LNShh cells induce osteoblast differentiation. (A) Effect on cell proliferation. MC3T3 cells were grown in 24-well plates with culture inserts containing either control LNCaP cells (squares), LNShh cells (triangles), or no cells (circles). Absorbance measurements are in direct proportion to the number of living cells. Each point is the mean ± SD of 3 replicate wells, and results are representative of two independent experiments. *P < 0.05 and **P < 0.001, compared to MC3T3 cells co-cultured with LNShh cells at indicated time points. (B) Effect on ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates. Quantitative ALP activity was determined in cell lysates at indicated time points. ALP activity in mixed cultures of MC3T3 and LNShh cells (filled bars) were compared to those in mixed cultures of MC3T3 and control LNCaP cells (open bars). Data are means ± SD of 6 replicate determinations from 3 independent samples for each group. *, P < 0.05. (C) Localization of ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates and stained for ALP activity at indicated time points. Magnification, 10×. (D) Effect on Akp2 expression. MC3T3 cells were grown in mixed culture with either LNCaP or LNShh cells. Expression of Akp2 in MC3T3 cells cultured with LNShh cells (filled bars) relative to those cultured with control LNCaP cells (open bars) were compared. (E) MC3T3 cells were cultured alone in the absence (open bar) or presence (filled bar) of 1 μg/ml Shh-N for 24 h, and their relative Akp2 expression was compared. In (D) and (E), values are means ± SD of 2–5 assays. *, P < 0.05.