Immunohistochemical (IHC) Staining of Prostate Tissues for CXCR4.

<p><b><i>A</i></b>, A human prostate tissue array, ranging from normal to high-grade prostate cancer, was evaluated by IHC for CXCR4 expression using standard methods. Samples were evaluated at magnification 40X, using a Q-Imaging camera of Olympus BX51 Microscope with Bioquant® Image Analysis Software (RtmBometrics). Normal prostate tissues demonstrated slightly weak or undetectable brown staining for CXCR4 (positive cells<5%), and no CXCR4 expression in the nucleus. Representative low grade prostate tissue (grade 2, stage II, T₂N₀M₀, adenocarcinoma) demonstrated random/focal positive staining for CXCR4 in the nucleus (positive cells >11%, but less than 50%), indicating low expression of CXCR4. Representative high grade metastatic prostate tissue (grade 4, stage IV, T₄N₁M₁, adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. <b><i>B</i></b>, CXCR4 IgG2B mouse monoclonal antibody was evaluated for specificity to CXCR4 protein by western blot analysis in PC3 (CXCR4 positive) or 293T (CXCR4 null) cell lines. <b><i>C</i></b>, CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. <b><i>D</i></b>, CXCR4 IgG2B antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation with Fibronectin IgG2B mouse monoclonal antibody (unrelated isotype control) and western blot analysis for CXCR4; expression of Fibronectin protein was confirmed by western blot analysis. Beta-actin was used as a loading control.</p

Nuclear CXCR4 was Functional at the Nucleus.

<p><b><i>A</i></b>, Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. <b><i>B</i></b>, Whole cells were treated with SDF1α prior to isolating and lysing intact nuclei. Nuclei lysates (1 mg) were immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed for G_αi (first row) or CXCR4 antibody (second row), respectively. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo1, nuclear) were used as markers for fractionation purity and as loading controls. <b><i>C</i></b>, PC3 nuclei were isolated, incubated with FluoForte dye Ca²⁺ probe, followed by incubation with AMD3100 or pertussis toxin (PTX) for 1 hr, then stimulated with SDF1α for 30 min. An increase in fluorescent-bound Ca²⁺ was measured on a microplate reader at ex = 490 nm/em = 525 nm.</p

CXCR4 and TRN1 Demonstrate an Interaction.

<p><b><i>A</i></b>, Sixty micrograms of total protein were analyzed for TRN1 expression by western blot analysis using a TRN1 specific antibody. Alpha-tubulin served as a loading control. <b><i>B</i></b>, One milligram of PC3 whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed with anti-TRN1 or anti-CXCR4 to ensure that CXCR4 interacted with TRN1 and was immunoprecipitated, respectively. Thirty micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4 immunoprecipitation. <b><i>C and D</i></b>, Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration <i>(</i><b><i>C</i></b><i>)</i>, prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 <i>(</i><b><i>D</i></b><i>)</i>. Images were taken using Zeiss Axio Imager.z1 fluorescence microscope at 40× magnification at excitation 470 nm for FITC and 551 nm for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bar represents 50 µm.</p

A Putative Functional NLS within CXCR4.

<p><b><i>A</i></b>, GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). <b><i>B</i></b>, Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4<b>R146A</b>,) and deleted NLS of CXCR4 (CXCR4<b>ΔNLS</b>) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. <b><i>C</i></b>, Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.</p

Nuclear CXCR4 Expression in Prostate Cancer Cell Lines.

<p><b><i>A</i></b>, Normal prostate epithelial (RWPE1) and PCa (PC3, DU145, 22RV1) cells were stimulated with SDF1α (100 ng/ µl) prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls. The bar graphs are quantitative results of the band density representing expression of CXCR4 in each fraction. Data were mean <u>+</u>S.E. from three independent experiments. *, P<0.05. <b><i>B</i></b>, Immunocytochemistry of PCa cell lines for CXCR4. PCa cells were stimulated with SDF1α (100 ng/ µl), fixed with methanol, blocked then incubated with an antibody mixture containing a mouse anti-CXCR4 monoclonal antibody and a rabbit polyclonal anti-Lamin A/C antibody, followed by secondary mixture containing a Cy3-conjugated anti-mouse antibody and FITC-conjugated anti-rabbit antibody. Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at excitation 488 nm for FITC and 543 nm for Cy3. Confocal images demonstrating the plasma membrane and cytosolic localization of CXCR4 (red), intact nuclear membrane (green), and nuclear-associated localization of CXCR4 (yellow/orange) are shown. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bars represent 50 µm.</p

Population-Based Prevalence of Chlamydia trachomatis Infection and Antibodies in Four Districts with Varying Levels of Trachoma Endemicity in Amhara, Ethiopia.

The Trachoma Control Program in Amhara region, Ethiopia, scaled up the surgery, antibiotics, facial cleanliness, and environmental improvement (SAFE) strategy in all districts starting in 2007. Despite these efforts, many districts still require additional years of SAFE. In 2017, four districts were selected for the assessment of antibody responses against Chlamydia trachomatis antigens and C. trachomatis infection to better understand transmission. Districts with differing endemicity were chosen, whereby one had a previous trachomatous inflammation-follicular (TF) prevalence of ≥ 30% (Andabet), one had a prevalence between 10% and 29.9% (Dera), one had a prevalence between 5% and 10% (Woreta town), and one had a previous TF prevalence of &lt; 5% (Alefa) and had not received antibiotic intervention for 2 years. Survey teams assessed trachoma clinical signs and took conjunctival swabs and dried blood spots (DBS) to measure infection and antibody responses. Trachomatous inflammation-follicular prevalence among children aged 1-9 years was 37.0% (95% CI: 31.1-43.3) for Andabet, 14.7% (95% CI: 10.0-20.5) for Dera, and &lt; 5% for Woreta town and Alefa. Chlamydia trachomatis infection was only detected in Andabet (11.3%). Within these districts, 2,195 children provided DBS. The prevalence of antibody responses to the antigen Pgp3 was 36.9% (95% CI: 29.0-45.6%) for Andabet, 11.3% (95% CI: 5.9-20.6%) for Dera, and &lt; 5% for Woreta town and Alefa. Seroconversion rate for Pgp3 in Andabet was 0.094 (95% CI: 0.069-0.128) events per year. In Andabet district, where SAFE implementation has occurred for 11 years, the antibody data support the finding of persistently high levels of trachoma transmission