Journal: International Journal of Molecular Medicine

Article Title: Leptin regulates the proliferation and apoptosis of human endometrial epithelial cells

doi: 10.3892/ijmm_00000073

Figure Lengend Snippet: Figure 1. Expression of leptin receptor isoforms in HHUA cells. (A) Expression of leptin receptor isoform mRNAs in HHUA cells. (B) Cell surface expression of leptin receptors in HHUA cells.

Article Snippet: The primary antibodies used were: mouse anti-human leptin receptor monoclonal antibody (clone 52208, R&D Systems Inc., Minneapolis, MO); mouse anti-human Fas (CD95) monoclonal antibody (clone UB2, MBL); and mouse anti-human Fas ligand (CD178) monoclonal antibody (clone NOK-1, BD Biosciences, San Jose, CA).

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Leptin regulates the proliferation and apoptosis of human endometrial epithelial cells

doi: 10.3892/ijmm_00000073

Figure Lengend Snippet: Figure 2. Effects of leptin on the proliferation and Fas-mediated apoptosis of HHUA cells. (A) Leptin stimulates HHUA cell proliferation in a dose-dependent manner. The arrow indicates the normal serum leptin concentration. (B) Leptin enhances Fas-mediated growth inhibition of HHUA cells. The cells were pretreated with leptin at a final concentration of 50 ng/ml. The solid line with closed circles shows the combined effects of leptin and anti-Fas IgM. The dotted line with open circles shows the effects of anti-Fas IgM without leptin. (C) Leptin enhances Fas-mediated DNA fragmentation in HHUA cells. In this experiment, the cells were pretreated with leptin at a final concentration of 200 ng/ml for 1 h, and then stimulated with anti-Fas IgM at a final concentration of 75 ng/ml.

Article Snippet: The primary antibodies used were: mouse anti-human leptin receptor monoclonal antibody (clone 52208, R&D Systems Inc., Minneapolis, MO); mouse anti-human Fas (CD95) monoclonal antibody (clone UB2, MBL); and mouse anti-human Fas ligand (CD178) monoclonal antibody (clone NOK-1, BD Biosciences, San Jose, CA).

Techniques: Concentration Assay, Inhibition

Journal: International Journal of Molecular Medicine

Article Title: Leptin regulates the proliferation and apoptosis of human endometrial epithelial cells

doi: 10.3892/ijmm_00000073

Figure Lengend Snippet: Figure 3. Effects of leptin on the cell surface expression levels of Fas antigen and Fas ligand in HHUA cells. The thick lines show the expression levels of Fas or Fas ligand (FasL) while the thin lines are negative controls. (A and C) Cells with no leptin. (B and D) Cells treated with leptin at a final concentration of 200 ng/ml. No significant differences in Fas expression were observed after leptin stimulation (A vs. B). Leptin treatment did not induce Fas ligand expression (C vs. D).

Article Snippet: The primary antibodies used were: mouse anti-human leptin receptor monoclonal antibody (clone 52208, R&D Systems Inc., Minneapolis, MO); mouse anti-human Fas (CD95) monoclonal antibody (clone UB2, MBL); and mouse anti-human Fas ligand (CD178) monoclonal antibody (clone NOK-1, BD Biosciences, San Jose, CA).

Techniques: Expressing, Concentration Assay

Journal: International Journal of Molecular Medicine

Article Title: Leptin regulates the proliferation and apoptosis of human endometrial epithelial cells

doi: 10.3892/ijmm_00000073

Figure Lengend Snippet: Figure 4. Effects of leptin on anticancer drug-induced apoptosis. (A) Leptin enhances the Fas-mediated growth inhibition of HHUA cells. In this experiment, leptin, at a final concentration of 100 ng/ml, was added. The dotted lines with open circles show the combined effects of leptin and anticancer drugs while the solid lines with closed circles show the effects of the anticancer drugs without leptin. The leptin combination effects on the growth inhibition by TNF-α (B), CDDP (C), SN38 (D), NPL (E) or paclitaxel (F).

Article Snippet: The primary antibodies used were: mouse anti-human leptin receptor monoclonal antibody (clone 52208, R&D Systems Inc., Minneapolis, MO); mouse anti-human Fas (CD95) monoclonal antibody (clone UB2, MBL); and mouse anti-human Fas ligand (CD178) monoclonal antibody (clone NOK-1, BD Biosciences, San Jose, CA).

Techniques: Inhibition, Concentration Assay

Journal: Cell Death & Disease

Article Title: Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway

doi: 10.1038/s41419-023-05641-2

Figure Lengend Snippet: A RA-FLS were stimulated with vehicle, serum from RA patient and HC with or without anti-leptin antibody (500 ng/mL), together with serum from RA patient and isotype antibody (500 ng/mL). Cells were harvested at 24 h and mRNA expression of the indicated genes was determined with real-time PCR analysis ( n = 5). B Measurement of CPT-1A, CPT-1B and CPT-1C mRNA expressions in RA-FLS by real-time PCR (left, n = 5). RA-FLS were treated with vehicle, serum from HC with or without leptin, serum from rheumatoid arthritis with or without leptin antibody, isotype antibody, and levels of CPT-1A were determined by western blot ( n = 4). C Expression of leptin receptor was assessed in RA-FLS treated by vehicle, serum from HC and RA patient with real-time PCR analysis ( n = 5), western blot and immunofluorescence assay (scale bar = 200 μm, n = 4). D CPT-1A was tested with real-time PCR in RA-FLS stimulated by leptin (100 ng/mL), anti-leptin antibody (500 ng/mL) and isotype antibody (500 ng/mL) for 24 h, respectively ( n = 5). CPT-1A protein expression was also detected at 48 h after indicated stimulations ( n = 4). Data are means ± SEM of three independent experiments, statistical significance was determined as ** P < 0.01, * P < 0.05, ns means no significance compared with control.

Article Snippet: Incubate with anti-human leptin receptor antibody (cat. no. 130-125-241, Miltenyi Biotec, Germany, 1:100 diluted) at room temperature for 30 min.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Control

Journal: Cell Death & Disease

Article Title: Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway

doi: 10.1038/s41419-023-05641-2

Figure Lengend Snippet: A Protein–protein interactions (PPI) among leptin, leptin receptor and major metabolic pathways was consturcted. Only the PPI with the confidence of >0.4 was kept. B AMPK total and phosphorylated protein at T172 (pAMPK), LKB1 and CAMKK protein levels were compared in RA-FLS treated in the presence or absence of leptin by western blot ( n = 4). C Assessment of LKB1 protein level in RA-FLS processed with vehicle, anti-leptin antibody and isotype antibody by western blot ( n = 4). D AMPK total and pAMPK in NC or down-regulation of LKB1 with siRNA in the presence or absence of leptin was detected at 48 h ( n = 4). E CPT-1A gene ( n = 5) and protein ( n = 4) levels in RA-FLS stimulated by leptin with or without compound C (CC, 5 μM) was evaluated. Data are means ± SEM of three independent experiments, statistical significance was determined as ** P < 0.01, * P < 0.05, *** P < 0.001, **** P < 0.0001, ns means no significance compared with control.

Article Snippet: Incubate with anti-human leptin receptor antibody (cat. no. 130-125-241, Miltenyi Biotec, Germany, 1:100 diluted) at room temperature for 30 min.

Techniques: Protein-Protein interactions, Western Blot, Control

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