 cGy and treatment with IgG (10 µg/dose) or anti-NRP1 (10 µg/dose). Left image shows full view taken using 20× lens and right images display individual channels from magnified yellow box. Scale bar 50 µm; magnified view scale bar, 20 µm. c Quantification of VE-Cadherin vascular area from the images in a , b . Dotted line represents mean Non-IRR vascular area. Data presented as mean values +/− SEM. ( n = 2 independent experiments, d3 IgG n = 4 fields of view, d3 anti-NRP1 n = 5 fields, d7 IgG n = 7 fields, d7 anti-NRP1 n = 7 fields; IgG d3: p = 0.016, anti-NRP1 d3: p = 0.0467, IgG d7: p = 0.0349, anti-NRP1 d7: p = 0.9694. d BM cell counts at day +7 following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5 mice/group, p = 0.0025). Dotted line shows cell counts of non-IRR BM. e At left, representative flow cytometric analysis of CD45 − VE-cad + BM ECs within BM lin − cells from the groups shown. At right, percentages of BM ECs at day +7 following 500 cGy. Dotted line represents %VE-cad + ECs in non-IRR controls ( n = 5 mice/group). f At left, representative histograms of % activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs at day +7 following 500 cGy. At right, mean % activated caspase 3/7 + BM ECs at day +7 following 500 cGy ( n = 5/group). Dotted line represents % activated caspase 3/7 + ECs in non-IRR controls. g Levels of Evans Blue Dye (EBD) in the BM extracellular space at +24 h following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5–6 mice/group, non-IRR vs. IgG: p = 0.0024, IgG vs. anti-NRP1: p = 0.188, non-IRR vs. anti-NRP1 p = 0.2679). d – f Data assessed by Student’s two-tailed t -test, g One-way ANOVA with Holm-Sidak’s multiple comparison two-sided t -test, c Two-sided one-sample t -test and Wilcoxon test used to compare sample means to the mean of non-IRR samples, * p < 0.05, ** p < 0.01, **** p < 0.0001. Source data provided as a Source Data file.

Article Snippet: For Leptin receptor+(LepR + ) stromal cells, CD45 − LepR + cells (1:200, rabbit anti-PE leptin receptor antibody, bs-0961R-PE, BiosS, Woburn, MA) were isolated.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a PB WBC, neutrophil (NEU), and lymphocyte (LYMPH) counts at days +3, +7, and +10 following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5–10 mice/group, mean values +/− SEM; WBC: d7: p = 0.0016, d10 p = 0.002; NEU: d7: p = 0.0089, d10: p = 0.0035, LYMPH: d10: p = 0.0482). b BM cell counts at day +10 following 500 cGy and treatment with IgG or anti-NRP1 ( n = 5–10 mice/group, p = 0.0033). Dotted line represents BM cell counts in non-IRR controls. c At left, flow cytometry of BM c-kit + sca-1 − lin − progenitors and KSL cells at day +10 following 500 cGy; at right, numbers of BM c-kit + sca-1 − lin − cells and KSL at day +10 ( n = 5–10 mice/group, p < 0.0001). d At left, PB donor CD45.2 + cell engraftment at 20 weeks in CD45.1 + mice transplanted with BM cells collected at day +10 from 500 cGy—irradiated CD45.2 + mice treated with IgG or anti-NRP1, along with CD45.1 + BM competitor cells. At right, % total CD45.2 + cells, CD45.2 + Mac1 + Gr1 + myeloid cells, CD45.2 + B220 + B cells, and CD45.2 + CD3 + T cells at 20 weeks ( n = 12–13 mice/group, total: p = 0.0288, myeloid: p = 0.0266, B cells: p = 0.0051, T cells: p = 0.0001). e At left, histograms of donor CD45.2 + KSL cells in the BM of transplanted CD45.1 + mice at 20 weeks ( n = 5 mice/group, p = 0.0079). f Percent survival of C57BL/6J mice following 800 cGy TBI and treatment with anti-NRP1 or IgG for 10 days (Log-rank test, p = 0.001, n = 12 mice/group). g PB WBCs and hemoglobin (Hb) at day +7, +10 and +14 from C57BL/6J mice following 800 cGy and treatment with anti-NRP1 or IgG ( n = 8–10 mice/condition; WBC d7: p = 0.4045, WBC d10: p = 0.0415, WBC d14: p = 0.8714; Hb d7: p = 0.0154, Hb d10: p = 0.2741, Hb d14: p = 0.6216). h At left, flow cytometry of BM KSL cells 10 days following 800 cGy and treatment with anti-NRP1 or IgG; at right, %KSL cells ( n = 10 mice/condition; p = 0.0358). a Two-way ANOVA with Holm Sidak’s multiple comparison two-sided t -test. b , c Two-sided t -test, d , e Two-sided Mann–Whitney test, f Two-sided log-rank test, g Two-way ANOVA with Holm-Sidak’s multiple comparison two-sided t -tests, and h Student’s two-sided unpaired t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: For Leptin receptor+(LepR + ) stromal cells, CD45 − LepR + cells (1:200, rabbit anti-PE leptin receptor antibody, bs-0961R-PE, BiosS, Woburn, MA) were isolated.

Techniques: Flow Cytometry, Irradiation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a qRT-PCR analysis of Sema3a and Nrp1 expression in BM ECs from mice at +24 h following 5-FU chemotherapy. For each gene, expression is normalized to vehicle treatment ( n = 5/condition; Sema3a : p < 0.0001, Nrp1 : p = 0.1291). b Representative images of VE-cadherin + (magenta) , CD31 + (green) BM vessels in femur sections from untreated control mice and at day +7 following 5-FU chemotherapy and treatment with anti-NRP1 or IgG. Nuclei are stained with DAPI (blue). Scale bar, 50 µm; magnified view scale bar, 20 µm. c BM cell counts at day +7 following 5-FU chemotherapy and treatment with anti-NRP1 or IgG ( n = 5 mice/group, p = 0.0219). d At left, representative flow cytometric analysis of CD45 − VE-cad + BM ECs within BM lin − cells from control mice and at day +7 following 5-FU chemotherapy and anti-NRP1 or IgG treatment. At right, percentages and numbers of BM ECs at day +7 following 5-FU chemotherapy and the treatments shown. Dotted lines represent numbers in control mice ( n = 5 mice/group; % BM ECs p < .0001; number BM ECs p = 0.0008). e At left, representative histograms show % activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs at day +7 following 5-FU. At right, % activated caspase 3/7 + BM ECs at day +7 ( n = 5/group; p < .0001). Dotted line represents percentages of activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs in non-IRR controls. f PB WBC, NEU, and LYMPHs at day +7 following 5-FU and the treatments shown ( n = 5 mice/group; p < 0.0001). g At left, flow cytometry of BM KSL cells at day +7 following 5-FU and the treatments shown. At right, % BM KSL cells ( n = 5 mice/group; p = 0.011). h Colony forming unit quantification from 30,000 BM cells isolated from mice at day +7 following 5-FU and IgG or anti-NRP1 treatment ( n = 4 replicates, mean values +/− SEM; GM: p < 0.0001; GEMM: p = 0.0201). Statistics show GM and GEMM comparisons. a , f , h Two-way ANOVA followed by Holm-Sidak’s multiple comparison two-sided t -tests; c , d , g Student’s unpaired two-sided t -test. * p < 0.05, *** p < 0.001, **** p < 0.0001. Source data provided as a Source Data file.

Article Snippet: For Leptin receptor+(LepR + ) stromal cells, CD45 − LepR + cells (1:200, rabbit anti-PE leptin receptor antibody, bs-0961R-PE, BiosS, Woburn, MA) were isolated.

Techniques: Quantitative RT-PCR, Expressing, Staining, Flow Cytometry, Isolation

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a qRT-PCR analysis for Nrp1 expression in VE-cadherin + BM ECs from Nrp1 fl/fl mice and Cdh5-Cre-ERT2;Nrp1 fl/fl mice following tamoxifen treatment ( n = 4–5 mice/group, p < 0.0001). Nrp1 expression was normalized to Gapdh levels and then replicates were normalized relative to the Nrp1 fl/fl group. b Representative microscopic images of VE-cad + BM vessels (red) in femur sections from Nrp1 fl/fl mice and Cdh5-Cre-ERT2;Nrp1 fl/fl mice at day +7 following 500 cGy TBI. Scale bar, 100 µm. c BM cell counts in Nrp1 fl/fl mice and Cdh5-Cre-ERT2;Nrp1 fl/fl mice at day +10 following 500 cGy ( n = 10 mice/group; p = 0.001). d Representative flow cytometric analysis of CD45 − CD31 + BM ECs within BM lin − cells from Nrp1 fl/fl mice and Cdh5-Cre-ERT2;Nrp1 fl/fl mice are shown at day +10. At right, representative percentages of Annexin + 7AAD − and Annexin + 7AAD + cells within the BM EC population are shown. e At left, mean percentages of CD31 + BM ECs within the BM lin − population at day +10 and at right, numbers of femoral CD31 + BM ECs at day +10 following 500 cGy TBI ( n = 5 mice/group; %BM ECs: p < 0.0001; number BM ECs: p = 0.0056). f Mean percentages of live, apoptotic, and necrotic BM ECs at day +10 following 500 cGy in the groups shown ( n = 5 mice/group; p < 0.0001). g PB WBC, NEU, and LYMPHS at day + 10 following 500 cGy TBI in Nrp1 fl/fl mice and Cdh5-Cre-ERT2;Nrp1 fl/fl mice ( n = 10 mice/group; WBC: p < 0.0001; NEU: p = 0.0014; LYMPH: p = 0.0327). h At left, representative flow cytometric analysis of BM c-kit + sca1 − lin − progenitors and KSL stem/progenitor cells at day +10 following 500 cGy TBI. At right, number of c-kit + sca1 − lin − progenitors and KSL cells at day +10 in the groups shown ( n = 10 mice/group, p < 0.0001). i CFC quantification from 10,000 BM cells isolated from mice at day +10 following IR ( n = 10 replicates, data are presented as mean values +/− SEM, p < 0.0001). a , c , e , h Student’s unpaired two-sided t -test, f , g , i Two-way ANOVA with Holm-Sidak’s multiple comparison two-sided t -tests. * p < 0.05, ** p < 0.01, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: For Leptin receptor+(LepR + ) stromal cells, CD45 − LepR + cells (1:200, rabbit anti-PE leptin receptor antibody, bs-0961R-PE, BiosS, Woburn, MA) were isolated.

Techniques: Quantitative RT-PCR, Expressing, Isolation

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a (Left) flow cytometry of activated caspase 3/7 + BM ECs in wild-type (WT) and Nrp1 VEGF− mice at day +5 following 500 cGy and the treatments shown; (Right) % activated caspase 3/7 + BM ECs ( n = 5/group; **** p < 0.0001; *** p = 0.0003). b (Left) Donor CD45.2 + cell engraftment at 20 weeks in the PB of CD45.1 + mice competitively transplanted with BM cells from 500 cGy—irradiated Nrp1 VEGF− mice treated with anti-NRP1 or IgG; (Right) % donor CD45.2 + , Mac1 + /Gr1 + , B220 + , and CD3 + cells at 20 weeks ( n = 5–6/group; total: p = 0.0346; myeloid: p = 0.0325; T cells: p = 0.0195). c (Left) Histograms of p-Cdk5 + BM ECs at 1 h following 800 cGy and the treatments shown; (Right) %p-p53 + BM ECs. Horizontal lines show gating for p-Cdk5 + cells ( n = 5 replicates, Non-IRR; n = 6 replicates for IRR, IRR + SEMA3A, IRR + SEMA3A + anti-NRP1; non-IRR vs. IRR: p < 0.0001; IRR vs. IRR + SEMA3A: p = 0.0005; IRR vs. IRR + SEMA3A + Anti-NRP1 p < 0.0001). d % activated caspase 3/7 + BM ECs at 24 h following 800 cGy and treatment with SEMA3A, SEMA3A + anti-NRP1 or SEMA3A + roscovitine (ROSC), 10 ng/ml ( n = 4–8 replicates; non-IRR vs. IRR: p < 0.0001; IRR vs. IRR + SEMA3A: p = 0.0007; IRR vs. IRR + SEMA3A + Anti-NRP1 p < 0.0001; IRR vs. IRR + SEMA3A + Anti-NRP1 + ROSC p < 0.0001). e At left, histograms of p-p53 + BM ECs at 1 h following 800 cGy and treatment with SEMA3A, SEMA3A + anti-NRP1, or SEMA3A + ROSC. At right, % p-p53 + BM ECs are shown. Black horizontal lines show gating for p-p53 + cells ( n = 6 replicates, Non-IRR; n = 9 replicates for IRR and IRR + SEMA3A; n = 5 replicates for IRR + SEMA3A + anti-NRP1; n = 4 replicates for IRR + SEMA3A + ROSC; p < 0.0001 for all comparisons). f At left, flow cytometry for activated caspase 3/7 + BM ECs from p53 −/− and p53 + /+ mice at 24 h following irradiation with 800 cGy and the treatments shown. At right, % activated caspase 3/7 + BM ECs are shown ( n = 5 replicates/group, p < 0.0001). g Puma expression in BM ECs at 6 h following 800 cGy and the treatments shown ( n = 4/condition; **** p < 0.0001, * p = 0.0142, ** p = 0.0025, *** p = 0.0009). a , c , d , e One-way ANOVA followed by Holm-Sidak’s two-sided unpaired t -tests, f Two-way ANOVA followed by Holm-Sidak’s two-sided unpaired t -test, g Brown–Forsythe ANOVA followed by two-sided unpaired t -tests with Welch’s correction, b Two-sided Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source Data file provided.

Article Snippet: For Leptin receptor+(LepR + ) stromal cells, CD45 − LepR + cells (1:200, rabbit anti-PE leptin receptor antibody, bs-0961R-PE, BiosS, Woburn, MA) were isolated.

Techniques: Flow Cytometry, Irradiation, Expressing, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells

doi: 10.1038/s41598-023-30446-w

Figure Lengend Snippet: LepR/Tom + PDL cells scarcely differentiate into hard tissue-forming cells in juvenile stages. ( A–E ) Histological analysis of maxillary first molar of 4-week-old LepR-cre ; R26-tdTomato mice. ( A ) Representative confocal images (Z stack) in thick sections; n = 9; scale bar = 500 μm. Numbered panels represent the magnified views of the boxed areas; scale bar = 50 μm. Asterisks: LepR/Tom + cells in gingival tissue, white arrows: LepR/Tom + cells in PDL, yellow arrows: LepR/Tom + cells in bone marrow space or dental pulp, white arrowheads: LepR/Tom − cementocytes, yellow arrowheads: LepR/Tom + osteocytes. Nuclei were visualized using Hoechst. ( B–D ) Quantification of LepR/Tom + cell localization in PDL. Representative image of boxed areas (0.05 mm 2 ) used for quantification of LepR/Tom + cells in PDL. 1: Root furcation, 2: Distal surface, 3: Root apex, 4: Mesial surface ( B ). Nuclei were visualized using Hoechst. Absolute number ( C ) and frequency ( D ) of LepR/Tom + cells at indicated area in ( B ). ( C , One-way ANOVA followed by Tukey’s test) and ( D , One-way ANOVA followed by Kruskal–Wallis test); n = 9. *p < 0.05, **p < 0.01. Data are represented as mean ± SD. ( E ) Representative confocal images (Z stack) in thick sections stained with anti-EMCN and -CD31 antibodies; n = 3; scale bar = 500 μm. Numbered panels represent the magnified views of the boxed area; scale bar = 50 μm. Nuclei were visualized using Hoechst. ( F–H) FACS analysis of LepR/Tom + cells in PDL from 4-week-old LepR-cre ; R26-tdTomato mice. ( F ) Representative FACS plots showing the frequency of CD45 − Ter119 − cells (left panel) and LepR/Tom + cells in the stromal PDL population (right panel); n = 6. Quantification of the frequency and absolute number of the CD45 − Ter119 − stromal population in PDL ( G , Two-tailed Student’s t -test) and the LepR/Tom + cells in the CD45 − Ter119 − stromal PDL population ( H , frequency: Two-tailed Student’s t -test; absolute number: two-tailed Mann–Whitney U -test); n = 6. NS: not significant. Data are represented as mean ± standard deviation (SD). P: pulp, D: dentin, AB: alveolar bone, PDL: periodontal ligament, CM: cementum, BM: bone marrow, DIC: differential interference contrast, EMCN: endomucin.

Article Snippet: The following primary antibodies were used: rat anti-endomuchin antibody (V. 7C7) (Santa Cruz Biotechnology, CA, USA) (1:100), goat anti-mouse leptin receptor (R&D SYSTEMS, Minneapolis, MN, USA) (1:100), goat anti-mouse-CD31antibody coupled to Alexa Fluor (AF) 488 (BD Biosciences, San Jose, CA, USA) (1:50), rat anti-mouse osteocalcin antibody (R21C-01A) (1:200) (Takara, Shiga, Japan), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F11) (1:500) and rat anti-Ter119 (TER-119) antibody coupled to APC (1:500) (both from Thermo Fisher Scientific, Waltham, MA, USA), rat anti-Ly-6A/E (Sca-1) antibody coupled to AF 488 (D7) (1:500), hamster anti-CD29 antibody coupled to AF 488 (HMβ1-1) (1:500), rat anti-CD90.2 antibody coupled to AF 488 (30-H12) (1:500), rat anti-CD146 antibody coupled to AF 488 (ME-9F1), isotype control rat IgG2a, κ coupled to AF 488 (RTK2758) (1:500) and isotype control hamster IgG coupled to AF 488 (HTK888) (1:500) (all from BioLegend, San Diego, CA, USA), rabbit anti-adiponectin antibody (1:10) (Novus Biologicals, Centennial, CO, USA), rabbit anti-osterix antibody (1:100) (Abcam, Cambridge, UK) and normal rabbit IgG (60024B) (R&D SYSTEMS).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, Standard Deviation

Journal: Scientific Reports

Article Title: Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells

doi: 10.1038/s41598-023-30446-w

Figure Lengend Snippet: LepR/Tom + PDL cell subpopulation expands and contributes to the AB and cementum during aging. (A–I) Time-course analysis of LepR/Tom + cells around dental tissue. ( A–C ) Representative confocal images (Z stack) in thick maxillary first molar sections of 4-week- ( A , n = 6), 4-month- ( B , n = 5), and 1-year-old ( C , n = 4) LepR-cre ; R26-tdTomato mice; scale bar = 500 μm. Numbered panels represent the magnified views of the boxed areas; scale bar = 50 μm. White arrows: LepR/Tom + osteocytes, White arrowheads: LepR/Tom + cementcytes, yellow arrows: LepR/Tom + PDL cells. P: pulp, D: dentin, AB: alveolar bone, PDL: periodontal ligament, CM: cementum, BM: bone marrow, DIC: differential interference contrast. Nuclei were visualized using Hoechst. ( D–I ) Quantification of LepR/Tom + cells around dental tissue. ( D ) Quantification areas in dental tissue. 1: Root apex (0.05 mm 2 ), 2: Root furcation (0.05 mm 2 ), 3: Mesial root surface (0.05 mm 2 ), 4: Alveolar bone (0.25 mm 2 ), 5: The dotted line, the cementum between the mesial and distal cemento-enamel junction. ( E–I ) Frequency and absolute number of LepR/Tom + cells in the areas as shown in D at the indicated time point. ( E ) Root apex (left panel: Welch’s one-way ANOVA; right panel: One-way ANOVA followed by Tukey’s test). ( F ) Root furcation (left panel: One-way ANOVA followed by Tukey’s test; right panel: One-way ANOVA followed by Kruskal–Wallis test). ( G ) Mesial root surface (One-way ANOVA followed by Tukey’s test). ( H ) Alveolar bone (left panel: One-way ANOVA followed by Tukey’s test; right panel: One-way ANOVA followed by Kruskal–Wallis test). ( I ) Cementum (Two-tailed Welch’s t -test between 4-week- and 4-month-old mice); n = 4–6. * p < 0.05, ** p < 0.01. NS: not significant. Data are represented as mean ± standard deviation (SD).

Article Snippet: The following primary antibodies were used: rat anti-endomuchin antibody (V. 7C7) (Santa Cruz Biotechnology, CA, USA) (1:100), goat anti-mouse leptin receptor (R&D SYSTEMS, Minneapolis, MN, USA) (1:100), goat anti-mouse-CD31antibody coupled to Alexa Fluor (AF) 488 (BD Biosciences, San Jose, CA, USA) (1:50), rat anti-mouse osteocalcin antibody (R21C-01A) (1:200) (Takara, Shiga, Japan), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F11) (1:500) and rat anti-Ter119 (TER-119) antibody coupled to APC (1:500) (both from Thermo Fisher Scientific, Waltham, MA, USA), rat anti-Ly-6A/E (Sca-1) antibody coupled to AF 488 (D7) (1:500), hamster anti-CD29 antibody coupled to AF 488 (HMβ1-1) (1:500), rat anti-CD90.2 antibody coupled to AF 488 (30-H12) (1:500), rat anti-CD146 antibody coupled to AF 488 (ME-9F1), isotype control rat IgG2a, κ coupled to AF 488 (RTK2758) (1:500) and isotype control hamster IgG coupled to AF 488 (HTK888) (1:500) (all from BioLegend, San Diego, CA, USA), rabbit anti-adiponectin antibody (1:10) (Novus Biologicals, Centennial, CO, USA), rabbit anti-osterix antibody (1:100) (Abcam, Cambridge, UK) and normal rabbit IgG (60024B) (R&D SYSTEMS).

Techniques: Two Tailed Test, Standard Deviation

Journal: Scientific Reports

Article Title: Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells

doi: 10.1038/s41598-023-30446-w

Figure Lengend Snippet: Self-renew stem cell activity is exerted in both LepR/Tom + and LepR/Tom − PDL cell populations. (A–E) Sphere formation assay of bone marrow (BM)- and periodontal ligament (PDL)-derived LepR/Tom + or LepR/Tom − stromal populations. ( A ) Representative FACS plots showing sorted population of BM (left panel, 1: LepR/Tom − , 2: LepR/Tom + ) and PDL cells (right panel, 3: LepR/Tom − , 4: LepR/Tom + ) from LepR-cre ; R26-tdTomato mice. ( B,C ) Representative images of spheres derived from BM ( B ) and PDL ( C ). Arrows: LepR/Tom-mixed spheres, arrowheads: LepR/Tom − spheres; scale bar = 1000 μm. Right panels are the magnified views of the boxed areas; scale bar = 50 μm. The panel numbers correspond to the gated number in ( A ). ( D ) Frequency of spheroid types; n = 3. Two-tailed Student’s t -test between LepR/Tom − and LepR/Tom-mixed spheres derived from sorted LepR/Tom − PDL cells. ** p < 0.01. ( E ) Frequency of sphere forming cells in the sorted cells; n = 3. One-way ANOVA followed by Tukey’s test among the sphere-detected groups. * p < 0.05. Data are represented as mean ± standard deviation (SD). BM: bone marrow, PDL: periodontal ligament.

Article Snippet: The following primary antibodies were used: rat anti-endomuchin antibody (V. 7C7) (Santa Cruz Biotechnology, CA, USA) (1:100), goat anti-mouse leptin receptor (R&D SYSTEMS, Minneapolis, MN, USA) (1:100), goat anti-mouse-CD31antibody coupled to Alexa Fluor (AF) 488 (BD Biosciences, San Jose, CA, USA) (1:50), rat anti-mouse osteocalcin antibody (R21C-01A) (1:200) (Takara, Shiga, Japan), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F11) (1:500) and rat anti-Ter119 (TER-119) antibody coupled to APC (1:500) (both from Thermo Fisher Scientific, Waltham, MA, USA), rat anti-Ly-6A/E (Sca-1) antibody coupled to AF 488 (D7) (1:500), hamster anti-CD29 antibody coupled to AF 488 (HMβ1-1) (1:500), rat anti-CD90.2 antibody coupled to AF 488 (30-H12) (1:500), rat anti-CD146 antibody coupled to AF 488 (ME-9F1), isotype control rat IgG2a, κ coupled to AF 488 (RTK2758) (1:500) and isotype control hamster IgG coupled to AF 488 (HTK888) (1:500) (all from BioLegend, San Diego, CA, USA), rabbit anti-adiponectin antibody (1:10) (Novus Biologicals, Centennial, CO, USA), rabbit anti-osterix antibody (1:100) (Abcam, Cambridge, UK) and normal rabbit IgG (60024B) (R&D SYSTEMS).

Techniques: Activity Assay, Tube Formation Assay, Derivative Assay, Two Tailed Test, Standard Deviation

Journal: Scientific Reports

Article Title: Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells

doi: 10.1038/s41598-023-30446-w

Figure Lengend Snippet: LepR/Tom + PDL cells express stem cell markers, while both LepR/Tom + and LepR/Tom − PDL cells possess in vitro pluripotency. (A) Representative FACS plots (gated on live LepR/Tom + cells) showing the frequency of stem cell marker expression in LepR/Tom + PDL cells; n = 3. Blue and red lines represent isotype controls and antibodies against the stem cell markers indicated, respectively. ( B, C ) Pluripotency of LepR/Tom + and LepR/Tom − PDL cells. Representative images of differentiated osteoblasts ( B ) and adipocytes ( C ) derived from PDL cells of LepR-cre; R26-tdTomato mice; n = 3. Osteoblasts or adipocytes were detected via staining for osterix ( B , green) or adiponectin ( C , green), respectively. Right panels are stained for control rabbit IgG. White arrows: LepR/Tom + /osterix + osteoblasts, white arrowheads: LepR/Tom − /osterix + osteoblasts, yellow arrows: LepR/Tom + /adiponectin + adipocytes, yellow arrowheads: LepR/Tom − /adiponectin + adipocytes. Nuclei were visualized using Hoechst; scale bar = 50 μm ( B ) and 20 μm ( C) .

Article Snippet: The following primary antibodies were used: rat anti-endomuchin antibody (V. 7C7) (Santa Cruz Biotechnology, CA, USA) (1:100), goat anti-mouse leptin receptor (R&D SYSTEMS, Minneapolis, MN, USA) (1:100), goat anti-mouse-CD31antibody coupled to Alexa Fluor (AF) 488 (BD Biosciences, San Jose, CA, USA) (1:50), rat anti-mouse osteocalcin antibody (R21C-01A) (1:200) (Takara, Shiga, Japan), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F11) (1:500) and rat anti-Ter119 (TER-119) antibody coupled to APC (1:500) (both from Thermo Fisher Scientific, Waltham, MA, USA), rat anti-Ly-6A/E (Sca-1) antibody coupled to AF 488 (D7) (1:500), hamster anti-CD29 antibody coupled to AF 488 (HMβ1-1) (1:500), rat anti-CD90.2 antibody coupled to AF 488 (30-H12) (1:500), rat anti-CD146 antibody coupled to AF 488 (ME-9F1), isotype control rat IgG2a, κ coupled to AF 488 (RTK2758) (1:500) and isotype control hamster IgG coupled to AF 488 (HTK888) (1:500) (all from BioLegend, San Diego, CA, USA), rabbit anti-adiponectin antibody (1:10) (Novus Biologicals, Centennial, CO, USA), rabbit anti-osterix antibody (1:100) (Abcam, Cambridge, UK) and normal rabbit IgG (60024B) (R&D SYSTEMS).

Techniques: In Vitro, Marker, Expressing, Derivative Assay, Staining, Control

Journal: Scientific Reports

Article Title: Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells

doi: 10.1038/s41598-023-30446-w

Figure Lengend Snippet: LepR/Tom + PDL cells contribute as some of the osteocytes in regenerative bone tissue of the extraction socket. (A–E) Lineage tracing analysis of LepR/Tom + cells in regenerative bone (RB) tissue of extraction socket. (A, B) Representative confocal images (Z stack) in thick sections of regenerated bone in the socket of maxillary first molars after 2 weeks post tooth extraction in 4-week-old LepR-cre ; R26-tdTomato mice stained with anti-OCN antibody ( A , n = 5) or in 4-week-old R26-tdTomato control mice ( B , n = 3). Scale bar = 200 μm. Lower panels represent the magnified views of the boxed areas; scale bar = 50 μm. Arrows: LepR/Tom + OCN + osteoblasts, arrowheads: LepR/Tom + osteocytes. Dotted lines indicate the border of the extraction socket. Nuclei were visualized using Hoechst. ( C–E ) Quantification of LepR/Tom + and LepR/Tom − osteocytes in RB tissue of the extraction socket. ( C ) Representative quantification area in RB (0.25 μm 2 ). Scale bar = 200 μm. Nuclei were visualized using Hoechst. ( D ) Absolute number of osteocytes in RB (Two-tailed Student’s t -test). ( E ) Frequency of LepR/Tom + and LepR/Tom − osteocytes in RB (two-tailed Mann–Whitney U -test); n = 5. ** p < 0.01, **** p < 0.0001. Data are represented as mean ± standard deviation (SD). OCN: osteocalcin, RB: regenerated bone, AB: alveolar bone, DIC: differential interference contrast.

Article Snippet: The following primary antibodies were used: rat anti-endomuchin antibody (V. 7C7) (Santa Cruz Biotechnology, CA, USA) (1:100), goat anti-mouse leptin receptor (R&D SYSTEMS, Minneapolis, MN, USA) (1:100), goat anti-mouse-CD31antibody coupled to Alexa Fluor (AF) 488 (BD Biosciences, San Jose, CA, USA) (1:50), rat anti-mouse osteocalcin antibody (R21C-01A) (1:200) (Takara, Shiga, Japan), rat anti-CD45 antibody coupled to allophycocyanine (APC) (30-F11) (1:500) and rat anti-Ter119 (TER-119) antibody coupled to APC (1:500) (both from Thermo Fisher Scientific, Waltham, MA, USA), rat anti-Ly-6A/E (Sca-1) antibody coupled to AF 488 (D7) (1:500), hamster anti-CD29 antibody coupled to AF 488 (HMβ1-1) (1:500), rat anti-CD90.2 antibody coupled to AF 488 (30-H12) (1:500), rat anti-CD146 antibody coupled to AF 488 (ME-9F1), isotype control rat IgG2a, κ coupled to AF 488 (RTK2758) (1:500) and isotype control hamster IgG coupled to AF 488 (HTK888) (1:500) (all from BioLegend, San Diego, CA, USA), rabbit anti-adiponectin antibody (1:10) (Novus Biologicals, Centennial, CO, USA), rabbit anti-osterix antibody (1:100) (Abcam, Cambridge, UK) and normal rabbit IgG (60024B) (R&D SYSTEMS).

Techniques: Extraction, Staining, Control, Two Tailed Test, MANN-WHITNEY, Standard Deviation

Journal: Mediators of Inflammation

Article Title: Intrauterine Malnutrition Reduced Long Leptin Receptor Isoform Expression and Proinflammatory Cytokine Production in Male Rat Pulmonary Endothelial Cells Stimulated by Lipopolysaccharide

doi: 10.1155/2018/8597361

Figure Lengend Snippet: Involvement of the p38 kinase and NF- κ B pathways in the regulation of Ob-R expression on LPS-induced Ob-R expression in pulmonary endothelial cells. Endothelial cells were harvested 6 h after stimulus with LPS and/or leptin to quantify the activation of the p38 MAPK and NF- κ B pathways using Western blotting. The graphs represent the band intensities determined by densitometric analyses and normalized to the total amount of β -actin (NF- κ B) and β -actinin (pp38) present in each lane. Cells were obtained from 8 male Wistar rats selected randomly from 5 different litters per group. The results are presented as the means ± SEM, ∗ P < 0.05.

Article Snippet: The membranes were incubated overnight with polyclonal antibodies against ObRb (sc-8325 (H-300) rabbit polyclonal IgG, Santa Cruz Biotechnology Inc., EUA), NF- κ B p65 (number 3987, Cell Signaling; rabbit polyclonal IgG Tech, USA), or phospho-p38 MAPK (number 9215, Cell Signaling; rabbit polyclonal IgG Tech, USA) at 1 : 1000 dilutions.

Techniques: Expressing, Activation Assay, Western Blot