Figure 5. Adipoq-Cre/ER⁺ bone marrow stromal cells form adipocytes, but rarely osteoblasts, in vivo.

(a–d) Percentages of whole bone marrow (WBM) cells (a; by flow cytometry), LepR⁺ stromal cells (b; by flow cytometry), or adipocytes (c; by microscopy in sections) that were Tomato⁺ in Adipoq-cre/ER; R26^tdTomato mice at 2–24 weeks after tamoxifen administration. Numbers of adipocytes/section (d). Two-way ANOVAs with Sidak’s multiple comparisons tests were used to assess differences among consecutive ages (*P<0.05, **P<0.01, ***P<0.001).

(e, f) Four months after tamoxifen treatment, Adipoq-cre/ER; R26^tdTomato; Col1a1*2.3-GFP mice had only rare GFP⁺ osteoblasts that were Tomato⁺ in bone marrow sections (e) or by flow cytometry (f).

(g, h) Based on flow cytometric analysis, the numbers of LepR⁺Tomato⁺ cells (g) and LepR⁺Tomato⁻ cells (h) declined in Adipoq-cre/ER; R26^tdTomato mice after irradiation and bone marrow transplantation. Differences between non-irradiated (Con) and irradiated mice (at 2 or 4 weeks) were assessed by one-way ANOVAs with Dunnett’s multiple comparisons tests (data from panel g were log2-transformed) (**p<0.01, ***P<0.001).

(i) Percentages of LepR⁺Tomato⁺ or LepR⁺Tomato⁻ cells that were also DAPI⁺Annexin V⁺ in enzymatically dissociated bone marrow cells from Adipoq-cre/ER; R26^tdTomato mice one day after irradiation. A two-way ANOVA with Sidak’s multiple comparisons test was used to assess differences among treatments.

(j, k) The percentages of LepR⁺ cells that were Tomato⁺ (j) declined in Adipoq-cre/ER; R26^tdTomato mice after irradiation. In contrast, the number of Tomato⁺ adipocytes in femur sections increased after irradiation (k). The statistical significance of differences between non-irradiated (Con) and irradiated mice was measured using one-way ANOVAs with Dunnett’s multiple comparisons tests (data of 5k were log2-transformed) (**P<0.01, ***P<0.001).

(l) The vast majority of bone marrow adipocytes were Tomato⁺ in non-irradiated and irradiated Adipoq-cre/ER; R26^tdTomato mice.

(m, n) Hoechst staining (m) and BrdU incorporation (14 day pulse, n) by Tomato⁺ bone marrow stromal cells in Adipoq-cre/ER; R26^tdTomato mice or Lepr^cre; R26^tdTomato mice that were non-irradiated (Con) or at 2 weeks after irradiation and bone marrow transplantation. Two-way repeated measures ANOVAs with Sidak’s multiple comparisons tests was used to assess differences among treatments.

All data in Figure 5 represent mean±SD from n=5 mice/time point from 3 independent experiments.