Figure 7.

PTHrP knockdown decreases the tumor-induced inhibition of osteoblast differentiation and bone formation in vivo. (A and B) Calcein double labeling of metaphyseal trabecular bone. This assay measures the osteoblast-mediated bone formation. 1x10⁶ NC, 995 or 996 cells in 20 μl PBS or 20 μl PBS (Ctrl) alone were injected into both left and right tibiae of 4-weeks-old male nude mice (10 mice or 20 tibiae per group). Four weeks later, mice were sacrificed. Sections of undecalcified tibiae (6 tibiae/group) were used for the MAR assay. Magnification: x200, *P<0.05 (vs. Ctrl), ^#P<0.05 (versus NC). (C and D) IHC staining. Five-μm sections of decalcified tibiae (6 tibiae/group) were immunohistochemically stained with an anti-Osx antibody. Representative lower magnification images (100x, top) and higher magnification images of the boxed areas (400x, bottom) are shown. Arrows in C indicate the Osx-positive osteoblasts that were stained brown. The numbers of Osx-positive cells located on trabecular bone surfaces were counted and normalized to trabecular bone perimeter (Osx (+) OBs/BPm) using an Image Pro Plus 7.0 software (G). *P <0.05 (vs. Ctrl), ^#P <0.05 (vs. NC).