Figure 4. Overexpression of flag-tagged LGR5 and its effect on clonogenicity in LIM 1899 cells.

LIM1899 cells were transfected with pTune vector containing human LGR5 flanked by a flag sequence and analysed 3 days after transfection. A) Confocal microscopy: LIM 1899 cells transfected with pTune/LGR5 were co-stained with anti-flag (M2) antibody followed by Alexa488 anti-mouse Ig (green), anti-LGR5 antibody HPA012530 followed by Alexa 546 anti-rabbit Ig (red) and the nuclear stain DAPI. Shown is a merged image (all channels) and greyscale images of the green and red channels, respectively. Confocal microscopy was performed as described in Methods. B) qRT-PCR: untransfected cells (parental) and cells transfected with the LGR5 expression vector were cultured for three days with or without IPTG (100 µM). RNA extraction and qRT-PCR were performed as described in Methods. The graph shows the level of expression of LGR5 in transfected relative to parental cells. Data are the mean +/−sd of triplicate samples, and are representative of >3 separate experiments. C) Clonogenic assay: untransfected cells and cells transfected with pTune/LGR5 or with Cy3-siRNA to LGR5 were seeded in soft-agar plates at 5×103 cells/plate as described in Methods. IPTG (100 µM). was added to triplicate plates for parental and LGR5-expressing cells only. Plates were incubated for 10 days then stained with crystal violet and colonies counted with a dissecting microscope. The graph shows means and standard deviation of three separate experiments, each normalized to the colony numbers for parental cells. The difference between parental and transfected cells is highly signficant (** = p<0.005 and *** = p<0.001 for the two culture conditions).