Journal: PLoS Genetics

Article Title: Congenital lipodystrophy induces severe osteosclerosis

doi: 10.1371/journal.pgen.1008244

Figure Lengend Snippet: A) Transplanted fat depots 3 months after surgery. B) Fat depot weight 3 months after transplantation. C) Serum leptin and adiponectin of FF mice 3 months after fat depot transplantation. WT and non-transplanted FF mice serve as control. D) μCT analysis of trabecular bone volume and bone mineral density of distal femurs of FF mice 3 months after sham operation or transplantation of fat derived from WT or adipokine-deficient mice. Data are presented as mean ± SD. **p<0.01; *** p<0.001 as determined by ANOVA with Holm-Sidak's post hoc analysis for multiple comparisons test. D) comparison with FF Sham except where detailed.

Article Snippet: The primary antibody cocktail contained rat anti-mouse CD45-BUV395 (BD Horizon, clone 30-F11, final dilution factor 1:200), rat anti-mouse TER-119-APC (BioLegend, clone TER-119, 1:200), rat anti-mouse CD41-BV421 (BioLegend, clone MWReg30, 1:300), rat anti-mouse/human CD11b (BioLegend, clone M1/70, 1:400), and rat-anti mouse Leptin receptor (LepR)-biotin (R&D Systems, polyclonal, 1:50) in Brilliant Stain Buffer (BD Biosciences) containing 10 μg/mL FcBlock.

Techniques: Transplantation Assay, Control, Derivative Assay, Comparison

Journal: Stem Cell Reports

Article Title: Mesenchymal loss of p53 alters stem cell capacity and models human soft tissue sarcoma traits

doi: 10.1016/j.stemcr.2023.03.009

Figure Lengend Snippet: p53-deficient MSCs show enhanced cell cycle progression, defective adipogenic differentiation, and impaired expression of stem cell markers (A and B) Cell cycle analysis of p53 +/+ and p53 Δ/Δ MSCs using intracellular Ki67 and Hoechst 33342 staining. Representative flow cytometric plots of CD45 - Ter119 − LepR + CD140a + BM MSCs (A) and the average percentage of MSCs in each phase of the cell cycle (B) (means ± SDs, n = 3, 36 weeks old). (C) Proliferation assay of p53 +/+ and p53 Δ/Δ MSCs in vitro . Representative images of p53 +/+ and p53 Δ/Δ MSCs on day 8 after culture (left panels) and growth curves of p53 +/+ and p53 Δ/Δ MSCs (right graph). The data are expressed as fold increases relative to the cell number on day 1 (n = 1; the data show representative results from three independent experiments). (D–F) Seahorse XF mitochondrial stress test of p53 +/+ and p53 Δ/Δ MSCs. The OCR (D) and ECAR (E) were measured. The basal OCR and maximal OCR were calculated according to the manufacturer’s instructions. The energy map is shown in (F) (means ± SDs, n = 4–5 technical replicates, the data show representative results from two independent experiments). (G) Adipogenic differentiation assay of p53 +/+ and p53 Δ/Δ MSCs. Representative images of Oil Red O staining (left) and quantitative analysis (right) without or with adipogenic induction are shown (means ± SDs, n = 3 technical replicates). (H) Osteogenic differentiation assay of p53 +/+ and p53 Δ/Δ MSCs. Representative images of alizarin red staining (left) and quantitative analysis (right) without or with osteogenic induction are shown (means ± SDs, n = 3 technical replicates). (I–K) Three-dimensional reconstructed μCT images of the distal femur of young (8–13 wk) and aged (31–45 wk) p53 +/+ and p53 Δ/Δ mice (Upper: longitudinal view; Lower: axial view of the metaphysis) (I). The bone volume in young (J) and aged (K) mice was calculated based on μCT analysis of the metaphyseal region 0.5 mm proximal to the growth plate (means ± SDs, n = 7–8). (L–N) GSEA plots for p53 +/+ MSCs, p53 Δ/Δ MSCs and p53 Δ/Δ tumors showing genes highly expressed in CXCL12 high and SCF high MSCs.

Article Snippet: For flow cytometry analysis, the following monoclonal antibodies (mAbs) were used in this study: rat mAbs against CD16/32 (93; eBioscience), CD45 (30-F11; BD Biosciences), TER-119 (TER119; TOMBO), CD31 (MEC13.3; Bolegend), CD140a (APA5; Invitrogen), and LepR (BAF497; R&D Systems).

Techniques: Expressing, Cell Cycle Assay, Staining, Proliferation Assay, In Vitro, Differentiation Assay

Journal: Obesity (Silver Spring, Md.)

Article Title: Adipocyte-derived lipoprotein lipase induces macrophage activation and monocyte adhesion: role of fatty acids.

doi: 10.1038/oby.2007.311

Figure Lengend Snippet: Figure 4: Effect of conditioned medium of insulin-treated adipo- cytes on J774 macrophage TNF and IL-6 secretion. Role of leptin. Confluent 3T3-L1 mature adipocytes were pre-incubated or not with 10 g/mL anti-leptin antibody, then incubated for 45 minutes in the presence or absence of insulin (100 nM). At the end of this incubation period, adipocyte conditioned medium was harvested and added to cultured macrophages for 48 hours. The amounts of TNF (A) and IL-6 (B) secreted by macrophages were measured by ELISA. Data represent the mean SE of six inde- pendent experiments. * p 0.05 vs. adipocyte conditioned me- dium. ** p 0.01 vs. adipocyte conditioned medium. CM, un- treated conditioned media; anti-leptin, anti-leptin antibody.

Article Snippet: Antibodies against mouse leptin and non-immune immunoglobulin G (IgG), recombinant mouse interferon- , and recombinant human leptin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Obesity (Silver Spring, Md.)

Article Title: Adipocyte-derived lipoprotein lipase induces macrophage activation and monocyte adhesion: role of fatty acids.

doi: 10.1038/oby.2007.311

Figure Lengend Snippet: Figure 5: Effect of macrophage-derived LPL on leptin-induced macrophage TNF and IL-6 secretion. Macrophages were pre-incubated or not with 10 g/mL LPL antibody or non-immune IgG antibody, then incubated for 24 to 48 hours in the presence of leptin (10 ng/mL). At the end of this incubation period, TNF (A) and IL-6 (B) secreted by macrophages were measured by ELISA. Data represent the mean SE of six independent experiments. * p 0.05 vs. medium. ** p 0.01 vs. medium. # p 0.05 vs. leptin. ## p 0.01 vs. leptin. Anti-LPL, LPL antibody; anti-IgG, non-immune IgG antibody.

Article Snippet: Antibodies against mouse leptin and non-immune immunoglobulin G (IgG), recombinant mouse interferon- , and recombinant human leptin were obtained from R&D Systems (Minneapolis, MN).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay