Extended Data Fig. 9: Ectopic Hepatic mCCN3 Expression in Control Esr1fl/fl Females Increase Bone Formation Without Affecting Bone Resorption.
a, Expression of mCCN3 protein in female liver transduced with a low dose of AAVdj-CAG-Ccn3 (0.5 * 1010 GC/mouse) 2 weeks post-injection; panels to the right represent digitally magnified images of individual positive cells. Scale bar = 100 µm. b, Relative levels of Ccn3 transcripts in liver tissue 5 weeks post-injection after transduction of 0.5 * 1010, 3 * 1010 and 15 * 1010 GC/mouse of AAVdj-CAG-Ccn3 viral vector and control AAV-empty vector (dj) into Esr1fl/fl male or female littermates, (N = 4–9 per group). c, Western blots of plasma and liver uncropped with Ponceau staining below used in Main Fig. 5d. The relative expression for Ccn3 by qPCR is listed for each sample in lanes. d, Bone volume and dynamic histomorphometry measurements after Calcein and Alizarin red double labeling (5 days apart) of female mice transduced with the lowest dose of AAVdj-CAG-Ccn3 viral vector compared to control vector (black) obtained in femurs from Esr1fl/fl control females. e, Bone formation rate and mineral apposition rate (MAR) from dynamic histomorphometry measurements with representative images of femur sections described above from aged Esr1fl/fl females (20–23 months of age) injected with AAVdj-CAG-CCN3. f, Bone volume of femur and L5 of female mice transduced with highest dose of AAVdj-CAG-Ccn3 viral vector. g, TRAP+ osteocytes (%) in femurs from young females (left). In vitro differentiation of osteoclasts from bone marrow isolated from young intact females treated with vehicle or recombinant CCN3 (right). h, Osteoblast surface (Ob.S), osteoclast surface (Oc.S) per bone surface (BS), and silver nitrate staining for lacunae. i. TRAP+ osteocytes (%) in femurs from aged females (left). In vitro differentiation of isolated osteoclasts from aged intact females treated with vehicle or recombinant CCN3 (right). Unpaired Student’s T test 2-tailed for low and high dose groups in panels e, d, g, h. *p < 0.05. ns = not significant. Error Bars ± SEM. Legend is provided above graphs. Abbreviations: MNCs mononucleated cells.
