Figure 3. Effects of PTHrP on anchorage-independent cell growth.

Assays to determine colony formation in soft agar were performed in 60 mm dishes containing a bottom layer consisting of 1.5 ml culture medium containing 0.4% (w/v) agar. Cells (1 × 10⁴) were plated in a top layer of 0.3% agar. (A) After 2 weeks in culture, the plates were photographed at 40 × magnification, and clone size was measured using the ImageJ software (NIH). (B) Photographs were also taken at 10 × magnification to measure clone frequency. All clones in focus > 50 μm in size were measured. (C) Colony size. Each bar is the mean ± SEM of 20 colonies for each of three independent clones overexpressing PTHrP (+), three independent empty vector-transfected clones (V), two independent cell lines (si1 and si2) generated by transfection with independent PTHrP siRNAs (si), or the respective scrambled siRNA sequences (sc), or parental cells (P). (D) Average number of colonies. Each bar is the mean ± SEM of three fields per plate for each of the cell lines described in (C). (C and D) * = Significantly different from the control value (P < 0.001).