DKK1 inhibits proliferation and migration of prostate cancer cells.

<p>(A) Tritiated thymidine incorporation in PC3 cultures in the absence or presence of Wnt3a or DKK1. While high concentration of Wnt3a (10nM) enhanced PC3 cell proliferation, DKK1 inhibited PC3 proliferation in a dose-dependent manner. (B) Regulation of PC3 cell migration by Wnt signaling. Data were collected from 6–8 cultures per group and are expressed as mean+SEM (t-test). Note that Wnt3a increased the number of migrated cells, whereas DKK1 inhibited cell migration.</p

Wnt signaling is activated in prostate cancer cells.

<p>TaqMan RT-PCR analysis of Axin2 expression in various human prostate epithelial cells and cancer cells. Data were collected from triplets and are expressed as mean+SEM (t-test). Note that Axin2 expression is much higher in prostate cancer cell lines (LNCaP, Du145, PC3) and human prostate tumor xenografts (LuCaP35, LuCaP77) as compared to primary prostate epithelial cells (PrEC) or non-tumorigenenic immortalized prostate epithelial cells (BPH1).</p

Wnt3a and DKK1 regulate prostatic epithelial branching morphogenesis.

<p>Whole mount ventral prostates were prepared from P2 rats and maintained for 7 days in serum-free medium in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C). Similar patterns were consistently seen in 3 repeat experiments of 5–6 prostate organs per group in each individual experiment. Note that addition of either Wnt3a or DKK1 to the culture resulted a change in epithelial branching morphogenesis. Quantification of the cultures by measuring the diameter of the cultured prostates (D) and the ductal tips (E) using Axiovision software and the branching points (F) were done by analyzing 4 randomly selected cultures per group. Data are expressed as mean + SEM (t-test, compared to control cultures). Bar, 400 µm.</p

Wnt signaling prevents prostatic epithelial cell differentiation. (A,B,C) p63 immunocytochemistry (red) of the P2 rat ventral prostate organ cultures maintained for 7 days in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C).

<p>The tissue sections were counterstained with DAPI (blue). While p63 positive cells (purple) represent basal cells where progenitor cells reside, the blue cells (arrows) that are negative to p63 in the epithelium are differentiated luminal cells. (D) Quantification of p63 positive cells over total epithelial cells. Data were collected from randomly selected 22–27 ductal units from sections of the organ cultures per group and are expressed as mean + SEM (t-test). Note that while Wnt3a led to a significant increase in the number of basal cells, DKK1 resulted in a reduction in basal cells. Bar, 50 µm for A-C.</p

TaqMan RT-PCR analyses of Axin2 expression during prostate development and regrowth following androgen deprivation and replacement.

<p>(A) A gradual downregulation of Axin2 from newborn to adulthood.nbsp;(B) Axin2 was upregulated following castration, but returned to normal low levels after androgen replacement. Data were collected from 4–6 samples per group and are expressed as mean+SEM (t-test, compared to normal adult prostates). Abbreviation: N, normal prostate; C3, 3 days after castration, C17; 17 days after castration; C14+T. 14 days after castration+3 days of testosterone treatment.</p

Activation of Wnt signaling enhances prostate epithelial cell proliferation.

<p>(A-F) Shown are anti-BrdU antibody (B,D,F) and DAPI (A,C,E) double labeling of the P3 rat ventral prostate organ cultures maintained for 3 days in the absence (A,B) or presence of 50 nM of Wnt3a (C,D) or 400 nM of DKK1(E,F). (G). Quantification of BrdU-positive cells in a given visual field of 434 µm×322 µm. (H) Quantification of Ki67-positive cells in the P2 prostate cultures maintained for 7 days, which was normalized to the control cultures. Cell counts (for G and H) were performed from randomly selected visual fields of 5 organ cultures per group and data were expressed as mean+SEM (t-test). Note that while Wnt3a significantly enhanced progenitor cell proliferation, DKK1 inhibited progenitor cell proliferation. (I). Expression level change of cyclin B2 in P2 rat prostate organ cultures.. Data were collected from 4 organ cultures maintained for 2 days per group and are expressed as mean+SEM (t-test). Note that expression of cyclin B2 was upregulated by wnt3a, but down-regulated by DKK1. Bar, 100 µm for A-F.</p

Expression and folding of CD20 in the <i>E. coli</i> inner membrane.

<p>Cell surface expression and orientation of CD20 was assessed from spheroplasts of <i>E. coli</i> cells expressing either Uni-CD20 (blue), LE-CD20 (green) or an empty control vector (red) treated with Alexa-488 conjugated anti-CD20 antibody to the extracellular loop of CD20 and analyzed by flow cytometry.</p

Leader dependent accumulation of CD20 and EG-VEGFR1 following induction of the <i>tphac</i> promoter.

<p>(<b>A</b>) Protein accumulation maximizes within thirty minutes of induction with the Uni leader. (<b>B</b>) Protein accumulation continues over 20 hours after induction with the LE leader. (<b>C</b>) The effect of C-terminal truncations on expression from the LE leader. The full 79 amino acid LE leader was truncated from the C-terminus to observe the importance of the translation initiation rate as compared to the length of the LE leader. Truncated leaders were fused to CD20 and the whole cell lysates were immunoblotted with HRP conjugated anti-His antibody. Two film exposures are shown. (<b>D</b>) Reduced promoter activity results in reversal of the relative expression levels from the Uni and the LE leaders fused to CD20. Cultures were grown under partial promoter induction and the whole cell lysates were probed with HRP conjugated anti-His antibody for detection.</p

Ligand binding to the GPCR, LE-EG-VEGFR1.

<p><i>E. coli</i> membrane proteoliposomes were treated with thrombin to remove the LE-leader and incubated overnight at 4°C with EG-VEGF in PBS. Pelleted membranes were separated by SDS-PAGE and developed by immuno-blot using an anti-EG VEGF antibody. Samples are: lane 1) pBR322 negative control; 2) LE-EG-VEGFR1, N-terminal FLAG; 3) LE-EG-VEGFR1, C-terminal FLAG. The location of EG-VEGF (molecular weight 9 kDa) is indicated by an arrow.</p

Improved cell growth and general accumulation of integral membrane proteins using a dually regulated promoter.

<p>(<b>A</b>) Restricted <i>E. coli</i> growth in LB with the <i>phoA</i>-RA1c construct is relieved by using the <i>tphac</i> promoter, which reduces basal level expression. A 24-hour growth curve shows the empty pBR322 vector control (blue triangles), <i>phoA</i>-RA1c expression construct (green diamonds), <i>tphac</i>-RA1c expression construct (red circles) and <i>phoA</i>-EGFL7 as a non-membrane protein control (brown squares). (<b>B</b>) A representative western blot of RA1c expression from the <i>phoA</i> promoter is shown following induction by phosphate depletion when the cells reach approximately 2 OD₆₀₀ (time 0). Maximum expression is reached within two hours post induction. By 6 hours, aggregation has begun and by twelve hours almost all the protein has moved from the monomer band to high molecular weight aggregate. Basal expression is shown after overnight growth in LB medium (LBON). The western blot was probed with an HRP coupled anti-his antibody. (<b>C</b>) A comparison of basal expression in LB of the GPCR proteins, RA1c and EG-VEGFR1, from the <i>phoA</i> and <i>tphac</i> promoters by western blot analysis. The <i>phoA</i> constructs show significant accumulation levels of the membrane proteins while the <i>tphac</i> constructs have reduced the accumulation to background levels. The arrow points to the monomer protein band.</p