Fig 1: eWAT-secreted OPN regulates bone resorption.a Representative western blots (left) and quantification (right, n = 3 biologically independent samples) of av and ß3 in BMSCs, BMDMs, and osteoclasts (Oc) in vitro. b Immunohistochemical staining for OPN and cathepsin K (CTSK) in serial sections of tibiae from mice fed for 12 weeks. Scale bar: 50 µm. c Representative western blots (left) and quantification (right, n = 3 biologically independent samples) of the bands of JNK and p-JNK in BMDMs, Oc, and Oc+rOPN after culture for 4 days in vitro. rOPN, 0.5 µg/ml. d Representative images of the osteo-erosion surface (top) and crystal violet staining of the unwashed Osteo Assay surface with or without rOPN treatment under osteoclastogenic induction for 12 days (bottom). Scale bar: 50 µm. e Quantification of the bone resorption area from d (n = 3 biologically independent samples). f Relative expression of Mmp9 in BMDMs with or without rOPN treatment under osteoclastogenic induction for 3 and 6 days, respectively (n = 3 biologically independent samples). g Representative western blots of av, ß3, and MMP9 in the bone marrow of NFD- and HFD-fed mice at week 12. h Immunohistochemical staining of MMP9 from the proximal tibiae of mice after injection of saline or OPN-neutralizing antibody (Neu Ab) into bilateral eWAT for 8 weeks. Scale bar: 100 µm (the bottom row was magnified from the top row, scale bar: 50 µm). i, j Representative µCT images (i) and quantification (n = 5 biologically independent samples) (j) of proximal tibiae after mice were injected with saline or Neu Ab. Scale bar: 500 µm. k, l Representative µCT images (k) and quantification (n = 4 biologically independent samples) (l) of rBMAT of tibiae after mice were injected with saline or Neu Ab. Scale bar: 500 µm. Images are representative of 3 independent experiments. All data are presented as mean ± SD. One-way ANOVA with Tukey’s post-hoc test (a, c), two-tailed Welch’s t-test (e), by two-way ANOVA with Sidak’s post-hoc test (f), and two-way ANOVA with Tukey’s post-hoc test (j, l) were used. Source data are provided as a Source Data file. See also Supplementary Tables 7 and 8.
Fig 2: Schematic diagram showing that macrophages in epididymal adipose tissue secrete OPN to regulate bone homeostasis.eWAT-secreted OPN accumulates in the bone marrow compartment where OPN promotes bone resorption and lipophagocytic mobilization, but the excess accumulation of lactate inhibits pre-osteoclast fusion and neutral lipid hydrolysis via lysosomal-dependent ATP6V0d2 in BMDMs.
Fig 3: Mucus secretion in response to rA2-L19F is altered in OPN-/- mice.WT C57BL/6 and OPN-/- mice were intranasally infection with a high dose of mucogenic rA2-L19F (1×106 pfu/mouse, n = 4) or a mock-inoculum, and at 5 and 8 dpi tissues were harvested. (A) Lung sections (5 µm) were stained with PAS per the manufacturer's instructions and counterstained with hematoxlin and eosin, then analyzed to calculate area of staining with ImageJ using Color Deconvolution plugin using single-stained customized vector settings. (B) The areas obtained through ImageJ analysis were related as a ratio of PAS-staining to hematoxylin staining, and displayed on a graph. A minimum of 10 fields of view were obtained at 200X per mouse and the images shown are a representation of the images collected. The experiment was performed in triplicate and data is displayed as means ±SEM.
Fig 4: Infiltrating ATMs in eWAT are related to bone marrow metabolism.a Immunofluorescence staining of macrophages (F4/80) in eWAT after mice were injected with clodronate liposomes (CL) or control liposomes (Ctrl) into bilateral eWAT for 8 weeks. Green, F4/80; blue, DAPI stain for cell nuclei. Scale bar: 75 µm. b Quantification of the F4/80-positive area percentage with immunofluorescence staining from A (n = 5 biologically independent samples). c, d Representative µCT image (c) and quantification (n = 5 biologically independent samples) (d) of proximal tibiae after mice were injected with control liposomes or CL for 8 weeks. Scale bar: 500 µm. e µCT quantification of rBMAT in tibiae after mice were injected with control liposomes or CL for 8 weeks (n = 4 biologically independent samples). f Heatmap summarizing the fold changes in mRNA expression of different biomarkers in eWAT from NFD- and HFD-fed mice at week 12 (n = 4 biologically independent samples). g Relative expression of Spp1 in eWAT (n = 4 biologically independent samples) and iWAT (n = 4 biologically independent samples) over the course of feeding. Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s post-hoc test (b, d, e) and two-way ANOVA with Sidak’s post-hoc test (g) were used. Source data are provided as a Source Data file. See also Supplementary Tables 4 and 5.
Fig 5: OPN is primarily secreted by F4/80 + and CD11b+ macrophages in eWAT.a Representative images of immunofluorescence staining of OPN in eWAT from NFD- and HFD-fed mice at weeks 8, 12, and 16. Scale bar: 100 µm. b Quantification of the F4/80-positive or OPN-positive stained area percentage in eWAT from a (n = 4 biologically independent samples). c Relative expression of Spp1 in the adipocyte fraction (AF) and stromal vascular fraction (SVF) from eWAT or iWAT of HFD-fed mice at week 12 (n = 3 biologically independent samples). d ATMs gated by FACS for F4/80 and CD11b expression from eWAT at week 12 of NFD (left) or HFD feeding (right). Q2 represents the F4/80 + CD11b + (FC + ) ATM population, and Q3 represents the F4/80-CD11b- (FC-) ATM population. e Percentage of FC + ATMs from NFD- and HFD-fed mice at week 12 (n = 4 biologically independent samples). f Relative expression of Spp1 in FC+ and FC- ATMs from HFD-fed mice at week 12 (n = 3 biologically independent samples). Images are representative of 3 independent experiments. All data are presented as mean ± SD. Two-way ANOVA with Sidak’s post-hoc test (b) and two-tailed Welch’s t-test (c, e, f) were used. Source data are provided as a Source Data file.
Supplier Page from Abcam for Mouse Osteopontin ELISA Kit