Figure 3.

Activation of Kras oncogene in Col2 cells at perinatal age increases stromal cell numbers (see also Supplementary figure S3). (a-b) Hematoxylin/eosin-stained paraffin sections showing the stromal cells between the trabeculae in P28 mice (secondary spongiosa) control (a) and Kras^G12D (b) after Kras activation at E18.5. × 40 magnification. (c-f) In situ hybridization for collagen 1 (Col1 ISH) (c,d) and osteocalcin (Oc ISH) (e,f) in the secondary spongiosa of P28-old mice control (c,e) or Kras^G12D (d,f) after tamoxifen injection at E18.5. Stromal cells are not stained for these markers. Black arrows, osteoblasts; red asterisks, stromal cells. (g-j) Immunohistochemistry for p-ERK at P1.5 (g,h) and P10 (i,j) in the tibia secondary spongiosa of control (g, i) and Kras^G12D (h, j) after tamoxifen injection at E18.5. Arrows show representative cells stained with anti- p-ERK. (k-l) Immunohistochemistry for p-Akt at P10 in the tibia secondary spongiosa of control (k) or Kras^G12D (l) after tamoxifen injection at E18.5. Arrows show representative cells stained with anti- p-Akt antibody. (m-p) BrdU (Bromodeoxyuridine) labeling of proliferating cells in the metaphysis of mice (tibia) control (m,o) and Kras^G12D (n,p) at P10 after Kras activation at E18.5. BrdU-positive cells are stained brown. (q) The BrdU index, calculated as the percentage of BrdU-labeled stromal cells, was significantly increased upon Kras^G12D activation. Stromal cells were defined as the non-hematopoietic cells that were not attached to the bone matrix. Data are represented as mean±S.E.M.; n=3, *P=0.002