Fig. 5: PIM1-mediated NDRG1 pS330 is androgen-dependent, and reduces its stability.

A LNCaP cells stably expressing vector only (LNCaP V.O.), WT PIM1 (LNCaP-WT PIM1), or kinase-dead PIM1 (LNCaP KD PIM1) were steroid-starved for 48 h, and Western blot was performed for NDRG1 pS330, total NDRG1, PIM1 (FLAG), and HSP90 (loading control). B LNCaP and VCaP cells were steroid-starved for 48 h, treated with 10 nM DHT, along with indicated amounts of PIM1 kinase inhibitor SGI-1776 for 24 h, and western blot was performed for NDRG1 pS330, total NDRG1, and HSP90. The ratio of NDRG1 pS330/total NDRG1 was quantified using ImageJ software. C Under the same conditions as Fig. 2B, PIM1 was knocked down using siRNA and western blot was performed for NDRG1 pS330, total NDRG1, PIM1, and HSP90. Ratios were quantified using ImageJ software. D Effect of PIM1-dependent phosphorylation on NDRG1 stability. NDRG1 WT and S330A were expressed in 293 cells, with and without PIM1. Cells were treated with cycloheximide for the indicated times, lysates prepared and blotted for NDRG1 and HSP90. Additionally, WT NDRG1 + PIM1 was treated with MG-132. E Extrapolated half-lives of NDRG1 under the various conditions are shown. F PIM1 overexpression in LNCaP cells results in loss of endogenous NDRG1.