Fig 2: NDRG1-AR interaction is modulated by NDRG1 phosphorylation.A LNCaP and VCaP cells were fractionated into membrane, cytoplasmic, soluble nuclear and chromatin-bound fractions and blotted for NDRG1 pS330 and NDRG1. Markers for each compartment were also blotted for to ensure the fidelity of the fractionation. B Endogenous NDRG1 co-immunoprecipitates with AR in LNCaP cells. C AR interacts more robustly with non-phosphorylated NDRG1. 293 T cells were transfected with either NDRG1 WT or NDRG1 S330A mutant (FLAG-tagged) and WT-AR (HA-tagged). AR was immunoprecipitated using HA beads, and Western blot performed for AR (HA) and NDRG1 (FLAG). D LNCaP transfected with indicated genes were steroid-starved for 48 h, and subsequently treated with 10 nM DHT. mRNA levels for PSA were measured relative to RPL19 by qRT-PCR. n = 3 and error bars represent the standard deviation. E–F Migration and matrigel invasion assays. LNCaP-PIM1 and VCaP-PIM1-expressing cells were transfected with vector only (VO), NDRG1 WT, or NDRG1 S330A. Cell migration was determined using a scratch assay. Micrographs of the cells at 0 and 72 h are shown, and percent area remaining after 72 h was quantitated. Invasion assay through matrigel was performed in LNCaP-PIM1 and VCaP-PIM1 cells expressing the indicated NDRG1 constructs, and cells invading through matrigel were stained and shown as micrographs. The stain was extracted from the cells that migrated through the matrigel and quantitated spectrophotometrically at 595 nm wavelength. n = 3 independent samples for each experiment. T test; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. G Model illustrating the effect of PIM1 phosphorylation on NDRG1 and its co-repression of AR.

Fig 3: NDRG1 phosphorylation is positively associated with PIM1 expression and high Gleason grade in primary prostate cancer.A Immunohistochemistry (IHC) of PIM1, AR pS213, and NDRG1 pS330 in primary prostate cancer samples (n = 67) with low PIM1 and high PIM1 levels. B IHC of NDRG1 pS330 in normal prostate, low Gleason grade, and high Gleason cases. C IHC analysis comparing NDRG1 pS330 and total NDRG1 in low Gleason tumors versus high Gleason tumors (n = 76). D Western blot analysis of NDRG1 pS330, total NDRG1, AR, PIM1 in different prostate cancer cell lines. HSP90 was used as loading control.

Fig 4: 14-3-3 ? and pS213 AR extensively co-occupy chromatin, and PIM1 over-expression shifts their chromatin occupancy.a PIM1 over-expression results in gain and loss of AR occupied sites on chromatin. b PIM1 over-expression results in increased pS213 AR occupancy on chromatin. c) pS213 AR occupancy sites almost completely overlap with AR occupancy sites in both control and PIM1 over-expressing cells. d PIM1 over-expression shifts 14-3-3 ? occupancy on chromatin. e The majority of pS213 AR sites on chromatin are also co-occupied by 14-3-3 ?. f The top 2000 sites co-occupied by pS213 AR and 14-3-3 ? in PIM1 over-expressing LNCaP cells are related to biological adhesion, locomotion, and immune system processes.

Fig 5: Mir-16 mimic down-regulates Pim-1 and decelerates FD-FLT3/ITD cell growth.(A) Using QPCR, Pim-1 mRNA level was quantified from FD-FLT3/ITD cells upon miR-16 mimic transfection. The graph is presented to show relevant gene expression fold differences in FD-FLT3/ITD cells after miR-16 mimic transfection compared to scrambled miR-16 transfection (fold = 1) as a control. The results show the mean and standard deviation for triplicate QPCR results from two independent experiments (p<0.01). The inset shows the results from immunoblotting of Pim-1 protein upon transfection of miR-16 mimic. Total protein extracts were obtained from FD-FLT3/ITD cells and then subjected to size fractionation in 10% poly-acrylamide gels. Immunoblotting was performed and probed with anti-Pim-1 antibody. Pim-1 protein expression has been reduced in FD-FLT3/ITD cells upon miR-16 mimic transfection (lane 2) when compared to control Pim-1 protein expression (lane 1). The membrane was stripped and re-probed with anti- ß-actin antibody to demonstrate equal loading of total protein extract. (B) Overexpression of miR-16 mimic decelerates growth of FD-FLT3/ITD cells. A quantity of 1×106 FD-FV and FD-FLT3/ITD cells were cultured after transfection with synthetic miR-16 mimics for 4 days. Viable cells were counted daily on the basis of trypan blue exclusion. Results shown are the means from triplicate assays. Error bars indicate SD.