Assessing Prostate Cancer Cell Proliferation

5,000 to 7,500 cells were plated in microculture devices as previously described (32 (link)). LNCaP or LNCaP-C4 cells were cultured with either UGSM-2, UGSM-2 + exogenous sonic hedgehog (Shh, Curis), or Gli3-/- UGSM cells (UGli3^-./-)(33 (link)). Drugs were added on day 1 and replenished until day 5. Following the growth period, cell proliferation was quantified using an EdU assay kit (Invitrogen) or RNA was extracted using DynaBeads (Invitrogen) (33 (link)). EdU assay images were obtained on an inverted Nikon Elipse Ti using a 4x objective. Fluorescent nuclear counts and GPF intensities were determined using ImageJ v1.38 (NIH). % EdU (+) cells were obtained by dividing total EdU (+) cells to total cell number (Hoescht-nuclear stain) X 100. Cell proliferation was assessed for significant differences by Wilcoxon Mann-Whitney test. Significant differences have a p-value <0.05.