Figure 7.
E6AP Represses the Levels of the Metastatic Suppressor NDRG1
(A) Heatmap of the top 20 significantly commonly upregulated transcripts and proteins upon E6AP KD in DU145 cells from the combined transcriptomics and proteomics screen depicted in Figure 2A.
(B) RT-PCR (left) and immunoblotting (right) of NDRG1 mRNA and protein levels in DU145-shE6AP and control cells (shCtr), treated with Doxy for three days or remained untreated. mRNA data are ΔΔCt±SD of technical triplicates of one of three independent experiments. Immunoblot is representative of three independent experiments. **p < 0.01, ***p < 0.001, unpaired t test.
(C) RT-PCR of NDRG1 mRNA in DU145 transduced with two different shRNA against E6AP than the one used in the screen (shE6AP#4 on the left and shE6AP#5 on the right) treated with Doxy for three days and remained untreated. mRNA data are ΔΔCt±SD of technical triplicates of one of two independent experiments. ***p < 0.001, ****p < 0.0001, unpaired t test.
(D and E) (D) Western blot analysis of E6AP, NDRG1, and Tubulin levels in BPH cells (D) and LNCaP xenograft cells (E) following Doxy-induced shE6AP expression (top panels). Bottom panels show densitometry quantification of results and statistical analysis p values where appropriate (paired t test).
(F) Endpoint tumor samples were analyzed for NDRG1 mRNA by RT-PCR (left) and immunoblotting (right). β-actin was used as a loading control in the immunoblots. mRNA data are ΔΔCt±SD of three technical replicates of three mice.