Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , 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, T 2 N 0 M 0 , 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, T 4 N 1 M 1 , adenocarcinoma) demonstrated diffuse/intense staining (positive cells >50%), indicating high expression for CXCR4 in the nucleus. Scale bar represents 50 µm. 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. C , CXCR4 antibody was evaluated for specificity to CXCR4 protein by immunoprecipitation for CXCR4 and western blot analysis for CXCR4. D , 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.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Expressing, Imaging, Microscopy, Software, Staining, Western Blot, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , 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 + S.E. from three independent experiments. *, P<0.05. 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.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Fractionation, Western Blot, Expressing, Immunocytochemistry, Incubation, Imaging, Microscopy, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , 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 , Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4 R146A ,) and deleted NLS of CXCR4 (CXCR4 ΔNLS ) 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. C , 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.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Transfection, Incubation, Staining, Software, Fluorescence, Microscopy, Mutagenesis, Immunocytochemistry, Imaging, Fractionation, Western Blot

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , 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 , 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. C and D , Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration ( C ) , prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 ( D ) . 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.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, SDS Page, Transfection, Concentration Assay, Immunohistochemistry, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

doi: 10.1371/journal.pone.0057194

Figure Lengend Snippet: A , Representative light images of whole cells and isolated nuclei confirmed the integrity of nuclear isolation at 20× magnification. 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. C , PC3 nuclei were isolated, incubated with FluoForte dye Ca 2+ 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 Ca 2+ was measured on a microplate reader at ex = 490 nm/em = 525 nm.

Article Snippet: One milligram of PC3 whole cell lysates were immunoprecipitated for CXCR4 (Santa Cruz; 1 μg per 250 μg of protein) overnight at 4°C, followed by incubation with Protein A/G Plus-Agarose beads (Santa Cruz) for 2 hrs at 4°C.

Techniques: Isolation, Immunoprecipitation, SDS Page, Fractionation, Incubation

Journal:

Article Title: Endocytosis of Titanium Dioxide Nanoparticles in Prostate Cancer PC-3M Cells

doi: 10.1016/j.nano.2010.09.004

Figure Lengend Snippet: Concentration and Competitor Dependent Uptake of TiO2-ARS Nanoconjugates. A. Serum starved PC-3M cells were treated with a range of concentrations of fluorescently labeled nanoconjugates (0-200 nM). After one hour, the cells were washed to minimize surface bound nanoparticles, and analyzed by flow cytometry. There was a statistically significant increase in signal for cells treated with 50 nM nanoparticles (4 ng/ml) compared to lower nanoparticle concentrations (*p<0.001) (n=3). There was also a significant difference in uptake between cells treated with 50 and 200 nM (‡p<0.05). B. GFP expressing cells were treated with 50 nM of TiO2-ARS nanoconjugates alone (left) or in combination with a 135 fold excess of unlabeled nanoparticles (6.7 μM) (right). Due to the significance of surface bound nanoparticles for this experiment, neither cell sample was glycine washed. Cells were fixed in 4% paraformaldehyde and imaged by laser scanning confocal microscopy. Cytoplasm and nucleus were both filled with GFP (green) while TiO2-ARS nanoparticles(maroon) could be seen only in the absence of TiO2 nanoparticle competition.

Article Snippet: PC-3M metastatic prostate cancer cells with stable GFP expression were a gift from Dr. Raymond Bergan, Northwestern University 34 ; these cells were used in our prior work establishing intracellular stability of nanoconjugates 15 .

Techniques: Concentration Assay, Labeling, Flow Cytometry, Expressing, Confocal Microscopy

Journal: Heliyon

Article Title: Exploring the therapeutic potential of triterpenoid saponins from Gymnema sylvestre : Mechanistic insights into hepatoprotection, immunomodulation, anticancer activities, molecular docking, and pharmacokinetics

doi: 10.1016/j.heliyon.2024.e40850

Figure Lengend Snippet: Effect of triterpenoid saponin extract of G. sylvestre on cell viability of (a) COLO205 (Human Colon Cancer) and PC3 (Human prostate carcinoma) cells (b) cell viability of SKOV3 (Human ovarian cancer) and B16F10 (Mouse Melanoma) cell lines. The data is represented in the form of a bar graph and plotted using means ± SD of 3 individual experiments. Con: Control; GST: Triterpenoid saponin extract of G. sylvestre .

Article Snippet: All cancer cell lines, including COLO205 (Human colon cancer), PC3 (Human prostate carcinoma), SKOV3 (Human ovarian carcinoma), B16F10 (Mouse melanoma), MCF-7, and MDA-MB-231 as well as the normal CHO and HUVEC cell lines, were obtained from the National Centre for Cell Science (NCCS) in Pune, India.

Techniques: Control