Fig 2: Mice with Nsd1 knockout in mesenchymal progenitors showed impaired fracture healing. a Radiographs of fractured femurs from WT and Nsd1f/f;Prx1-Cre mice at different days post fracture (dpf). b Quantitative analysis of formed calluses at different days post fracture (dpf). n = 5. Alcian blue/eosin staining (c) and quantitative results (d) of callus sections. The dashed black lines show the location of the callus. Scale bar = 500 μm. n = 5. Immunofluorescence staining (e) and quantitative results (f) of type II collagen in callus sections. The dashed white lines show the location of the callus. Scale bar= 50 µm. n = 5. g Micro-CT images of calluses in WT and Nsd1f/f;Prx1-Cre mice at 18 dpf. h Quantitative statistics of micro-CT results of calluses. n = 3. i Radiographs of fractured femurs in WT and Nsd1f/f;Col2-Cre mice at different days post fracture (dpf). j Quantitative analysis of formed calluses at different days post fracture (dpf). n = 5. Alcian blue/eosin staining (k) and quantitative results (l) of callus sections. The dashed black lines show the location of the callus. Scale bar = 500 μm. n = 5. m Micro-CT images of calluses in WT and Nsd1f/f;Col2-Cre mice at 21 dpf. n Quantitative statistics of micro-CT results of calluses. BV bone volume, BS bone surface, Tb.N trabecular bone number. n = 3. The values are presented as the means ± SEMs. *P < 0.05, **P < 0.01, ns means not significant

Fig 3: Nsd1 deficiency in mesenchymal progenitors led to skeletal growth defects in mice. Gross images of 1-month-old WT, Nsd1f/f;Prx1-Cre (a) and Nsd1f/f;Col2-Cre (b) mice. Pictures of hindlimbs (left) and quantitative statistics (right) of hindlimb length in 1-month-old WT, Nsd1f/f;Prx1-Cre (c) and Nsd1f/f;Col2-Cre (d) mice. Scale bar = 2 mm. The values are presented as the means ± SEMs, n = 6. *P < 0.05, ns means not significant. Safranin O (SO) staining results (e, g) and growth plate quantification data (f, h) of tibia sections from 1-month-old WT, Nsd1f/f;Prx1-Cre (e, f) and Nsd1f/f;Col2-Cre (g, h) mice. Scale bar (top) = 100 μm. Scale bar (bottom) = 50 μm. The values are presented as the means ± SEMs, n = 6. *P < 0.05, **P < 0.01, ns means not significant

Fig 4: Mice with Nsd1 knockout in mesenchymal progenitors showed impaired cartilage development. a mRNA levels of H3K36 methyltransferases and chondrocyte differentiation marker genes were determined by qRT-PCR in micromasses at different differentiation time points. The values are presented as the means ± SEMs, n = 4. *P < 0.05, **P < 0.01, ns means not significant. The inset shows Alcian blue staining results of micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. Scale bar = 2 mm. b SO staining results of E11.5 limb buds. Scale bar = 100 µm. c Whole-mount in situ hybridization (WISH) results for Col2 in E12.5 embryos (top) and sections of forelimbs (bottom). The dashed purple lines show the digits already present. Scale bar (top) = 500 µm, scale bar (bottom) = 200 µm. d SO staining results of E13.5 (first line), E14.5 (second line), E15.5 (third line), and E16.5 (fourth line) femur sections from WT and Nsd1f/f;Prx1-Cre mice. The dashed black lines show the borders between hypertrophic chondrocytes and the primary ossification center. Scale bar = 200 µm. SO staining of P7 (e) and P14 (f) hindlimb sections from WT and Nsd1f/f;Prx1-Cre mice. Scale bar (top) = 200 µm. Scale bar (bottom) = 500 µm. g Whole-mount in situ hybridization (WISH) results for Col2 in E12.5 embryos (top) and sections of forelimbs (bottom). The dashed purple lines show the digits already present. Scale bar (top) = 500 µm, scale bar (bottom) = 200 µm. h SO staining results of E13.5 (top) and E15.5 (bottom) femur sections from WT, Nsd1f/f;Col2-Cre mice. The dashed black lines show the borders between hypertrophic chondrocytes and the primary ossification center. Scale bar = 200 µm. SO staining of P7 (i) and P14 (j) hindlimb sections from WT and Nsd1f/f;Col2-Cre mice. Scale bar (top) = 200 µm, Scale bar (bottom) = 500 µm

Fig 5: Chondroprogenitor cells with Nsd1 knockout showed impaired chondrocyte differentiation and increased proliferation. a Gross images of pellets formed by chondroprogenitor cells from neonatal mice. Scale bar = 1 mm. b HE staining (top), SO staining (middle), and Col2 in situ hybridization (bottom) results of sections from pellets formed by chondroprogenitor cells. Scale bar = 20 µm. c Alcian blue staining results of micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. Scale bar = 2 mm. d Quantitative analysis of Alcian blue staining. The values are presented as the means ± SEMs, n = 4. **P < 0.05, ns means not significant. qRT-PCR results for Nsd1 (e) and chondrocyte differentiation marker genes, including Sox9 (f), Col2 (g), and Acan (h), in micromasses cultured for 1, 4, and 7 days with chondroprogenitor cells. The values are presented as the means ± SEMs, n = 4. *P < 0.05, **P < 0.01, ns means not significant. i Crystal violet staining results of chondroprogenitor cells cultured for 1, 3, 5, and 7 days. Scale bar = 5 mm. j Quantification of crystal violet staining. The values are presented as the means ± SEMs, n = 6. **P < 0.01, ns means not significant