Figure 3.
Impaired bone formation and BM stromal niche in th3 mice. (A) Histological analysis of representative BM sections stained with hematoxylin and eosin. Metaphyses from WT and th3 bones are depicted. WT, n = 10; th3, n = 10. Original magnification ×40. (B) Representative sections of the proximal metaphysis of the tibiae, analyzed by pQCT in WT and th3 mice. Trabecular BMD values of mice tibiae. WT, n = 16; th3, n = 16. (C) IHC evaluation of collagen-1 deposition in subchondral trabecular bone area of th3 and control mice. (i-ii) Original magnification ×200, inset ×40; (iii-iv) original magnification ×200. Representative quantitative analysis of collagen-1–stained area by software analysis calculating the IHC-positive area as a percentage of the total nuclear area. WT, n = 4; th3, n = 4. IHC on BM sections by staining with anti-mouse Collagen-I antibody was performed. (D) Evaluation of soluble markers of bone turnover in WT and th3 sera and BM extracellular fluid by enzyme-linked immunosorbent assay. WT, n ≥ 4; th3, n ≥ 6. (E) IHC analysis of JAG1 in BM stromal niche of th3 and control mice (left). Original magnification ×400. Representative quantitative analysis of the JAG1-stained area, calculated with image-analysis software (right). WT, n = 6; th3 n = 6. IHC on BM sections by staining with anti-mouse JAG1 antibody was performed. (F) IHC staining of JAG1 in BM sections of th3 and control mice, highlighting the difference in staining of the endosteal compartment (arrows). Original magnification ×400. (G) Frequencies of ex vivo isolated MSCs expressing the CD73 and Sca-1 markers in WT and th3 BM. Data are represented as the percentage of the Lin−CD45−CD31−CD105− population. WT, n = 10; th3, n = 10. (H) RNA-seq normalized expression of Cxcl12, Scf, and Notch ligands in MSCs from WT and th3 mice. WT, n = 3 pools; th3, n = 3 pools. (I) Median fluorescence intensity (MFI) of NICD+ HSCs in WT and th3 mice. WT, n = 9; th3, n = 8. (J) RNA-seq normalized expression of Hes1 and Hey1 Notch target genes in sorted HSCs from WT and th3 mice. WT, n = 3 pools; th3, n = 3 pools. (K) Evaluation of PTH in WT and th3 sera by enzyme-linked immunosorbent assay. WT, n = 6; th3, n = 7. *P < .05; **P < .01; ***P < .001; ****P < .0001. FPKM, fragments per kilobase million.

Impaired bone formation and BM stromal niche in th3 mice. (A) Histological analysis of representative BM sections stained with hematoxylin and eosin. Metaphyses from WT and th3 bones are depicted. WT, n = 10; th3, n = 10. Original magnification ×40. (B) Representative sections of the proximal metaphysis of the tibiae, analyzed by pQCT in WT and th3 mice. Trabecular BMD values of mice tibiae. WT, n = 16; th3, n = 16. (C) IHC evaluation of collagen-1 deposition in subchondral trabecular bone area of th3 and control mice. (i-ii) Original magnification ×200, inset ×40; (iii-iv) original magnification ×200. Representative quantitative analysis of collagen-1–stained area by software analysis calculating the IHC-positive area as a percentage of the total nuclear area. WT, n = 4; th3, n = 4. IHC on BM sections by staining with anti-mouse Collagen-I antibody was performed. (D) Evaluation of soluble markers of bone turnover in WT and th3 sera and BM extracellular fluid by enzyme-linked immunosorbent assay. WT, n ≥ 4; th3, n ≥ 6. (E) IHC analysis of JAG1 in BM stromal niche of th3 and control mice (left). Original magnification ×400. Representative quantitative analysis of the JAG1-stained area, calculated with image-analysis software (right). WT, n = 6; th3 n = 6. IHC on BM sections by staining with anti-mouse JAG1 antibody was performed. (F) IHC staining of JAG1 in BM sections of th3 and control mice, highlighting the difference in staining of the endosteal compartment (arrows). Original magnification ×400. (G) Frequencies of ex vivo isolated MSCs expressing the CD73 and Sca-1 markers in WT and th3 BM. Data are represented as the percentage of the Lin−CD45−CD31−CD105− population. WT, n = 10; th3, n = 10. (H) RNA-seq normalized expression of Cxcl12, Scf, and Notch ligands in MSCs from WT and th3 mice. WT, n = 3 pools; th3, n = 3 pools. (I) Median fluorescence intensity (MFI) of NICD+ HSCs in WT and th3 mice. WT, n = 9; th3, n = 8. (J) RNA-seq normalized expression of Hes1 and Hey1 Notch target genes in sorted HSCs from WT and th3 mice. WT, n = 3 pools; th3, n = 3 pools. (K) Evaluation of PTH in WT and th3 sera by enzyme-linked immunosorbent assay. WT, n = 6; th3, n = 7. *P < .05; **P < .01; ***P < .001; ****P < .0001. FPKM, fragments per kilobase million.

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