Figure 2. WIP1 enhances hedgehog signaling independent of p53.
(A) Twenty-four hours after plating 1×105 Shh-EGFP cells, media was changed to serum-free media containing vehicle or Shh (3µg/mL). Cells were transduced with lentivirus containing negative control (shNC) or Trp53 shRNA (shTrp53) in combination with lentivirus containing an empty vector or YFP-tagged WIP1 cDNA (LentiORF-YFP-WIP1), and incubated for another 48 hours. Shown is real-time, RT-PCR for Gli1 and Trp53, relative to Gapdh and normalized to vehicle-treated, shNC and empty vector-transduced controls, *p<0.005. (B) Twenty-four hours after plating 1×105 Shh-EGFP cells, media was changed to serum-free media containing vehicle, 8µM Nutlin-3a, and/or Shh (3µg/mL). Cells were also transduced with lentivirus containing pLKO.1 empty vector or LentiORF-YFP-WIP1. 48 hours later, cells were fixed in 4% paraformaldehyde, permeabilized, incubated with α-WIP1 or α-Ki-67 antibody, and mounted using media containing DAPI. Scale bar, 100µm. (C) Quantitation of cells from (B) that express green fluorescent protein (GFP) or Ki-67, per high power field (HPF). Bars, mean cell counts from 10 representative fields for each condition, *p<0.005. (D) mRNA was used to determine Gli1, relative to Gapdh and normalized to expression in empty vector-transduced, vehicle-treated cells, by real-time, RT-PCR, *p<0.005. Error bars, standard deviation (SD) among replicates of at least three per treatment. All experiments were repeated at least three times. ns, not significant.