Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Activity Assay, Expressing, Functional Assay, Gene Expression, Quantitative RT-PCR, esiRNA

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Negative Control, Quantitative RT-PCR, Knockdown, IF-P, ChIP-qPCR, Control, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive 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 , 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: Oncology Reports

Article Title: Enrichment and mutation detection of circulating tumor cells from blood samples

doi: 10.3892/or.2018.6342

Figure Lengend Snippet: Nested PCR amplification of the TP53 gene from PC3 cells and the androgen receptor gene from LNCaP cells. (A) Electrophoresis gel imaging of nested PCR amplicons from 0, 28, 280, 400, 800 and 1,200 PC3 cells. (B) Electrophoresis gel imaging of nested PCR amplicons from 0, 5, 10, 20, 25, 100 and 200 LNCaP cells.

Article Snippet: Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

Techniques: Nested PCR, Amplification, Electrophoresis, Imaging

Journal: Oncology Reports

Article Title: Enrichment and mutation detection of circulating tumor cells from blood samples

doi: 10.3892/or.2018.6342

Figure Lengend Snippet: Sanger sequencing results of the nested PCR amplicons from PC3 and LNCaP cells. (A) Sequencing results of TP53 from control gDNA (upper panel) and PC3 cells (lower panel), revealing a deletion in TP53 from PC3 cells. (B) Sequencing results of AR from control gDNA (upper panel) and LNCaP cells (lower panel), revealing a missense mutation in AR from LNCaP cells.

Article Snippet: Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

Techniques: Sequencing, Nested PCR, Control, Mutagenesis

Journal: Oncology Reports

Article Title: Enrichment and mutation detection of circulating tumor cells from blood samples

doi: 10.3892/or.2018.6342

Figure Lengend Snippet: Efficiency of CTC retrieval using Celsee PREP100.

Article Snippet: Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

Techniques:

Journal: Oncology Reports

Article Title: Enrichment and mutation detection of circulating tumor cells from blood samples

doi: 10.3892/or.2018.6342

Figure Lengend Snippet: Efficiency of CTC enrichment from blood samples using Celsee PREP100.

Article Snippet: Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

Techniques:

Journal: Oncology Reports

Article Title: Enrichment and mutation detection of circulating tumor cells from blood samples

doi: 10.3892/or.2018.6342

Figure Lengend Snippet: PCR products of the TP53 gene from the enriched PC3 cells from blood and the androgen receptor gene from enriched LNCaP cells from blood. (A) Electrophoresis gel imaging of nested PCR amplicons of the enriched cells from blood samples with spiked-in 50, 200, 300, 1,000 PC3 cells. (B) Electrophoresis of nested PCR amplicons of the enriched cells from blood samples with spiked-in 25, 50, 100 and 250 LNCaP cells.

Article Snippet: Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

Techniques: Electrophoresis, Imaging, Nested PCR

Journal: Oncology Reports

Article Title: Enrichment and mutation detection of circulating tumor cells from blood samples

doi: 10.3892/or.2018.6342

Figure Lengend Snippet: Sanger sequencing results of PCR amplicons of the TP53 and androgen receptor genes of the enriched PC3 and LNCaP cells from blood. (A) The upper panel is the sequencing result from the control gDNA and the lower panel is the representative sequencing result of nested PCR amplicons of the enriched PC3 cells. (B) The representative Sanger sequencing result of nested PCR amplicons of the enriched LNCaP cells. Arrows indicate the mutations.

Article Snippet: Furthermore, the enriched PC3 and LNCaP cells accounted for ~40 and 25% of the total retrieved cells and the ratio of captured CTCs to the background cells reached 1:1.5 for PC3 and 1:2.9 for LNCaP cells, suggesting that the removal of blood cells by the Celsee PREP100 was nearly complete and the level of remaining leukocytes in the enriched sample was very low.

Techniques: Sequencing, Control, Nested PCR

Journal: Contrast Media & Molecular Imaging

Article Title: High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26

doi: 10.1155/2017/6873684

Figure Lengend Snippet: (a) In vitro specificity test of 57 Co-NOTA-PEG 2 -RM26 binding to GRPR on PC-3 (human prostate cancer) cells. Presaturation of receptors with unlabeled NOTA-PEG 2 -RM26 caused significant ( p < 0.05) reduction of cell-bound 57 Co-NOTA-PEG 2 -RM26 radioactivity. (b) Binding and internalization of 57 Co-NOTA-PEG 2 -RM26 at 37°C by PC-3 cells. Data are normalized to a maximum cell-bound radioactivity and presented as average value from 3 cell dishes ± SD. Not all error bars are visible due to the small standard deviations.

Article Snippet: PC-3 human prostate cancer cells expressing GRPR were purchased form ATCC via LGC Promochem.

Techniques: In Vitro, Binding Assay, Radioactivity

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. Prostate cancer cell lines were lysed and subjected to SDS-PAGE, followed by immunoblotting with antibodies against PG and GAPDH. PG levels were determined by normalizing to GAPDH levels for each cell line using Image J. The immunoblot shows that the expression of PG in prostate cancer cell lines inversely correlates with the degree of aggressiveness of the cell line. B. Prostate cancer cell lines were plated onto coverslips and allowed to attach and spread. Immunofluorescence was performed using antibodies against PG to demonstrate the localization of PG in ARCaP E , ARCaP M , LNCaP, C4-2B, DU145, PC-3, and PC3-M cell lines. The immunofluorescence shows weaker staining and a loss of cell-cell border localization of PG in the more aggressive cell lines (ARCaP M , C4-2B, and PC3-M). Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG protein levels for the blot shown. All the PG protein levels were normalized to ARCaP E levels within each blot. The average level and standard deviation of PG protein levels in panel A is as follows: ARCaP E 1.0, ARCaP M 0.3+/−0.2, LNCaP 1.5+/−0.5, C4-2B 0.5+/−0.2, DU145 3.4+/−1.2, PC-3 1.0+/−0.4, PC3-M 0.5+/−0.2.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: SDS Page, Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. Representative image of a dispase assay in PC3-M cells after transduction with PG OE adenovirus or control GFP adenovirus. B. Quantitations of dispase assays performed in triplicate; LNCaP, C4-2B, or PC3-M cells were plated in 6-well plates, and allowed to attach and spread. Cultures were transduced with GFP-containing adenovirus (GFP) or PG-containing adenovirus (PG OE), and after 24 hours the dispase assay was performed. Overexpression of PG in all 3 cell lines strengthens cell-cell adhesion. C. LNCaP cells were plated in 6-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool. The dispase assay was performed 96 hours after transfection. Suppression of PG expression results in a weakening of cell-cell adhesion in LNCaP cells. D. Representative image of a hanging drop assay in DU145 cells after transfection with control or PG siRNA pool. E–F. Quantitation of hanging drop assays performed in triplicate; C4-2B and PC3-M cells were transduced with GFP- or PG-containing adenoviruses (E), ARCaP E, LNCaP, DU145 and PC3 cells were transfected with control siRNA or PG siRNA pool (F). Aggregation assays were done 24 h (E), or 96 h (F) after the treatment. Graphs represent averages +/− SEM. *P<0.06; **P<0.005; ***P<0.0007, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Control, Over Expression, Transfection, Expressing, Quantitation Assay

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. PC3-M and ARCaP M were plated in 24-well plates and allowed to attach and spread. The cells were then transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours a scratch wound was made. Images were taken at 0 and 24 hours and the % wound closure was calculated by comparing the size of the wound at 24 hours to the 0 hour time point for each condition. Overexpression of PG suppresses motility in these two cell lines. B. ARCaP E and C4-2B cells were plated in 24-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, the scratch wounds were made. Suppression of PG leads to an increase in motility in ARCaP E and C4-2B cells. Scratch wound assay done in triplicate in prostate cancer cell lines after overexpression or knockdown of PG. C. ARCaP M , C4-2B, DU145 and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours plated onto Matrigel coated transwell membranes for an invasion assay. Overexpression of PG suppresses invasion in these four cell lines. D. ARCaPE, LNCaP, C4-2B, DU145, PC-3 and PC3-M cells were transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, plated onto Matrigel coated transwell membranes for an invasion assay. Suppression of PG leads to an increase in invasion in these six cell lines. Invasion assays were done in triplicate. Average of the total number of invading cells is presented. Graphs represent averages +/− SEM. *P<0.1; **P<0.05; ***P<0.001, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Over Expression, Transfection, Control, Scratch Wound Assay Assay, Knockdown, Invasion Assay

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A–B. PC3-M, C4-2B (A), and LNCaP cells (B) were plated in 6 well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA (a pool of 4 sequences). PC3-M, C4-2B, and LNCaP cells were treated with medium containing PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the dispase assay. Inhibition of Src strengthens cell-cell adhesion in PCa cells in general and is able to rescue cell-cell adhesion in PG-deficient cells. C. PC3-M and ARCaP M cells were plated in 24-well plates and allowed to attach and spread. The cells were treated with medium containing the selective Src- family kinase inhibitor PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the scratch wound assay. D. ARCaP E cells were plated in 24-well plates and allowed to attach and spread. The cells were transduced with GFP-containing adenovirus or caSrc-containing adenovirus, and after 24 hours a scratch wound was made. Activity of Src is directly correlated with motility of PCa cells. E. ARCaP M , PC3-M, and LNCaP cells were plated in 6-well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA pool and after 96 hours, the cells were lysed. ARCAP M and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours the cells were lysed. The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against PG, pSrc, Src, and GAPDH. Levels of pSrc were determined by normalizing to Src levels for each cell line using Image J. Phosphorylation (activation) of Src is inversely correlated with PG levels. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized pSrc/Src ratio for the blot shown. The average ratio and standard deviation of pSrc/Src normalized for loading by GAPDH is as follows: ARCaP M 0.6+/−0.2, LNCaP 1.7+/−0.2, PC3M 0.7+/−0.1. Graphs represent averages +/− SEM. *P<0.04; **P<003; ****P<0.0001, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transfection, Control, Solvent, Inhibition, Scratch Wound Assay Assay, Transduction, Activity Assay, SDS Page, Western Blot, Phospho-proteomics, Activation Assay, Standard Deviation

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A–E. ARCaP E , ARCaP M , LNCaP, C4-2B, and PC3-M cells were plated in 6-well plates and allowed to attach and spread. The cells were either transduced with GFP- or PG-containing adenovirus and were lysed after 24 hours (PC3-M, C4-2B, ARCaP M ); or the cells were transfected with control siRNA or PG siRNA pool and after 96 hours lysed (ARCaP E , LNCaP, C4-2B). The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against VN (A), FN (B), LN (C), Col. I (D), Col IV (E) and GAPDH. Levels of ECM proteins were determined by normalizing to GAPDH levels for each cell line using Image J. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG siRNA/Con siRNA or PG OE/GFP ratio for the blot shown. The average ratio and standard deviation of GAPDH normalized PG siRNA/Con siRNA and PG OE/GFP is as follows: (A) ARCaP E 3.1+/−1.3, LNCaP 2.0+/−0.9, PC3-M 0.3+/−0.1; (B) LNCaP 0.7+/−0.05, C4-2B KD 0.8+/−0.1, C4-2B OE 1.2+/−0.2, PC3-M 1.5+/−0.05; (C) ARCaP M 0.6+/−0.1, PC3-M 0.4+/−0.3; (D) 0.3+/−0.1; (E) 0.4+/−0.2.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Transfection, Control, SDS Page, Western Blot, Standard Deviation