Dominant 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.