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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Nat Metab. 2019 Jan 7;1(1):111–124. doi: 10.1038/s42255-018-0005-8

Figure 3. Deficiency of Tfr2 in osteogenic cells increases bone mass.

Figure 3

(a, d) Immunohistochemical analysis of Tfr2 on vertebral bone of WT mice. One representative image is shown out of three. Arrows indicate Tfr2 expression in multinucleated osteoclasts and osteoblasts/osteocytes (osteoblasts: black arrows; osteocytes: orange arrows). Scale bar: 20 µm. (b, e) Tfr2α mRNA expression during osteoclast (b) and osteoblast (e) differentiation of WT cells (n=4). (c, f) Immunofluorescence staining for Tfr2 in mature osteoclasts (c) and immature osteoblasts (f). Scale bar: 20 µm. These staining were repeated twice with similar results. (g) Subcellular fractioning of day 7 osteoblast protein lysates from WT mice. One representative blot is shown out of three. CP: cytoplasm; M: membrane; Nuc: nuclear fraction. Cx-43: connexin-43. (h) Bone marrow was transplanted from 12-week-old male WT or Tfr2-/- mice into lethally irradiated 9-week-old male WT and Tfr2-/- recipient (recip) mice. After 16 weeks, the bone volume/total volume (BV/TV) was measured using µCT. (L4, WT-WT n=11, Tfr2-/--WT n=11, Tfr2-/--Tfr2-/- n=7, WT-Tfr2-/- n=9; Femur, WT-WT n=12, Tfr2-/--WT n=10, Tfr2-/--Tfr2-/- n=8, WT-Tfr2-/- n=10). Two-way ANOVA with Bonferroni post-hoc test was used for statistical analysis. (i) BV/TV of the fourth lumbar vertebrae of 10-week-old male cre-positive and cre-negative Tfr2f/f;Lysm-Cre and Tfr2f/f;Ctsk-Cre mice. (Tfr2f/f; Lysm-cre, Cre- n=16, Cre+ n= 9; Tfr2f/f; CtskCre, Cre- n=8, Cre+ n=12). (j-o) Bone analysis of 10-week-old male cre-positive and cre-negative littermate control Tfr2f/f;Osx-Cre mice. (j) BV/TV (L4, Cre- n=18, Cre+ n=12; Femur, Cre- n=18, Cre+ n=13). (k) Trabecular number (Tb.N) and trabecular separation (Tb.Sp) (Tb.N., Cre- n=18, Cre+ n=13; Tb.Sp., Cre- n=19, Cre+ n=12). (l) Serum levels of P1NP. (n=6 per group). (m) Mineralizing surface per bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate per bone surface (BFR/BS) determined at the lumbar spine. (MS/BS, Cre- n=11, Cre+ n=10; MAR, Cre- n=9, Cre+ n=6; BFR, Cre- n=10, Cre+ n=6). (n) Osteoclast surface per bone surface (Oc.S/BS) analyzed at the lumbar spine. (Cre- n=7, Cre+ n=6). (o) Serum CTX levels (n=6 per group). (i-o) A two-tailed t-test was used for statistical analysis. All data are presented as mean±SD. *p<0.05; **p<0.01; ***p<0.001.