Fig. 2.

Hh signaling and expression, and changes in proliferation in wild-type and spontaneously transformed mesenchymal stem cells (MSCs). A: immunoblot of Shh and Gli2 protein expression in wild-type MSCs (wtMSC), wild-type MSCs overexpressing Shh (wtMSC^Shh), spontaneously transformed MSCs (stMSC^vect), and spontaneously transformed MSCs with Shh knockdown (stMSC^ShhKO) demonstrating elevated Hedgehog (Hh) signaling and expression. B: quantitative RT-PCR was performed on RNA prepared from wtMSC, wtMSC^Shh, stMSC^vect, and stMSC^ShhKO cells. Shown is the expression of Shh, Gli1, Patched (Ptch), and Hedgehog interacting protein (Hhip) mRNA relative to the wtMSC group showing elevated Hh signaling. C: quantitative RT-PCR was performed on RNA prepared from wtMSC, wtMSC^Shh, stMSC^vect, and stMSC^ShhKO cells. Shown is the elevated expression of cyclin B1 and cyclin D1 mRNA relative to the wtMSC group. *P < 0.05, compared with wtMSC group. D: cells were stained with propidium iodide and cell cycle analyzed by flow cytometry. The data were calculated from the cell cycle phase analysis by MODFIT software showing changes in distribution of G₀/G₁, S, and G₂/M phases in wtMSC, wtMSC^Shh, stMSC^vect, and stMSC^ShhKO cells. E: mean values from 3 individual experiments of wtMSC, wtMSC^Shh, stMSC^vect, and stMSC^ShhKO cells.