Restoration of N-CoR function attenuates the proliferative properties of AML-M5 cells regardless of the Flt3 receptor mutational status.

<p><i>A</i>, Genistein stabilized N-CoR in all 5 AML-M5 cell lines at 50 µM with a concurrent down-regulation of the Flt3 receptor expression as determined via western blotting. <i>B</i>, Restoration of N-CoR function in AML-M5 cells attenuated the proliferative potential of AML-M5 cells. Proliferative capacity of N-CoR positive AML cells HL-60 and N-CoR negative AML-M5 cells after treatment with Genistein at 72 hours was determined via growth proliferation assay (MTT). Growth inhibition was most pronounced at 50 µM in AML-M5 cells while at the same dose, growth inhibitory effects on N-CoR positive HL-60 was less pronounced. Results are representative of 3 independent experiments and asterisks represent p<0.05. <i>C</i>, Morphology of N-CoR positive HL-60 and N-CoR null AML-M5 cells (THP-1, Nomo-1, MV-4-11, SigM5, MM1) as determined via Wright-Giemsa staining after treatment with 50 µM Genistein for 72 hours.</p

Repression of Flt3 by N-CoR protein.

<p><i>A</i>, Relative activity of a luciferase reporter driven by the Flt3 promoter was determined in various leukemic cell lines. The cells were transfected with reporter and reference plasmids using electroporation. The values presented in each bar represent the average of three independent experiments. <i>B</i>, Effect of ectopic N-CoR on the activity of the Flt3 promoter in THP-1 cells electroporated with Flag-tagged N-CoR plasmid in a dose dependent manner was determined via luciferase assay (left panel). The values presented in each bar represent the average of three independent experiments. In parallel, levels of ectopic N-CoR protein in THP-1 cells used in the luciferase assay were determined in western blotting assay with anti-Flag antibody (right panel). <i>C</i>, Effect of ectopic N-CoR on the activity of the Flt3 promoter in 293T cells transfected with N-CoR or control siRNA was determined using luciferase assay. In pGL3-Flt3 (−901) reporter plasmid, luciferase reporter was placed under the control of the full length Flt3 promoter. The values presented in each bar represent the average of three independent experiments. <i>D</i>, The dose dependent fold repression by ectopic N-CoR in N-CoR ablated or non-ablated 293T cells was calculated by dividing the mean relative luciferase activity in N-CoR siRNA transfected cells with that of control siRNA transfected cells of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034501#pone-0034501-g003" target="_blank">Fig. 3C</a>. <i>E</i>, N-CoR is associated with the Flt3 promoter. Relative amounts of Flt3 promoter sequence associated with N-CoR protein in HL-60 or NB4 cells were determined through ChIP assay. The antibody used in the ChIP assay is mentioned at the bottom. N-CoR association with CD36 promoter, a known N-CoR target gene, was determined as positive control.</p

Restoration of N-CoR function in THP-1 cells, down-regulated Flt3 levels and induced differentiation.

<p><i>A</i>, Flt3 levels were down-regulated at both the protein and mRNA levels after Genistein treatment. Level of Flt3 expression was determined via western blotting and RT-PCR analysis. <i>B</i>, Genistein inhibited the proliferation of THP-1 cells in a dose dependent manner when determined via growth proliferation assay (MTT). Results are representative of 3 independent experiments and asterisks represent p<0.05. <i>C</i>, Nuclear morphology of THP-1 cells treated with Genistein for the duration of 72 hours in a dose dependent manner was determined in Wright–Giemsa assay. The arrowheads mark the indented-shaped nucleus of differentiated cells. <i>D</i> & <i>E</i>, CD14 levels in THP-1 cells treated with Genistein for 72 hours in a dose dependent manner was determined by FACS (<i>D</i>) and RT-PCR (<i>E</i>) analysis. <i>F</i>, N-CoR, Flt3 and CD14 levels in THP-1 cells transfected with N-CoR siRNA and treated with 50 µM Genistein for 72 hours was determined by western blotting assay and RT-PCR analysis.</p

N-CoR loss is associated with the up-regulation of Flt3.

<p><i>A</i>, Relative expressions of selected hematopoietic genes in AML-M5 and non-AML-M5 (HL-60 and U937) cells were determined by real time PCR analysis. Data was analyzed via the comparative C_t method with the expression level of each gene in HL-60 cells set as the reference value, and the level of expression of the HPRT gene used as the endogenous control. The graph was plotted on a logarithmic scale with a base of 10. Expression levels in HL-60 cells for all genes were set to 0 while genes which were up-regulated relative to their expression levels in HL-60 cells was given a positive value, and those which were down-regulated relative to expression level in HL-60 cells were given a negative value. Raw C_t values that registered as undetermined were given a value of 40. (Results presented are the averages of 3 independent experiments.).</p

N-CoR loss promotes growth potential, which is amplified by Flt3 signaling activation.

<p><i>A</i>, Levels of N-CoR and Flt3 protein in Ba/F3 cells transfected with control or N-CoR siRNA were determined via western blotting assay with an anti-Flt3 antibody that recognizes the murine form of the receptor and anti-N-CoR antibody. <i>B</i>, N-CoR loss mediated Flt3 expression enhanced the IL-3 independent growth potential of BA/F3 cells. Effect of IL-3 independent growth of Ba/F3 cells transfected with control or N-CoR siRNA (population from 4A) with and without Flt3 ligand stimulation were determined by cell proliferation assay. The Y-axis of the graph represents the proliferation index of viable cells, and the duration of culture is plotted on the X-axis. The symbols used in the graph are as follows: Control siRNA transfected cells treated with vehicle (<>\raster="rg1"<>), Control siRNA transfected cells stimulated with Flt3 ligand (<>\raster="rg2"<>), N-CoR siRNA transfected cells treated with vehicle (<>\raster="rg3"<>) and N-CoR siRNA transfected cells stimulated with Flt3 ligand (<>\raster="rg4"<>). The values presented in each graph are average of three independent experiments. <i>C</i>, Flt3 stimulation leads to N-CoR loss. Levels of N-CoR and Flt3 proteins in 293T cells treated with vehicle or Flt3 ligand (30 ng/ml) was determined by western blotting assay. <i>D</i>, Blocking Flt3 stimulation leads to N-CoR stabilization in THP-1 cells. Effect of Flt3 antibody on the levels of N-CoR and Flt3 proteins in THP-1 cells treated with vehicle or Flt3 ligand (30 ng/ml) was determined by western blotting assay.</p

Flt3 expression is inversely related to N-CoR protein status.

<p><i>A</i>, N-CoR and Flt3 levels in various human leukemia cells were determined in western blotting and RT-PCR analysis respectively. <i>B & C</i>, Flt3 and N-CoR levels in AML-M5 derived cell lines (<i>B</i>) and in multiple histologically confirmed human primary AML-M5 samples (<i>C</i>) were determined through western blotting assay using the respective antibodies. Levels of N-CoR and Flt3 in HL-60 cells were used as a reference. <i>D</i>, Levels of Flt3, HoxA9 and Scl/Tal in HL-60 cells transfected with N-CoR or control siRNA were determined by RT-PCR analysis (left panel). N-CoR knockdown efficiency at protein (middle panel) and transcript level (right panel) in HL-60 cells transfected with N-CoR or control siRNA was determined through western blotting assay and RT-PCR analysis. <i>E</i>, Ectopic expression of Flag-tagged N-CoR in THP-1 cells resulted in the loss of Flt3 expression as determine via western blotting assay with anti-Flt3 antibody. Levels of ectopic N-CoR expression were determined via western blotting assay with anti-Flag antibody.</p

Sumoylation is essential for Stra13-dependent growth inhibition.

<p>(A) Lysates of NIH3T3 cells transfected with Myc-Stra13, Stra13 2KR and SENP1 were immunoblotted with anti-Myc antibody. (B–C) After selection, colony assays were performed and colonies were stained with crystal violet. Representative plates are shown (B). The mean relative absorbance after extraction of crystal violet stain from plates in shown in C. Error bars indicate mean ±SD.</p

Stra13 is sumoylated.

<p>(A) Schematic representation of the Stra13 domain structure (upper panel). The basic and HLH domains are shown along with three α-helices in the C-terminal repression domain. Potential sumoylation acceptor lysines at 159 and 279 (K159 and K279) are indicated. Numbers indicate amino acid residues in the mouse Stra13 cDNA. Alignment of Stra13 cDNA from various species revealed a highly conserved SUMO consensus motif IKQE, and a somewhat less conserved motif AKHE that are highlighted. K159 and K279 are indicated by arrowheads (lower panel). (B) Cells were co-transfected with Myc-Stra13, SUMO1 and SENP1 as indicated. Lysates were immunoprecipitated with Myc-agarose beads followed by immunoblotting with anti-SUMO1 antibody. Input shows expression of Stra13 using anti-Myc antibody. β-actin served as a loading control. (C) Cells were co-transfected with Myc-Stra13, or point mutants (Stra13 K279R, Stra13 K159R, Stra13 2KR) together with SUMO1. Cell lysates were immunoprecipitated with Myc-agarose beads and the immunoprecipitates were subjected to western blotting with anti-SUMO1 antibody. (D) Myc-Stra13 and SUMO1 were expressed along with Flag-PIAS1, PIAS3, PIASxα, or PIASy as indicated. Lysates were immunoprecipitated with Myc- agarose beads followed by western blotting with anti-SUMO1 antibody. Lysates (input) were probed for Stra13 and PIAS.</p

Mutation of sumoylation sites abrogates Stra13-mediated growth suppression.

<p>(A) NIH3T3 cells were co-transfected with Stra13 or Stra13 2KR together with a puromycin resistance plasmid. Empty vector (pCS2) was transfected in control cells (Vector). Stra13 expression was determined by western blotting using anti-Myc antibody. (B–C) Colony forming assays were performed with control, Stra13 and Stra13 2KR cells. Colonies were stained with crystal violet 14 days later. Data are representative of three independent experiments (B). Crystal violet dye was extracted and the absorbance measured at a wavelength of 570 nm. The error bars indicate standard deviations for triplicate wells in each experiment (C). (D) Growth of NIH3T3 cells expressing vector alone, Stra13 and Stra13 2KR was evaluated over a five-day period. Cell numbers at each time are represented as mean ±SD. (E) Stra13^−/− MEFs were transfected at passage 5 with equivalent amounts of Stra13 and Stra13 2KR. Cell viability was measured three days later by MTT assays. (F) Cell cycle profile of control (Vector), Stra13 and Stra132KR cells was determined by PI staining and FACS analysis. Representative histograms of cell cycle profiles in cells expressing vector alone, Stra13 and Stra13 2KR. The result shown is representative of three independent experiments.</p

Sumoylation regulates Stra13 transcriptional activity but not its subcellular localization.

<p>(A) mRNA levels of cyclin D1, p21^Cip/WAF, cyclin B1, and cyclin E1 were analyzed by Q-PCR in vector, Stra13 and Stra13 2KR cells. (B) Cells were transfected with the cyclin D1 promoter reporter pD1luc (100 ng) together with Stra13 (25 ng), Stra13 2KR (25 ng), SUMO1 (25 ng) or SENP1 (25 ng), as indicated. Cells were harvested 48 hr after transfection, and assayed for luciferase activity. (C) COS-7 cells were transfected with Stra13 and Stra13 2KR alone or together with SUMO1. Cells were stained with anti-Myc antibody. Nuclei were stained with DAPI. Error bars indicate mean ±SD. (D) NIH3T3 cells were left untreated or treated with TSA. ChIP assays were done to determine Stra13 occupancy on the cyclin D1 promoter.</p