Figure 4From: Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiationDominant negative Gli1 inhibits paracrine Shh signalling and osteoblast differentiation. (A) MC3T3 cells were stably transfected with human GLI1 cDNA with deleted region containing the t rans a ctivation d omain: pCMV-GL1(-)TAD. Expression of mouse endogenous Gli1 (mGli1) and human GLI1-TAD transgene in parental MC3T3 cells and transfected cells, designated M-TAD cells, were determined by conventional RT-PCR analysis. The mouse gene for ribosomal protein 19 (mrpl19) was used as housekeeping gene. (B) Hematoxylin staining of single cultures of MC3T3 and M-TAD cells are shown in panels a and b, respectively. Fluorescent microscopy images of 14-day mixed cultures of MC3T3 and LNShh cells or M-TAD and LNShh cells are shown in panels c and d, respectively. The photomicrograph of mixed culture of MC3T3 and LNShh cells shown in panel c is the same as that shown in Figure 1 panel c, except only phalloidin staining and GFP expression are revealed here. Scale bars: 25 μm (a, b) and 50 μm (c, d). (C) MC3T3 cells or M-TAD cells were grown for 14 days in 6-well plates with culture inserts containing either LNCaP or LNShh cells (separate co-cultures). Expression of Gli1 and Ptc1 in MC3T3 or M-TAD cells co-cultured with LNShh cells (filled bars) relative to those co-cultured with control LNCaP cells (open bars) were compared. Results are representative of 2 independent experiments. (D) Mixed cultures of MC3T3 and M-TAD cells with either LNCaP or LNShh cells were grown in chamber slides for 14 days and stained for ALP activity (panels a, b and c) followed by hematoxylin staining (panels d, e and f). Scale bars: 50 μm.Back to article page