rwpe 1  (ATCC)


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    Structured Review

    ATCC rwpe 1
    Western blotting of mAbs 2-7A50 and 2-5C11 against cell extracts from tumoral and non-tumoral cell lines. MW: molecular weight marker; RW: <t>RWPE-1</t> cell line extract; PC-3: PC-3 cell line extract; β-A RW: β-actin antibody against RWPE-1 cell line extract; β-A PC-3: β-actin antibody against PC-3 cell line extract. Both mAbs recognized bands present only at the PC-3 cell line. No reactivity is shown against the RWPE-1 cell line. mAb 2-7A50 specifically detected a 45kDa band while mAb 2-5C11 specifically detected a 62 kDa band. Detection was performed using chemiluminescent method.
    Rwpe 1, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rwpe 1/product/ATCC
    Average 97 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    rwpe 1 - by Bioz Stars, 2022-10
    97/100 stars

    Images

    1) Product Images from "Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity"

    Article Title: Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.760817

    Western blotting of mAbs 2-7A50 and 2-5C11 against cell extracts from tumoral and non-tumoral cell lines. MW: molecular weight marker; RW: RWPE-1 cell line extract; PC-3: PC-3 cell line extract; β-A RW: β-actin antibody against RWPE-1 cell line extract; β-A PC-3: β-actin antibody against PC-3 cell line extract. Both mAbs recognized bands present only at the PC-3 cell line. No reactivity is shown against the RWPE-1 cell line. mAb 2-7A50 specifically detected a 45kDa band while mAb 2-5C11 specifically detected a 62 kDa band. Detection was performed using chemiluminescent method.
    Figure Legend Snippet: Western blotting of mAbs 2-7A50 and 2-5C11 against cell extracts from tumoral and non-tumoral cell lines. MW: molecular weight marker; RW: RWPE-1 cell line extract; PC-3: PC-3 cell line extract; β-A RW: β-actin antibody against RWPE-1 cell line extract; β-A PC-3: β-actin antibody against PC-3 cell line extract. Both mAbs recognized bands present only at the PC-3 cell line. No reactivity is shown against the RWPE-1 cell line. mAb 2-7A50 specifically detected a 45kDa band while mAb 2-5C11 specifically detected a 62 kDa band. Detection was performed using chemiluminescent method.

    Techniques Used: Western Blot, Molecular Weight, Marker

    2) Product Images from "Hydroxytyrosol Induces Apoptosis, Cell Cycle Arrest and Suppresses Multiple Oncogenic Signaling Pathways in Prostate Cancer Cells"

    Article Title: Hydroxytyrosol Induces Apoptosis, Cell Cycle Arrest and Suppresses Multiple Oncogenic Signaling Pathways in Prostate Cancer Cells

    Journal: Nutrition and cancer

    doi: 10.1080/01635581.2017.1339818

    Hydroxytyrosol (HT) specifically inhibits the growth of prostate cancer cells. (A) Chemical structure of HT/3,4-dihydroxyphenylethanol (HT). (B) Normal prostate epithelium cells, RWPE1 and RWPE2, and prostate cancer cells, C4–2 and LNCaP, grown in 96-well plates, were treated with increasing dose of HT. Percent viability of these cells was measured using the WST-1 assay after 48 or 72 h of HT treatment. (C) Dose dependent HT treatment of LNCaP and C4–2 cell lines significantly altered cell morphology and the cells became round and shrunken in a dose-dependent manner, as examined under phase-contrast microscope. Representative images are shown.
    Figure Legend Snippet: Hydroxytyrosol (HT) specifically inhibits the growth of prostate cancer cells. (A) Chemical structure of HT/3,4-dihydroxyphenylethanol (HT). (B) Normal prostate epithelium cells, RWPE1 and RWPE2, and prostate cancer cells, C4–2 and LNCaP, grown in 96-well plates, were treated with increasing dose of HT. Percent viability of these cells was measured using the WST-1 assay after 48 or 72 h of HT treatment. (C) Dose dependent HT treatment of LNCaP and C4–2 cell lines significantly altered cell morphology and the cells became round and shrunken in a dose-dependent manner, as examined under phase-contrast microscope. Representative images are shown.

    Techniques Used: WST-1 Assay, Microscopy

    3) Product Images from ""

    Article Title:

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M111.014845

    Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different androgen independent and androgen sensitive prostate cancer cell lines including PC3, DU145, VCaP, LNCaP and C4–2 versus benign epithelial prostate cell line RWPE-1. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–100 nm size were identified as being exosomes.
    Figure Legend Snippet: Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different androgen independent and androgen sensitive prostate cancer cell lines including PC3, DU145, VCaP, LNCaP and C4–2 versus benign epithelial prostate cell line RWPE-1. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–100 nm size were identified as being exosomes.

    Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Derivative Assay, Staining

    Exosome lipidomic data. The lipid content of four major lipid classes was measured in PC3, DU145 and VCaP, LNCaP, C4–2 and RWPE-1 cells and compared with their derived exosomes, using LC-MS. The bar diagrams are representative of A , glycerolipid, B , glycerophospholipid, C , sphingolipid, D , glycosphingolipid in cell lysates and exosomes. Relative amounts of each lipid group were calculated as (sum of all AUC's for a particular lipid group) ÷ (sum of AUC's for all lipid groups) ×100%. * denotes a significant difference ( p
    Figure Legend Snippet: Exosome lipidomic data. The lipid content of four major lipid classes was measured in PC3, DU145 and VCaP, LNCaP, C4–2 and RWPE-1 cells and compared with their derived exosomes, using LC-MS. The bar diagrams are representative of A , glycerolipid, B , glycerophospholipid, C , sphingolipid, D , glycosphingolipid in cell lysates and exosomes. Relative amounts of each lipid group were calculated as (sum of all AUC's for a particular lipid group) ÷ (sum of AUC's for all lipid groups) ×100%. * denotes a significant difference ( p

    Techniques Used: Derivative Assay, Liquid Chromatography with Mass Spectroscopy

    Cholesterol concentration. The bar diagrams show the cholesterol concentration of A , lysates of PC3, DU145 and VCaP, LNCaP, C4–2, and RWPE-1 cells B , exosomes derived from the six different prostate cell lines. Cholesterol results were normalized to protein concentration of each sample and expressed as μg Cholesterol/μg Protein. * indicate significantly difference ( p
    Figure Legend Snippet: Cholesterol concentration. The bar diagrams show the cholesterol concentration of A , lysates of PC3, DU145 and VCaP, LNCaP, C4–2, and RWPE-1 cells B , exosomes derived from the six different prostate cell lines. Cholesterol results were normalized to protein concentration of each sample and expressed as μg Cholesterol/μg Protein. * indicate significantly difference ( p

    Techniques Used: Concentration Assay, Derivative Assay, Protein Concentration

    4) Product Images from "DLC1 suppresses NF-?B activity in prostate cancer cells due to its stabilizing effect on adherens junctions"

    Article Title: DLC1 suppresses NF-?B activity in prostate cancer cells due to its stabilizing effect on adherens junctions

    Journal: SpringerPlus

    doi: 10.1186/2193-1801-3-27

    DLC1 suppresses NF-κB activation. (A) Relative luciferase activity of NF-κB reporter in RWPE-1, C4-2-B2 and PC-3 cells, (B) Western blot showing the level of DLC1, pNF-κB (p65), NF-κB (p65), RhoAGTP, and total RhoA in RWPE-1, C4-2-B2 and PC-3 cells and level of DLC1 in RWPE-1 and Ad-DLC1 transduced (50MOI) C4-2-B2 and PC-3cells. (C) Western blot showing level of phosphorylated p65 subunit of NF-κB in the Ad-Lac Z and Ad-DLC1 transduced C4-2-B2 and PC-3 cells. (D) Relative luciferase activity of NF-κB reporter in AD-LacZ and DLC1 transduced C4-2-B2 and PC-3 cells. All values are means ± SD of three independent counts. (E) Western blot showing the level of phosphorylated p65 subunit of NF-κB in DLC1- and α-catenin-knock down RWPE-1 cells.
    Figure Legend Snippet: DLC1 suppresses NF-κB activation. (A) Relative luciferase activity of NF-κB reporter in RWPE-1, C4-2-B2 and PC-3 cells, (B) Western blot showing the level of DLC1, pNF-κB (p65), NF-κB (p65), RhoAGTP, and total RhoA in RWPE-1, C4-2-B2 and PC-3 cells and level of DLC1 in RWPE-1 and Ad-DLC1 transduced (50MOI) C4-2-B2 and PC-3cells. (C) Western blot showing level of phosphorylated p65 subunit of NF-κB in the Ad-Lac Z and Ad-DLC1 transduced C4-2-B2 and PC-3 cells. (D) Relative luciferase activity of NF-κB reporter in AD-LacZ and DLC1 transduced C4-2-B2 and PC-3 cells. All values are means ± SD of three independent counts. (E) Western blot showing the level of phosphorylated p65 subunit of NF-κB in DLC1- and α-catenin-knock down RWPE-1 cells.

    Techniques Used: Activation Assay, Luciferase, Activity Assay, Western Blot

    5) Product Images from "The long non-coding RNA GHSROS reprograms prostate cancer cell lines toward a more aggressive phenotype"

    Article Title: The long non-coding RNA GHSROS reprograms prostate cancer cell lines toward a more aggressive phenotype

    Journal: bioRxiv

    doi: 10.1101/682203

    Overview of the lncRNA GHSROS and its expression in cancer. (a) Overview of the GHSR and GHSROS gene loci. GHSR exons (black), GHSROS exon (red), repetitive elements (orange), introns (lines). (b) GHSROS expression in 19 cancers (TissueScan Cancer Survey Tissue qPCR panel). N (black) denotes normal tissue; T tumor (red). For each cancer, data are expressed as mean fold change using the comparative 2 -ΔΔCt method against a non-malignant control tissue. Normalized to β-actin ( ACTB ). (c) Relative gene expression of GHSROS in OriGene cDNA panels of tissues from normal prostate ( n =24; blue), primary prostate cancer ( n =88; red), and other prostatic diseases ( n =31; orange). Determined by qRT-PCR, normalized to ribosomal protein L32 ( RPL32 ), and represented as standardized expression values ( Z -scores). (d) GHSROS expression in an Andalusian Biobank prostate tissue cohort. Absolute expression levels were determined by qRT-PCR and adjusted by a normalization factor calculated from the expression levels of three housekeeping genes ( HPRT , ACTB , and GAPDH ). NP denotes non-malignant prostate. * P ≤0.05, Mann-Whitney-Wilcoxon test. (e) Expression of GHSROS in immortalized, cultured cell lines and patient-derived xenograft (PDX) lines. Mean ± s.e.m. ( n =3). * P ≤0.05, ** P ≤0.01, *** P ≤0.001, Student’s t -test. Normalized as in (b) to the RWPE-1 non-malignant cell line. Androgen-independent lines are labeled in orange.
    Figure Legend Snippet: Overview of the lncRNA GHSROS and its expression in cancer. (a) Overview of the GHSR and GHSROS gene loci. GHSR exons (black), GHSROS exon (red), repetitive elements (orange), introns (lines). (b) GHSROS expression in 19 cancers (TissueScan Cancer Survey Tissue qPCR panel). N (black) denotes normal tissue; T tumor (red). For each cancer, data are expressed as mean fold change using the comparative 2 -ΔΔCt method against a non-malignant control tissue. Normalized to β-actin ( ACTB ). (c) Relative gene expression of GHSROS in OriGene cDNA panels of tissues from normal prostate ( n =24; blue), primary prostate cancer ( n =88; red), and other prostatic diseases ( n =31; orange). Determined by qRT-PCR, normalized to ribosomal protein L32 ( RPL32 ), and represented as standardized expression values ( Z -scores). (d) GHSROS expression in an Andalusian Biobank prostate tissue cohort. Absolute expression levels were determined by qRT-PCR and adjusted by a normalization factor calculated from the expression levels of three housekeeping genes ( HPRT , ACTB , and GAPDH ). NP denotes non-malignant prostate. * P ≤0.05, Mann-Whitney-Wilcoxon test. (e) Expression of GHSROS in immortalized, cultured cell lines and patient-derived xenograft (PDX) lines. Mean ± s.e.m. ( n =3). * P ≤0.05, ** P ≤0.01, *** P ≤0.001, Student’s t -test. Normalized as in (b) to the RWPE-1 non-malignant cell line. Androgen-independent lines are labeled in orange.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY, Cell Culture, Derivative Assay, Labeling

    6) Product Images from "Inhibition of Polo-like Kinase 1 (Plk1) Enhances the Antineoplastic Activity of Metformin in Prostate Cancer *"

    Article Title: Inhibition of Polo-like Kinase 1 (Plk1) Enhances the Antineoplastic Activity of Metformin in Prostate Cancer *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.596817

    Plk1 status affects the cellular response to metformin. A , RWPE-1 cells were infected with adenovirus expressing GFP or GFP-Plk1 for 2 days and harvested for immunoblotting with the indicated antibodies. CTRL , control. B , RWPE-1 cells expressing GFP or
    Figure Legend Snippet: Plk1 status affects the cellular response to metformin. A , RWPE-1 cells were infected with adenovirus expressing GFP or GFP-Plk1 for 2 days and harvested for immunoblotting with the indicated antibodies. CTRL , control. B , RWPE-1 cells expressing GFP or

    Techniques Used: Infection, Expressing

    7) Product Images from "Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC"

    Article Title: Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78774-5

    Characterization of OLFM4 -GFP reporter RWPE1 cells in 3D Matrigel culture. Prostate sphere-formation and organoids assays were performed with OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells grown in Matrigel. ( a ) Representative GFP-field sphere images using single-cell tracing over time (scale bar: 20 µm). ( b ) Representative GFP-field images of sphere, colony and branch formation after 10 days in Matrigel culture. Scale bar: 100 µm. Bar graph presents mean number (± standard deviation; SD, n = 6) of spheres ( > 50 µm in diameter), colonies, or branches formed after 10 days in culture. *** p
    Figure Legend Snippet: Characterization of OLFM4 -GFP reporter RWPE1 cells in 3D Matrigel culture. Prostate sphere-formation and organoids assays were performed with OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells grown in Matrigel. ( a ) Representative GFP-field sphere images using single-cell tracing over time (scale bar: 20 µm). ( b ) Representative GFP-field images of sphere, colony and branch formation after 10 days in Matrigel culture. Scale bar: 100 µm. Bar graph presents mean number (± standard deviation; SD, n = 6) of spheres ( > 50 µm in diameter), colonies, or branches formed after 10 days in culture. *** p

    Techniques Used: Knock-Out, Standard Deviation

    Identification of OLFM4 -expressing RWPE1 cells. ( a ) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. ( b ) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. ( c ) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.
    Figure Legend Snippet: Identification of OLFM4 -expressing RWPE1 cells. ( a ) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. ( b ) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. ( c ) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.

    Techniques Used: Expressing, RNA Sequencing Assay, Marker, Staining

    Reactomes and pathways enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Enrichment of reactomes in OLFM4 -knockout GFP reporter RWPE1 cells. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways from Wikipathways. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_KEGG. ( d ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_Panther.
    Figure Legend Snippet: Reactomes and pathways enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Enrichment of reactomes in OLFM4 -knockout GFP reporter RWPE1 cells. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways from Wikipathways. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_KEGG. ( d ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_Panther.

    Techniques Used: Knock-Out

    Characterization of OLFM4 -knockout GFP reporter RWPE1 cells in 2D culture. ( a ) Representative FACS analysis of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells using antibodies to GFP combined with antibodies to stem/progenitor-cell markers CD49F, CD44, CD26, or CD24. Inset values indicate the percentage of double-marker—positive cell populations. ( b ) Representative GFP- and light-field images generated using single-cell tracing of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells at 4 and 7 days in 2D culture. Scale bar: 10 µm. ( c ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 1, 2, 4, and 7 days in 2D culture. Scale bar: 10 µm. ( d ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 4 days in 2D culture. Scale bar: 10 µm. ( e ) Illustration of OLFM4 -expressing GFP-positive RWPE1 stem/progenitor-like cell symmetric and asymmetric division. OLFM4-KO, knockout; OLFM4-W, wild.
    Figure Legend Snippet: Characterization of OLFM4 -knockout GFP reporter RWPE1 cells in 2D culture. ( a ) Representative FACS analysis of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells using antibodies to GFP combined with antibodies to stem/progenitor-cell markers CD49F, CD44, CD26, or CD24. Inset values indicate the percentage of double-marker—positive cell populations. ( b ) Representative GFP- and light-field images generated using single-cell tracing of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells at 4 and 7 days in 2D culture. Scale bar: 10 µm. ( c ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 1, 2, 4, and 7 days in 2D culture. Scale bar: 10 µm. ( d ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 4 days in 2D culture. Scale bar: 10 µm. ( e ) Illustration of OLFM4 -expressing GFP-positive RWPE1 stem/progenitor-like cell symmetric and asymmetric division. OLFM4-KO, knockout; OLFM4-W, wild.

    Techniques Used: Knock-Out, FACS, Marker, Generated, Expressing

    GSEA analysis for OLFM4 -knockout GFP reporter RWPE1 cells from bulk-cell RNA sequencing data. ( a ) Strategy of bulk-cell RNA sequencing for FACS cell sorting by GFP-marker expression of OLFM4 -GFP reporter RWPE1 cells. ( b ) GSEA showing enrichment of stem-cell-like gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( c ) GSEA showing enrichment of WNT -signaling and APC / MYC -signaling target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( d ) GSEA showing enrichment of MYC target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells from three different data resources. ( e ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days in 6-well plates, then stained with the Diff-Quik Stain Set. ( f ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM, 5 µM, or 10 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days. Scale bar: 100 µm. ( g ) Representative GFP- and light-field images of spheres of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 10 days in 3D culture. Scale bar: 100 µm. Bar graph presents mean number of spheres (± SD, n = 6) from these experiments using 100 nM of (+)-JQ1 (or DMSO, vehicle control). W, wild; KO, knockout. *** p
    Figure Legend Snippet: GSEA analysis for OLFM4 -knockout GFP reporter RWPE1 cells from bulk-cell RNA sequencing data. ( a ) Strategy of bulk-cell RNA sequencing for FACS cell sorting by GFP-marker expression of OLFM4 -GFP reporter RWPE1 cells. ( b ) GSEA showing enrichment of stem-cell-like gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( c ) GSEA showing enrichment of WNT -signaling and APC / MYC -signaling target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( d ) GSEA showing enrichment of MYC target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells from three different data resources. ( e ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days in 6-well plates, then stained with the Diff-Quik Stain Set. ( f ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM, 5 µM, or 10 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days. Scale bar: 100 µm. ( g ) Representative GFP- and light-field images of spheres of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 10 days in 3D culture. Scale bar: 100 µm. Bar graph presents mean number of spheres (± SD, n = 6) from these experiments using 100 nM of (+)-JQ1 (or DMSO, vehicle control). W, wild; KO, knockout. *** p

    Techniques Used: Knock-Out, RNA Sequencing Assay, FACS, Marker, Expressing, Cell Culture, Staining, Diff-Quik

    Gene ontology enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for biological processes using WebGestalt 2019. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for cellular components. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways for molecular functions.
    Figure Legend Snippet: Gene ontology enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for biological processes using WebGestalt 2019. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for cellular components. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways for molecular functions.

    Techniques Used: Knock-Out

    8) Product Images from "Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC"

    Article Title: Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78774-5

    Characterization of OLFM4 -GFP reporter RWPE1 cells in 3D Matrigel culture. Prostate sphere-formation and organoids assays were performed with OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells grown in Matrigel. ( a ) Representative GFP-field sphere images using single-cell tracing over time (scale bar: 20 µm). ( b ) Representative GFP-field images of sphere, colony and branch formation after 10 days in Matrigel culture. Scale bar: 100 µm. Bar graph presents mean number (± standard deviation; SD, n = 6) of spheres ( > 50 µm in diameter), colonies, or branches formed after 10 days in culture. *** p
    Figure Legend Snippet: Characterization of OLFM4 -GFP reporter RWPE1 cells in 3D Matrigel culture. Prostate sphere-formation and organoids assays were performed with OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells grown in Matrigel. ( a ) Representative GFP-field sphere images using single-cell tracing over time (scale bar: 20 µm). ( b ) Representative GFP-field images of sphere, colony and branch formation after 10 days in Matrigel culture. Scale bar: 100 µm. Bar graph presents mean number (± standard deviation; SD, n = 6) of spheres ( > 50 µm in diameter), colonies, or branches formed after 10 days in culture. *** p

    Techniques Used: Knock-Out, Standard Deviation

    Identification of OLFM4 -expressing RWPE1 cells. ( a ) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. ( b ) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. ( c ) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.
    Figure Legend Snippet: Identification of OLFM4 -expressing RWPE1 cells. ( a ) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. ( b ) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. ( c ) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.

    Techniques Used: Expressing, RNA Sequencing Assay, Marker, Staining

    Reactomes and pathways enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Enrichment of reactomes in OLFM4 -knockout GFP reporter RWPE1 cells. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways from Wikipathways. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_KEGG. ( d ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_Panther.
    Figure Legend Snippet: Reactomes and pathways enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Enrichment of reactomes in OLFM4 -knockout GFP reporter RWPE1 cells. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways from Wikipathways. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_KEGG. ( d ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_Panther.

    Techniques Used: Knock-Out

    Characterization of OLFM4 -knockout GFP reporter RWPE1 cells in 2D culture. ( a ) Representative FACS analysis of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells using antibodies to GFP combined with antibodies to stem/progenitor-cell markers CD49F, CD44, CD26, or CD24. Inset values indicate the percentage of double-marker—positive cell populations. ( b ) Representative GFP- and light-field images generated using single-cell tracing of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells at 4 and 7 days in 2D culture. Scale bar: 10 µm. ( c ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 1, 2, 4, and 7 days in 2D culture. Scale bar: 10 µm. ( d ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 4 days in 2D culture. Scale bar: 10 µm. ( e ) Illustration of OLFM4 -expressing GFP-positive RWPE1 stem/progenitor-like cell symmetric and asymmetric division. OLFM4-KO, knockout; OLFM4-W, wild.
    Figure Legend Snippet: Characterization of OLFM4 -knockout GFP reporter RWPE1 cells in 2D culture. ( a ) Representative FACS analysis of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells using antibodies to GFP combined with antibodies to stem/progenitor-cell markers CD49F, CD44, CD26, or CD24. Inset values indicate the percentage of double-marker—positive cell populations. ( b ) Representative GFP- and light-field images generated using single-cell tracing of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells at 4 and 7 days in 2D culture. Scale bar: 10 µm. ( c ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 1, 2, 4, and 7 days in 2D culture. Scale bar: 10 µm. ( d ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 4 days in 2D culture. Scale bar: 10 µm. ( e ) Illustration of OLFM4 -expressing GFP-positive RWPE1 stem/progenitor-like cell symmetric and asymmetric division. OLFM4-KO, knockout; OLFM4-W, wild.

    Techniques Used: Knock-Out, FACS, Marker, Generated, Expressing

    GSEA analysis for OLFM4 -knockout GFP reporter RWPE1 cells from bulk-cell RNA sequencing data. ( a ) Strategy of bulk-cell RNA sequencing for FACS cell sorting by GFP-marker expression of OLFM4 -GFP reporter RWPE1 cells. ( b ) GSEA showing enrichment of stem-cell-like gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( c ) GSEA showing enrichment of WNT -signaling and APC / MYC -signaling target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( d ) GSEA showing enrichment of MYC target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells from three different data resources. ( e ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days in 6-well plates, then stained with the Diff-Quik Stain Set. ( f ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM, 5 µM, or 10 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days. Scale bar: 100 µm. ( g ) Representative GFP- and light-field images of spheres of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 10 days in 3D culture. Scale bar: 100 µm. Bar graph presents mean number of spheres (± SD, n = 6) from these experiments using 100 nM of (+)-JQ1 (or DMSO, vehicle control). W, wild; KO, knockout. *** p
    Figure Legend Snippet: GSEA analysis for OLFM4 -knockout GFP reporter RWPE1 cells from bulk-cell RNA sequencing data. ( a ) Strategy of bulk-cell RNA sequencing for FACS cell sorting by GFP-marker expression of OLFM4 -GFP reporter RWPE1 cells. ( b ) GSEA showing enrichment of stem-cell-like gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( c ) GSEA showing enrichment of WNT -signaling and APC / MYC -signaling target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( d ) GSEA showing enrichment of MYC target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells from three different data resources. ( e ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days in 6-well plates, then stained with the Diff-Quik Stain Set. ( f ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM, 5 µM, or 10 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days. Scale bar: 100 µm. ( g ) Representative GFP- and light-field images of spheres of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 10 days in 3D culture. Scale bar: 100 µm. Bar graph presents mean number of spheres (± SD, n = 6) from these experiments using 100 nM of (+)-JQ1 (or DMSO, vehicle control). W, wild; KO, knockout. *** p

    Techniques Used: Knock-Out, RNA Sequencing Assay, FACS, Marker, Expressing, Cell Culture, Staining, Diff-Quik

    Gene ontology enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for biological processes using WebGestalt 2019. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for cellular components. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways for molecular functions.
    Figure Legend Snippet: Gene ontology enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for biological processes using WebGestalt 2019. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for cellular components. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways for molecular functions.

    Techniques Used: Knock-Out

    9) Product Images from "Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC"

    Article Title: Olfactomedin 4 mediation of prostate stem/progenitor-like cell proliferation and differentiation via MYC

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-78774-5

    Characterization of OLFM4 -GFP reporter RWPE1 cells in 3D Matrigel culture. Prostate sphere-formation and organoids assays were performed with OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells grown in Matrigel. ( a ) Representative GFP-field sphere images using single-cell tracing over time (scale bar: 20 µm). ( b ) Representative GFP-field images of sphere, colony and branch formation after 10 days in Matrigel culture. Scale bar: 100 µm. Bar graph presents mean number (± standard deviation; SD, n = 6) of spheres ( > 50 µm in diameter), colonies, or branches formed after 10 days in culture. *** p
    Figure Legend Snippet: Characterization of OLFM4 -GFP reporter RWPE1 cells in 3D Matrigel culture. Prostate sphere-formation and organoids assays were performed with OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells grown in Matrigel. ( a ) Representative GFP-field sphere images using single-cell tracing over time (scale bar: 20 µm). ( b ) Representative GFP-field images of sphere, colony and branch formation after 10 days in Matrigel culture. Scale bar: 100 µm. Bar graph presents mean number (± standard deviation; SD, n = 6) of spheres ( > 50 µm in diameter), colonies, or branches formed after 10 days in culture. *** p

    Techniques Used: Knock-Out, Standard Deviation

    Identification of OLFM4 -expressing RWPE1 cells. ( a ) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. ( b ) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. ( c ) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.
    Figure Legend Snippet: Identification of OLFM4 -expressing RWPE1 cells. ( a ) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. ( b ) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. ( c ) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.

    Techniques Used: Expressing, RNA Sequencing Assay, Marker, Staining

    Reactomes and pathways enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Enrichment of reactomes in OLFM4 -knockout GFP reporter RWPE1 cells. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways from Wikipathways. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_KEGG. ( d ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_Panther.
    Figure Legend Snippet: Reactomes and pathways enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Enrichment of reactomes in OLFM4 -knockout GFP reporter RWPE1 cells. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways from Wikipathways. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_KEGG. ( d ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways from Pathway_Panther.

    Techniques Used: Knock-Out

    Characterization of OLFM4 -knockout GFP reporter RWPE1 cells in 2D culture. ( a ) Representative FACS analysis of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells using antibodies to GFP combined with antibodies to stem/progenitor-cell markers CD49F, CD44, CD26, or CD24. Inset values indicate the percentage of double-marker—positive cell populations. ( b ) Representative GFP- and light-field images generated using single-cell tracing of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells at 4 and 7 days in 2D culture. Scale bar: 10 µm. ( c ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 1, 2, 4, and 7 days in 2D culture. Scale bar: 10 µm. ( d ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 4 days in 2D culture. Scale bar: 10 µm. ( e ) Illustration of OLFM4 -expressing GFP-positive RWPE1 stem/progenitor-like cell symmetric and asymmetric division. OLFM4-KO, knockout; OLFM4-W, wild.
    Figure Legend Snippet: Characterization of OLFM4 -knockout GFP reporter RWPE1 cells in 2D culture. ( a ) Representative FACS analysis of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells using antibodies to GFP combined with antibodies to stem/progenitor-cell markers CD49F, CD44, CD26, or CD24. Inset values indicate the percentage of double-marker—positive cell populations. ( b ) Representative GFP- and light-field images generated using single-cell tracing of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells at 4 and 7 days in 2D culture. Scale bar: 10 µm. ( c ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 1, 2, 4, and 7 days in 2D culture. Scale bar: 10 µm. ( d ) Representative GFP- and light-field images of OLFM4 -knockout GFP reporter RWPE1 cells at 4 days in 2D culture. Scale bar: 10 µm. ( e ) Illustration of OLFM4 -expressing GFP-positive RWPE1 stem/progenitor-like cell symmetric and asymmetric division. OLFM4-KO, knockout; OLFM4-W, wild.

    Techniques Used: Knock-Out, FACS, Marker, Generated, Expressing

    GSEA analysis for OLFM4 -knockout GFP reporter RWPE1 cells from bulk-cell RNA sequencing data. ( a ) Strategy of bulk-cell RNA sequencing for FACS cell sorting by GFP-marker expression of OLFM4 -GFP reporter RWPE1 cells. ( b ) GSEA showing enrichment of stem-cell-like gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( c ) GSEA showing enrichment of WNT -signaling and APC / MYC -signaling target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( d ) GSEA showing enrichment of MYC target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells from three different data resources. ( e ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days in 6-well plates, then stained with the Diff-Quik Stain Set. ( f ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM, 5 µM, or 10 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days. Scale bar: 100 µm. ( g ) Representative GFP- and light-field images of spheres of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 10 days in 3D culture. Scale bar: 100 µm. Bar graph presents mean number of spheres (± SD, n = 6) from these experiments using 100 nM of (+)-JQ1 (or DMSO, vehicle control). W, wild; KO, knockout. *** p
    Figure Legend Snippet: GSEA analysis for OLFM4 -knockout GFP reporter RWPE1 cells from bulk-cell RNA sequencing data. ( a ) Strategy of bulk-cell RNA sequencing for FACS cell sorting by GFP-marker expression of OLFM4 -GFP reporter RWPE1 cells. ( b ) GSEA showing enrichment of stem-cell-like gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( c ) GSEA showing enrichment of WNT -signaling and APC / MYC -signaling target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells. ( d ) GSEA showing enrichment of MYC target gene signatures in OLFM4 -knockout GFP reporter RWPE1 cells from three different data resources. ( e ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days in 6-well plates, then stained with the Diff-Quik Stain Set. ( f ) Representative images of colonies of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM, 5 µM, or 10 µM (+)-JQ1 (or DMSO, vehicle control) for 7 days. Scale bar: 100 µm. ( g ) Representative GFP- and light-field images of spheres of OLFM4 -wild and OLFM4 -knockout GFP reporter RWPE1 cells cultured in the presence of 1 µM (+)-JQ1 (or DMSO, vehicle control) for 10 days in 3D culture. Scale bar: 100 µm. Bar graph presents mean number of spheres (± SD, n = 6) from these experiments using 100 nM of (+)-JQ1 (or DMSO, vehicle control). W, wild; KO, knockout. *** p

    Techniques Used: Knock-Out, RNA Sequencing Assay, FACS, Marker, Expressing, Cell Culture, Staining, Diff-Quik

    Gene ontology enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for biological processes using WebGestalt 2019. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for cellular components. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways for molecular functions.
    Figure Legend Snippet: Gene ontology enrichments for OLFM4 -knockout GFP reporter RWPE1 cells. ( a ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for biological processes using WebGestalt 2019. ( b ) Bar graph presents upregulated (blue; FDR ≤ 0.05) and downregulated (orange; FDR ≤ 0.05) pathways for cellular components. ( c ) Bar graph presents upregulated (blue; FDR ≤ 0.05) pathways for molecular functions.

    Techniques Used: Knock-Out

    10) Product Images from "Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment"

    Article Title: Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7052

    Exosomes increase RWPE-1 cell migration RWPE-1 cells were incubated for 48 hours with exosomes (100 μg/ml) derived from ( A ) LNCaP, ( B ) DU145 cells or corresponding serum-free medium and loaded into the upper chamber of a transwell. Exosomes concentrations (100 μg/ml) or serum free-medium were maintained in upper and lower chambers. After 24 h incubation, the migration activity was quantified by counting the migrated cells on the lower surface of the membrane of at least five fields per chamber using a x10 objective. Representative photographs are shown in the left panel. Quantification of migrating cells is shown in the right panel. All values are representative of at least two independent experiments with similar results, and are displayed as mean ± SD, where *** P
    Figure Legend Snippet: Exosomes increase RWPE-1 cell migration RWPE-1 cells were incubated for 48 hours with exosomes (100 μg/ml) derived from ( A ) LNCaP, ( B ) DU145 cells or corresponding serum-free medium and loaded into the upper chamber of a transwell. Exosomes concentrations (100 μg/ml) or serum free-medium were maintained in upper and lower chambers. After 24 h incubation, the migration activity was quantified by counting the migrated cells on the lower surface of the membrane of at least five fields per chamber using a x10 objective. Representative photographs are shown in the left panel. Quantification of migrating cells is shown in the right panel. All values are representative of at least two independent experiments with similar results, and are displayed as mean ± SD, where *** P

    Techniques Used: Migration, Incubation, Derivative Assay, Activity Assay

    Western blot analysis (WB) Three different prostate cell lines, including ( A ) DU145 (androgen independent), ( B ) LNCaP androgen sensitive (prostate cancer cell line) and ( C ) RWPE-1 (benign epithelial prostate cell line) were treated with one dose of 100 μg/mL of DU145 derived exosomes for 5, 10, 15, 30 and 60 min, 6 and 24 hr as indicated. Western blot was used to analyse cell lysates with the indicated antibodies.
    Figure Legend Snippet: Western blot analysis (WB) Three different prostate cell lines, including ( A ) DU145 (androgen independent), ( B ) LNCaP androgen sensitive (prostate cancer cell line) and ( C ) RWPE-1 (benign epithelial prostate cell line) were treated with one dose of 100 μg/mL of DU145 derived exosomes for 5, 10, 15, 30 and 60 min, 6 and 24 hr as indicated. Western blot was used to analyse cell lysates with the indicated antibodies.

    Techniques Used: Western Blot, Derivative Assay

    Western blot analysis (WB) Five different prostate cell lines, including ( A ) PC3, ( B ) DU145 (androgen independent), ( C ) LNCaP, ( D ) C4–2 (androgen sensitive) and ( E ) RWPE-1 (benign epithelial prostate cell line) were treated with one dose of 100 μg/mL of LNCaP derived exosomes for 5, 10, 15, 30 and 60 min, 6 and 24 hr. as indicated. Western blot was used to analyse cell lysates with the indicated antibodies.
    Figure Legend Snippet: Western blot analysis (WB) Five different prostate cell lines, including ( A ) PC3, ( B ) DU145 (androgen independent), ( C ) LNCaP, ( D ) C4–2 (androgen sensitive) and ( E ) RWPE-1 (benign epithelial prostate cell line) were treated with one dose of 100 μg/mL of LNCaP derived exosomes for 5, 10, 15, 30 and 60 min, 6 and 24 hr. as indicated. Western blot was used to analyse cell lysates with the indicated antibodies.

    Techniques Used: Western Blot, Derivative Assay

    Confocal microscopy Confocal microscopy was used to visualize freshly isolated exosomes derived from a CLUGFP stably over-expressing LNCaP cell line, which contains CLUGFP, being taken up by ( A ) and ( D ) PC3 (AR-ve) and ( B ) and ( E ) LNCaP (AR +ve) PCa cell lines versus ( C ) and ( F ) benign epithelial prostate cell line RWPE-1, after overnight incubation. Both cell lines were further fixed and stained with DAPI and E-Cadherin/Caveolin-1 prior to imaging of the cells by confocal microscopy.
    Figure Legend Snippet: Confocal microscopy Confocal microscopy was used to visualize freshly isolated exosomes derived from a CLUGFP stably over-expressing LNCaP cell line, which contains CLUGFP, being taken up by ( A ) and ( D ) PC3 (AR-ve) and ( B ) and ( E ) LNCaP (AR +ve) PCa cell lines versus ( C ) and ( F ) benign epithelial prostate cell line RWPE-1, after overnight incubation. Both cell lines were further fixed and stained with DAPI and E-Cadherin/Caveolin-1 prior to imaging of the cells by confocal microscopy.

    Techniques Used: Confocal Microscopy, Isolation, Derivative Assay, Stable Transfection, Expressing, Incubation, Staining, Imaging

    Apoptosis assay Analysis of apoptosis in PCa Cell lines (LNCaP and DU145) and benign epithelial prostate cell (RWPE-1) after treatment with 100 μg/mL of exosome derived from LNCaP or DU145 cells after 0, 5, 10, 15, 30 min, 1, 6, 24 hours. ( A ) Both DU145 and LNCaP-derived exosomes significantly reduced the caspase 3/7 activity in LNCaP cell line in most of the time points. ( B ) Exosomes derived from DU145 or LNCaP cells did not significantly influence the caspase 3/7 activity in DU145 cells. ( C ) Treatment with DU145-derived exosomes led to a significant reduction of apoptosis in RWPE-1 cells whereas the RWPE-1 cells did not display significant decrease of apoptosis after treatment with exosomes derived from LNCaP cells. All values are representative of at least two independent experiments with similar results, and are presented as the percentage of caspase 3/7 activity, where non-treated cells were regarded as 100% ( P
    Figure Legend Snippet: Apoptosis assay Analysis of apoptosis in PCa Cell lines (LNCaP and DU145) and benign epithelial prostate cell (RWPE-1) after treatment with 100 μg/mL of exosome derived from LNCaP or DU145 cells after 0, 5, 10, 15, 30 min, 1, 6, 24 hours. ( A ) Both DU145 and LNCaP-derived exosomes significantly reduced the caspase 3/7 activity in LNCaP cell line in most of the time points. ( B ) Exosomes derived from DU145 or LNCaP cells did not significantly influence the caspase 3/7 activity in DU145 cells. ( C ) Treatment with DU145-derived exosomes led to a significant reduction of apoptosis in RWPE-1 cells whereas the RWPE-1 cells did not display significant decrease of apoptosis after treatment with exosomes derived from LNCaP cells. All values are representative of at least two independent experiments with similar results, and are presented as the percentage of caspase 3/7 activity, where non-treated cells were regarded as 100% ( P

    Techniques Used: Apoptosis Assay, Derivative Assay, Activity Assay

    Real time proliferation and migration cell analysis Cell growth of ( A ) LNCaP, ( B ) DU145 and ( C ) RWPE-1 cells were analysed using the xCELLigence system which relies on the generation of electrical impedance as cell growth by 16-well plates were used in the impedance based system, cells were seeded at specific densities (LNCaP, RWPE-1 20,000/well, and DU145 7,000/well) after 24 hours, cells were treated with different final concentrations of the LNCaP exoxomes. All slopes were compared with the control (black bar, and red lines in the graphs at * P
    Figure Legend Snippet: Real time proliferation and migration cell analysis Cell growth of ( A ) LNCaP, ( B ) DU145 and ( C ) RWPE-1 cells were analysed using the xCELLigence system which relies on the generation of electrical impedance as cell growth by 16-well plates were used in the impedance based system, cells were seeded at specific densities (LNCaP, RWPE-1 20,000/well, and DU145 7,000/well) after 24 hours, cells were treated with different final concentrations of the LNCaP exoxomes. All slopes were compared with the control (black bar, and red lines in the graphs at * P

    Techniques Used: Migration

    Exosomes increase RWPE-1 cell migration on collagen (I) RWPE-1 spheroids were incubated for 48 hours with exosomes (100 μg/ml) derived from (A) LNCaP, (B) DU145 cells or corresponding serum-free medium and transferred to microwells coated with type I collagen The cell migration was scored at t = 12 hours and t = 24 hours by measuring the migrated cell area and normalizing to the migration seen at t = 0. Representative photographs are shown in the upper panel. Quantification of migration area is shown in the lower panel. All values are representative of at least two independent experiments with similar results, and are displayed as mean ± SD, where *** P
    Figure Legend Snippet: Exosomes increase RWPE-1 cell migration on collagen (I) RWPE-1 spheroids were incubated for 48 hours with exosomes (100 μg/ml) derived from (A) LNCaP, (B) DU145 cells or corresponding serum-free medium and transferred to microwells coated with type I collagen The cell migration was scored at t = 12 hours and t = 24 hours by measuring the migrated cell area and normalizing to the migration seen at t = 0. Representative photographs are shown in the upper panel. Quantification of migration area is shown in the lower panel. All values are representative of at least two independent experiments with similar results, and are displayed as mean ± SD, where *** P

    Techniques Used: Migration, Incubation, Derivative Assay

    11) Product Images from "SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells"

    Article Title: SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0141-x

    Knockdown of SULF2 decreased viability and migration of prostate cells. RWPE-1 epithelial prostate cells and DU-145 and PC3 prostate cancer cells were transfected with siRNA (Life Technologies) targeting SULF2. To silence SULF2 gene, trials with siRNA preset by the manufacturer (Life Technologies, CA, USA) were performed as described in Methods. Three shRNAs were used to each gene, in addition to the positive (GAPDH) and negative (scramble sequence) controls. The gene silencing was confirmed by Real Time PCR 48 h after transfection (A) . MTT viability assay (B) and wound healing assay (C) were performed as described previously. (CTRL NEG: scramble siRNA sequence). *P ≤ 0.05.
    Figure Legend Snippet: Knockdown of SULF2 decreased viability and migration of prostate cells. RWPE-1 epithelial prostate cells and DU-145 and PC3 prostate cancer cells were transfected with siRNA (Life Technologies) targeting SULF2. To silence SULF2 gene, trials with siRNA preset by the manufacturer (Life Technologies, CA, USA) were performed as described in Methods. Three shRNAs were used to each gene, in addition to the positive (GAPDH) and negative (scramble sequence) controls. The gene silencing was confirmed by Real Time PCR 48 h after transfection (A) . MTT viability assay (B) and wound healing assay (C) were performed as described previously. (CTRL NEG: scramble siRNA sequence). *P ≤ 0.05.

    Techniques Used: Migration, Transfection, Sequencing, Real-time Polymerase Chain Reaction, MTT Assay, Viability Assay, Wound Healing Assay

    SULF2 expression in prostate cancer cells. Normal prostate epithelial cell line RWPE-1, and prostate cancer cell lines LNCap, PC3 and DU-145 SULF2 mRNA level was analyzed by RT-PCR in agarose gel (A) . RWPE-1, PC3 and DU-145 cells were transfected with either SULF2 expressing plasmid pcDNA3.1/Myc-His(−)Hsulf-2 (Addgene plasmid 13004) or empty vector using Fugene reagent (Promega). The control cells (CTRL) were not transfected. SULF2 mRNA expression was confirmed with quantitative real-time PCR. The expression level of each gene was normalized by GAPDH expression (B) . The overexpression of SULF2 was also confirmed by protein western blotting under non-reducing conditions (C) and immunofluorescence analyzed in confocal microscope (D) . The data from each experiment was obtained in triplicate and are represented as the average ± standard deviation. (CTRL: not transfected cells; VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2 containing vector). Scale bars 20 μm. *P ≤ 0.05.
    Figure Legend Snippet: SULF2 expression in prostate cancer cells. Normal prostate epithelial cell line RWPE-1, and prostate cancer cell lines LNCap, PC3 and DU-145 SULF2 mRNA level was analyzed by RT-PCR in agarose gel (A) . RWPE-1, PC3 and DU-145 cells were transfected with either SULF2 expressing plasmid pcDNA3.1/Myc-His(−)Hsulf-2 (Addgene plasmid 13004) or empty vector using Fugene reagent (Promega). The control cells (CTRL) were not transfected. SULF2 mRNA expression was confirmed with quantitative real-time PCR. The expression level of each gene was normalized by GAPDH expression (B) . The overexpression of SULF2 was also confirmed by protein western blotting under non-reducing conditions (C) and immunofluorescence analyzed in confocal microscope (D) . The data from each experiment was obtained in triplicate and are represented as the average ± standard deviation. (CTRL: not transfected cells; VECTOR: cells transfected with the empty vector; SULF2: cells transfected with SULF2 containing vector). Scale bars 20 μm. *P ≤ 0.05.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Over Expression, Western Blot, Immunofluorescence, Microscopy, Standard Deviation

    12) Product Images from "Semaphorin 3 C drives epithelial-to-mesenchymal transition, invasiveness, and stem-like characteristics in prostate cells"

    Article Title: Semaphorin 3 C drives epithelial-to-mesenchymal transition, invasiveness, and stem-like characteristics in prostate cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11914-6

    SEMA3C promotes cell dissemination in vivo . RWPE-2 cells were made to stably overexpress SEMA3C and firefly luciferase by lentiviral transduction to generate RWPE-2-FUGWBW and RWPE-2-SEMA3C. SEMA3C overexpression and phosphorylated and total levels of Akt, EGFR, and MAPK were examined by Western blot analysis. ( a ) Western blot band intensity was quantitated by densitometry ( a , right); ‘F’ = FUGWBW, ‘S’ = SEMA3C. Cell viability of RWPE-1-FUGWBW, RWPE-1-SEMA3C, RWPE-2-FUGWBW, and RWPE-2-SEMA3C was compared; ( b ) data represent mean viability over day 1 ± SD; * p
    Figure Legend Snippet: SEMA3C promotes cell dissemination in vivo . RWPE-2 cells were made to stably overexpress SEMA3C and firefly luciferase by lentiviral transduction to generate RWPE-2-FUGWBW and RWPE-2-SEMA3C. SEMA3C overexpression and phosphorylated and total levels of Akt, EGFR, and MAPK were examined by Western blot analysis. ( a ) Western blot band intensity was quantitated by densitometry ( a , right); ‘F’ = FUGWBW, ‘S’ = SEMA3C. Cell viability of RWPE-1-FUGWBW, RWPE-1-SEMA3C, RWPE-2-FUGWBW, and RWPE-2-SEMA3C was compared; ( b ) data represent mean viability over day 1 ± SD; * p

    Techniques Used: In Vivo, Stable Transfection, Luciferase, Transduction, Over Expression, Western Blot

    Overexpression of SEMA3C causes an upregulation of EMT markers. Expression levels of a panel of EMT markers were compared between SEMA3C-overexpressing RWPE-1 cells (RWPE-1-SEMA3C) and control cells (RWPE-1-FUGWBW). Significant upregulation of N-cadherin, ZEB2, ZEB1, fibronectin, vimentin, and downregulation of E-cadherin was observed in RWPE-1-SEMA3C compared to control RWPE-1-FUGWBW as shown by qPCR. ( a ) Data represent mean, ± SD; *** p
    Figure Legend Snippet: Overexpression of SEMA3C causes an upregulation of EMT markers. Expression levels of a panel of EMT markers were compared between SEMA3C-overexpressing RWPE-1 cells (RWPE-1-SEMA3C) and control cells (RWPE-1-FUGWBW). Significant upregulation of N-cadherin, ZEB2, ZEB1, fibronectin, vimentin, and downregulation of E-cadherin was observed in RWPE-1-SEMA3C compared to control RWPE-1-FUGWBW as shown by qPCR. ( a ) Data represent mean, ± SD; *** p

    Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction

    SEMA3C increases migration and invasion in vitro . The migration of RWPE-1-SEMA3C was compared to that of control cells by wound-healing assay. ( a ) % Migration in wound-healing assay was quantitated by the formula [(pixels at T 0h ) − (pixels at T 24h )]/(pixels at T 0h ) × 100%. ( b ) In transwell migration assay, RWPE-1-SEMA3C cells were roughly 3.5 times more motile than control cells; ( c ) y-axis is fold increase in migration over control cells. In Matrigel Invasion assays, RWPE-1-SEMA3C cells were 2 times more invasive than control cells; ( d ) y-axis is fold increase in invasion over control cells. RWPE-1-FUGWBW cells migrated more strongly toward SEMA3C (1 μM) than to PBS; ( e ) y-axis is fold increase in migration over PBS. Data represent mean, ± SD; * p
    Figure Legend Snippet: SEMA3C increases migration and invasion in vitro . The migration of RWPE-1-SEMA3C was compared to that of control cells by wound-healing assay. ( a ) % Migration in wound-healing assay was quantitated by the formula [(pixels at T 0h ) − (pixels at T 24h )]/(pixels at T 0h ) × 100%. ( b ) In transwell migration assay, RWPE-1-SEMA3C cells were roughly 3.5 times more motile than control cells; ( c ) y-axis is fold increase in migration over control cells. In Matrigel Invasion assays, RWPE-1-SEMA3C cells were 2 times more invasive than control cells; ( d ) y-axis is fold increase in invasion over control cells. RWPE-1-FUGWBW cells migrated more strongly toward SEMA3C (1 μM) than to PBS; ( e ) y-axis is fold increase in migration over PBS. Data represent mean, ± SD; * p

    Techniques Used: Migration, In Vitro, Wound Healing Assay, Transwell Migration Assay

    Overexpression of SEMA3C promotes stem-like characteristics. RWPE-1-FUGWBW cells expressed detectable levels of the PCa stem cell marker CD44 as shown by flow cytometry. However, RWPE-1-SEMA3C contained two distinguishable CD44 populations, a CD44 low population and a CD44 high population. ( a ) The two CD44 cell populations within the RWPE-1-SEMA3C cell population were sorted on CD44 status; CD44 low cells were cobblestone in morphology ( b ) and remained CD44-low ( c ) whereas CD44 high cells were spindle-shaped ( b ) and reconstituted the CD44 low population. ( c ) Numbers in the top left corner of FACS plots refer to the number of passages following cell sorting. Levels of E-cadherin, N-cadherin, and fibronectin in CD44 low and CD44 high cells were examined by Western blot at one (+1) and thirteen (+13) passages following sorting. ( d ) Based on densitometry, ratios of E-cadherin, N-cadherin, and fibronectin between CD44 high and CD44 low are presented graphically ( d , right). Sphere-formation is used as an in vitro measure of stemness; while RWPE-1-FUGWBW cells were capable of forming modestly sized spheres which existed as solitary or aggregates of three or fewer spheres, RWPE-1-SEMA3C cells formed larger spheres which was likely the result of coalescence of many individual spheres. ( e ) To quantitate sphere-forming abilities, spheres were dissociated and constituent cells were counted by hemocytometer. RWPE-1-SEMA3C cells formed spheres roughly two times more efficiently than control cells when evaluated in this way; ( f ) results are representative of three independent experiments. Data represent mean, ± SD; *** p
    Figure Legend Snippet: Overexpression of SEMA3C promotes stem-like characteristics. RWPE-1-FUGWBW cells expressed detectable levels of the PCa stem cell marker CD44 as shown by flow cytometry. However, RWPE-1-SEMA3C contained two distinguishable CD44 populations, a CD44 low population and a CD44 high population. ( a ) The two CD44 cell populations within the RWPE-1-SEMA3C cell population were sorted on CD44 status; CD44 low cells were cobblestone in morphology ( b ) and remained CD44-low ( c ) whereas CD44 high cells were spindle-shaped ( b ) and reconstituted the CD44 low population. ( c ) Numbers in the top left corner of FACS plots refer to the number of passages following cell sorting. Levels of E-cadherin, N-cadherin, and fibronectin in CD44 low and CD44 high cells were examined by Western blot at one (+1) and thirteen (+13) passages following sorting. ( d ) Based on densitometry, ratios of E-cadherin, N-cadherin, and fibronectin between CD44 high and CD44 low are presented graphically ( d , right). Sphere-formation is used as an in vitro measure of stemness; while RWPE-1-FUGWBW cells were capable of forming modestly sized spheres which existed as solitary or aggregates of three or fewer spheres, RWPE-1-SEMA3C cells formed larger spheres which was likely the result of coalescence of many individual spheres. ( e ) To quantitate sphere-forming abilities, spheres were dissociated and constituent cells were counted by hemocytometer. RWPE-1-SEMA3C cells formed spheres roughly two times more efficiently than control cells when evaluated in this way; ( f ) results are representative of three independent experiments. Data represent mean, ± SD; *** p

    Techniques Used: Over Expression, Marker, Flow Cytometry, Cytometry, FACS, Western Blot, In Vitro

    Generation of RWPE-1 cells stably overexpressing SEMA3C. SEMA3C was cloned under the control of a human Ubiquitin C promoter in a modified FUGW lentiviral vector designated FUGWBW using Gateway technology ( a ) (Invitrogen). Immortalized normal prostate epithelia RWPE-1 cells were transduced with virus made from either a SEMA3C overexpression construct to achieve constitutive expression (SEMA3C) or empty parental vector to serve as a control (FUGWBW). Overexpression of SEMA3C was confirmed by Western blot analysis of cell lysate where actin served as loading control ( b ). Phospho- and total levels of Akt, EGFR, and MAPK were also examined where actin or vinculin served as loading control. Western blot band intensity was quantitated by densitometry ( b , right); ‘F’ = FUGWBW, ‘S’ = SEMA3C. Control cells showed cobblestone morphology which is characteristic of epithelia while SEMA3C-overexpressing cells showed cobblestone and spindle-like morphologies ( c ).
    Figure Legend Snippet: Generation of RWPE-1 cells stably overexpressing SEMA3C. SEMA3C was cloned under the control of a human Ubiquitin C promoter in a modified FUGW lentiviral vector designated FUGWBW using Gateway technology ( a ) (Invitrogen). Immortalized normal prostate epithelia RWPE-1 cells were transduced with virus made from either a SEMA3C overexpression construct to achieve constitutive expression (SEMA3C) or empty parental vector to serve as a control (FUGWBW). Overexpression of SEMA3C was confirmed by Western blot analysis of cell lysate where actin served as loading control ( b ). Phospho- and total levels of Akt, EGFR, and MAPK were also examined where actin or vinculin served as loading control. Western blot band intensity was quantitated by densitometry ( b , right); ‘F’ = FUGWBW, ‘S’ = SEMA3C. Control cells showed cobblestone morphology which is characteristic of epithelia while SEMA3C-overexpressing cells showed cobblestone and spindle-like morphologies ( c ).

    Techniques Used: Stable Transfection, Clone Assay, Modification, Plasmid Preparation, Transduction, Over Expression, Construct, Expressing, Western Blot

    Co-expression of EMT and stem markers on SEMA3C-overexpressing cells. Co-expression of EMT markers and CD44 was determined by co-staining studies followed by flow cytometry. Within the RWPE-1-SEMA3C cell population, high CD44 expression was associated with low E-cadherin, high N-cadherin, and high vimentin expression while low CD44 expression was associated with high E-cadherin, low N-cadherin, and low vimentin expression. ( a ) Co-expression of mesenchymal and stem markers was confirmed by immunofluorescence microscopy where an inverse staining relationship existed between CD44 and E-cadherin and a positive staining relationship existed between CD44 and N-cadherin and between CD44 and vimentin ( b ).
    Figure Legend Snippet: Co-expression of EMT and stem markers on SEMA3C-overexpressing cells. Co-expression of EMT markers and CD44 was determined by co-staining studies followed by flow cytometry. Within the RWPE-1-SEMA3C cell population, high CD44 expression was associated with low E-cadherin, high N-cadherin, and high vimentin expression while low CD44 expression was associated with high E-cadherin, low N-cadherin, and low vimentin expression. ( a ) Co-expression of mesenchymal and stem markers was confirmed by immunofluorescence microscopy where an inverse staining relationship existed between CD44 and E-cadherin and a positive staining relationship existed between CD44 and N-cadherin and between CD44 and vimentin ( b ).

    Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Microscopy

    13) Product Images from "In vitro evaluation of novel N-acetylalaninate prodrugs that selectively induce apoptosis in prostate cancer cells"

    Article Title: In vitro evaluation of novel N-acetylalaninate prodrugs that selectively induce apoptosis in prostate cancer cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-675

    LNCaP and COS-7-OPH cell viabilities were diminished more than RWPE-1 and COS-7 by S-NPAA. A) RWPE-1 and LNCaP and B) COS-7 and COS-7-OPH cells were treated with the indicated doses of S-NPAA and incubated at 37°C for 24 hours. Cell viability was measured as a percent of control using a MTS viability assay; *indicates a significant difference between cell lines at p
    Figure Legend Snippet: LNCaP and COS-7-OPH cell viabilities were diminished more than RWPE-1 and COS-7 by S-NPAA. A) RWPE-1 and LNCaP and B) COS-7 and COS-7-OPH cells were treated with the indicated doses of S-NPAA and incubated at 37°C for 24 hours. Cell viability was measured as a percent of control using a MTS viability assay; *indicates a significant difference between cell lines at p

    Techniques Used: Incubation, Viability Assay

    GSH depletion in cultured cells and prostate cell lysates treated with S-NPAA. A) LNCaP, RWPE-1, COS-7, and COS-7-OPH cell cultures were incubated with 25 μM S-NPAA for 30 min followed by a 30 min incubation with CMAC. The blue fluorescence indicates the presence of GSH. B) Microscopy images were analyzed with ImageJ to measure the relative fluorescence between cell lines. Percent area threshold was defined as the percent area of fluorescence that exceeded background; *indicates that the treatment was significantly different from control (vehicle) at P
    Figure Legend Snippet: GSH depletion in cultured cells and prostate cell lysates treated with S-NPAA. A) LNCaP, RWPE-1, COS-7, and COS-7-OPH cell cultures were incubated with 25 μM S-NPAA for 30 min followed by a 30 min incubation with CMAC. The blue fluorescence indicates the presence of GSH. B) Microscopy images were analyzed with ImageJ to measure the relative fluorescence between cell lines. Percent area threshold was defined as the percent area of fluorescence that exceeded background; *indicates that the treatment was significantly different from control (vehicle) at P

    Techniques Used: Cell Culture, Incubation, Fluorescence, Microscopy

    LNCaP and COS-7-OPH show increased oxidative stress and apoptosis after treatment with S-NPAA. A) RWPE-1, LNCaP, COS-7, and COS-7-OPH cell cultures were incubated with 25 μM S-NPAA for 6 hours. Protein carbonyl levels were measured in cellular lysates as described in the Methods Section. B) RWPE-1, LNCaP, DU145, PC3, COS-7, and COS-7-OPH cell cultures were incubated with 25 μM NPAA or 5 μM staurosporine (STS) as a positive control for 6 hours and caspase-3 activities in cellular lysates were measured as described in the Methods Section; *indicates that the S-NPAA treatment was significantly different from control (vehicle) at p
    Figure Legend Snippet: LNCaP and COS-7-OPH show increased oxidative stress and apoptosis after treatment with S-NPAA. A) RWPE-1, LNCaP, COS-7, and COS-7-OPH cell cultures were incubated with 25 μM S-NPAA for 6 hours. Protein carbonyl levels were measured in cellular lysates as described in the Methods Section. B) RWPE-1, LNCaP, DU145, PC3, COS-7, and COS-7-OPH cell cultures were incubated with 25 μM NPAA or 5 μM staurosporine (STS) as a positive control for 6 hours and caspase-3 activities in cellular lysates were measured as described in the Methods Section; *indicates that the S-NPAA treatment was significantly different from control (vehicle) at p

    Techniques Used: Incubation, Positive Control

    14) Product Images from "Per- and Polyfluoroalkyl Substance Exposure Combined with High-Fat Diet Supports Prostate Cancer Progression"

    Article Title: Per- and Polyfluoroalkyl Substance Exposure Combined with High-Fat Diet Supports Prostate Cancer Progression

    Journal: Nutrients

    doi: 10.3390/nu13113902

    WST-1 assay shows that PFOS ( A ) or PFBS ( B ) exposure for 1 week increases cell viability of prostate benign (RWPE-1) and cancerous (RWPE-kRAS) cells ( C ). Comparison of cell viability of RWPE1 and RWPE-kRAS cells, when they are treated with Veh, 1 nM DHT, or 10 nM of PFAS.
    Figure Legend Snippet: WST-1 assay shows that PFOS ( A ) or PFBS ( B ) exposure for 1 week increases cell viability of prostate benign (RWPE-1) and cancerous (RWPE-kRAS) cells ( C ). Comparison of cell viability of RWPE1 and RWPE-kRAS cells, when they are treated with Veh, 1 nM DHT, or 10 nM of PFAS.

    Techniques Used: WST-1 Assay

    Venn diagram analysis of up- ( A ) or downregulated ( B ) genes in different treatment groups compared to the control group. ( C ) RNA-seq data indicate that PFAS exposure combined with a high-fat diet increased PPAR signaling in RWPE-kRAS xenografts. ( D ) RNA-seq data also indicated epigenetic regulation of transcription-associated genes in RWPE-kRAS xenografts. ( E ) Western blots demonstrating PFAS exposure increased histone acetyl markers in RWPE-kRAS cells. Numbers above bands indicate quantitation of signal for marks in samples that are treated with DHT + PFAS that change over DHT treatments.
    Figure Legend Snippet: Venn diagram analysis of up- ( A ) or downregulated ( B ) genes in different treatment groups compared to the control group. ( C ) RNA-seq data indicate that PFAS exposure combined with a high-fat diet increased PPAR signaling in RWPE-kRAS xenografts. ( D ) RNA-seq data also indicated epigenetic regulation of transcription-associated genes in RWPE-kRAS xenografts. ( E ) Western blots demonstrating PFAS exposure increased histone acetyl markers in RWPE-kRAS cells. Numbers above bands indicate quantitation of signal for marks in samples that are treated with DHT + PFAS that change over DHT treatments.

    Techniques Used: RNA Sequencing Assay, Western Blot, Quantitation Assay

    PFAS treatment increases pyruvate and acetyl-CoA levels in RWPE-kRAS cells. ( A ) PFOS-induced metabolites in RWPE-kRAS cells identified by GC/MS analysis. ( B ) Pyruvate levels from ( A ), ( C ) GSEA of PFOS + HFD-induced genes in RWPE-kRAS xenografts identified by RNA-seq. ( D ) mRNA expression of PDHB and PDHX, components of PDC, were increased with PFOS and a high-fat diet (HFD) in RWPE-kRAS xenografts. ( E ) Acetyl-CoA levels in PFOS-treated RWPE-kRAS cells (10 nM PFOS ± DHT, 24 h) using a fluorescence-based assay. * p
    Figure Legend Snippet: PFAS treatment increases pyruvate and acetyl-CoA levels in RWPE-kRAS cells. ( A ) PFOS-induced metabolites in RWPE-kRAS cells identified by GC/MS analysis. ( B ) Pyruvate levels from ( A ), ( C ) GSEA of PFOS + HFD-induced genes in RWPE-kRAS xenografts identified by RNA-seq. ( D ) mRNA expression of PDHB and PDHX, components of PDC, were increased with PFOS and a high-fat diet (HFD) in RWPE-kRAS xenografts. ( E ) Acetyl-CoA levels in PFOS-treated RWPE-kRAS cells (10 nM PFOS ± DHT, 24 h) using a fluorescence-based assay. * p

    Techniques Used: Gas Chromatography-Mass Spectrometry, RNA Sequencing Assay, Expressing, Fluorescence

    PFAS and an HFD synergize to increase prostate cancer xenograft growth. The 1 × 10 6 RWPE-kRAS cells were injected subcutaneously in 4-week-old athymic nude male mice. Mice were fed an HFD or control diet and treated with 10 mg/kg oral PFOS or vehicle control 5 days/week for 40 days. Tumor volume was measured using electronic calipers three times/week. A two-way ANOVA model for the time dependent effects of treatments on tumor growth was fitted. When the change is significant, Tukey’s multiple comparison test was employed.
    Figure Legend Snippet: PFAS and an HFD synergize to increase prostate cancer xenograft growth. The 1 × 10 6 RWPE-kRAS cells were injected subcutaneously in 4-week-old athymic nude male mice. Mice were fed an HFD or control diet and treated with 10 mg/kg oral PFOS or vehicle control 5 days/week for 40 days. Tumor volume was measured using electronic calipers three times/week. A two-way ANOVA model for the time dependent effects of treatments on tumor growth was fitted. When the change is significant, Tukey’s multiple comparison test was employed.

    Techniques Used: Injection, Mouse Assay

    15) Product Images from "Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific"

    Article Title: Growth inhibition mediated by PSP94 or CRISP-3 is prostate cancer cell line specific

    Journal: Asian Journal of Andrology

    doi: 10.1038/aja.2010.56

    Cell line specific growth-inhibitory effect caused by CRISP-3 over-expression. (A): Expression of recombinant CRISP-3 was detected by Western blot analysis using anti-CRISP-3 antibody. TCA-precipitated proteins from the conditioned medium of PC3 cells that were transiently transfected with CRISP-3 expression construct (Lane 1) showed the presence of glycosylated and unglycosylated CRISP-3 similar to the positive control (Lane 3) that contained TCA-precipitated proteins from the conditioned medium of WPE1-NB26 cells. TCA-precipitated proteins from the conditioned medium of PC3 cells transfected with empty vector (Lane 2) as well as untransfected RWPE-1 (Lane 4) served as a negative control. (B): A representative composite picture of the crystal violet staining from the clonogenic survival assay. A single well (of the six-well plate) is shown for each cell line (PC3, WPE1-NB26 and LNCaP) that was transfected with either the empty vector, pEFIRES-P (left wells), or the CRISP-3 expression construct (right wells). At 24 h after transfection, the cells were split and stable clones were selected on puromycin. Clones obtained after 2 weeks of selection were stained and counted. (C): Effect of over-expression of CRISP-3 on the clonogenic survival of PC3, WPE1-NB26 and LNCaP cells. The percentage survival with respect to empty vector was calculated as described above. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as mean percentage survival ± SE. The reduction in percentage survival of PC3 and WPE1-NB26 cells was statistically significant when compared with LNCaP cells ( * P
    Figure Legend Snippet: Cell line specific growth-inhibitory effect caused by CRISP-3 over-expression. (A): Expression of recombinant CRISP-3 was detected by Western blot analysis using anti-CRISP-3 antibody. TCA-precipitated proteins from the conditioned medium of PC3 cells that were transiently transfected with CRISP-3 expression construct (Lane 1) showed the presence of glycosylated and unglycosylated CRISP-3 similar to the positive control (Lane 3) that contained TCA-precipitated proteins from the conditioned medium of WPE1-NB26 cells. TCA-precipitated proteins from the conditioned medium of PC3 cells transfected with empty vector (Lane 2) as well as untransfected RWPE-1 (Lane 4) served as a negative control. (B): A representative composite picture of the crystal violet staining from the clonogenic survival assay. A single well (of the six-well plate) is shown for each cell line (PC3, WPE1-NB26 and LNCaP) that was transfected with either the empty vector, pEFIRES-P (left wells), or the CRISP-3 expression construct (right wells). At 24 h after transfection, the cells were split and stable clones were selected on puromycin. Clones obtained after 2 weeks of selection were stained and counted. (C): Effect of over-expression of CRISP-3 on the clonogenic survival of PC3, WPE1-NB26 and LNCaP cells. The percentage survival with respect to empty vector was calculated as described above. The assay was repeated at least three times for each cell line. The graph is the average of three independent observations and is plotted as mean percentage survival ± SE. The reduction in percentage survival of PC3 and WPE1-NB26 cells was statistically significant when compared with LNCaP cells ( * P

    Techniques Used: Over Expression, Expressing, Recombinant, Western Blot, Transfection, Construct, Positive Control, Plasmid Preparation, Negative Control, Staining, Clonogenic Cell Survival Assay, Clone Assay, Selection

    Pattern of endogenous PSP94 and CRISP-3 expression in different prostate cell lines. (A): Reverse transcription-polymerase chain reaction (RT-PCR) results of the total RNA content that was extracted from the five tested cell lines. The panels from the top to the bottom show amplification of PSP94, CRISP-3 and GAPDH, respectively. Lane 1 is a 100-bp ladder. Lanes 2, 4, 6, 8 and 10 are RT-PCR products for PC3, DU145, LNCaP, RWPE-1 and WPE1-NB26, respectively. Lanes 3, 5, 7, 9 and 11 are PCR reactions for no RT enzyme controls for the same samples. Lane 12 is a water control. (B): Semi-quantitative Western blot analysis for detection of CRISP-3 protein. TCA-precipitated proteins from conditioned medium that was harvested at 48 h (left side) and 72 h (right side) from 5 × 10 5 PC3 cells, WPE1-NB26 cells and LNCaP cells were subjected to Western blot analysis using an anti-CRISP-3 polyclonal antibody. The presence of two bands at around 30 and 28 kDa in the WPE1-NB26 and LNCaP cells represent glycosylated and unglycosylated CRISP-3 protein, respectively.
    Figure Legend Snippet: Pattern of endogenous PSP94 and CRISP-3 expression in different prostate cell lines. (A): Reverse transcription-polymerase chain reaction (RT-PCR) results of the total RNA content that was extracted from the five tested cell lines. The panels from the top to the bottom show amplification of PSP94, CRISP-3 and GAPDH, respectively. Lane 1 is a 100-bp ladder. Lanes 2, 4, 6, 8 and 10 are RT-PCR products for PC3, DU145, LNCaP, RWPE-1 and WPE1-NB26, respectively. Lanes 3, 5, 7, 9 and 11 are PCR reactions for no RT enzyme controls for the same samples. Lane 12 is a water control. (B): Semi-quantitative Western blot analysis for detection of CRISP-3 protein. TCA-precipitated proteins from conditioned medium that was harvested at 48 h (left side) and 72 h (right side) from 5 × 10 5 PC3 cells, WPE1-NB26 cells and LNCaP cells were subjected to Western blot analysis using an anti-CRISP-3 polyclonal antibody. The presence of two bands at around 30 and 28 kDa in the WPE1-NB26 and LNCaP cells represent glycosylated and unglycosylated CRISP-3 protein, respectively.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Western Blot

    16) Product Images from "A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines"

    Article Title: A Modified Coupled Spectrophotometric Method to Detect 2-5 Oligoadenylate Synthetase Activity in Prostate Cell Lines

    Journal: Biological Procedures Online

    doi: 10.1186/s12575-016-0038-x

    OAS1 protein expression in crude and purified lysates of prostate cancer cell line. OAS1 p46 isoform expression in lysates from 293T, RwPE1, DU145 and LNCaP cell line. a OAS 1 expression in 20 μg crude lysates ( upper panel ) and GAPDH ( lower panel ) was used as a loading control. b OAS 1 expression in lysates purified from 20 μg of crude lysates with activated poly I:C bound sepharose beads ( upper panel ), GAPDH ( middle panel ) was used as a loading control and elute-1 collected during washing to remove the unbound OAS enzyme and OAS free cell lysates. DU145 lysates purified with unactivated sepharose beads with no poly I:C bound to it was used as a negative control
    Figure Legend Snippet: OAS1 protein expression in crude and purified lysates of prostate cancer cell line. OAS1 p46 isoform expression in lysates from 293T, RwPE1, DU145 and LNCaP cell line. a OAS 1 expression in 20 μg crude lysates ( upper panel ) and GAPDH ( lower panel ) was used as a loading control. b OAS 1 expression in lysates purified from 20 μg of crude lysates with activated poly I:C bound sepharose beads ( upper panel ), GAPDH ( middle panel ) was used as a loading control and elute-1 collected during washing to remove the unbound OAS enzyme and OAS free cell lysates. DU145 lysates purified with unactivated sepharose beads with no poly I:C bound to it was used as a negative control

    Techniques Used: Expressing, Purification, Negative Control

    Enzyme activity in prostate cancer cell line. OAS enzyme assay measurement with pyrophosphate assay in lysates purified from 293T, RwPE1, LNCaP, DU145 and control which is lysate from DU145 cell purified with unactivated sepharose beads with no poly I:C. Shift in absorbance at 360 nm after completion of reaction post addition of PNP and inorganic phosphatase. Assay was done thrice in triplicates. Statistical significance is shown as *** for p
    Figure Legend Snippet: Enzyme activity in prostate cancer cell line. OAS enzyme assay measurement with pyrophosphate assay in lysates purified from 293T, RwPE1, LNCaP, DU145 and control which is lysate from DU145 cell purified with unactivated sepharose beads with no poly I:C. Shift in absorbance at 360 nm after completion of reaction post addition of PNP and inorganic phosphatase. Assay was done thrice in triplicates. Statistical significance is shown as *** for p

    Techniques Used: Activity Assay, Enzymatic Assay, Pyrophosphate Assay, Purification, Phosphatase Assay

    17) Product Images from "Nrdp1-mediated regulation of ErbB3 expression by the androgen receptor in androgen-dependent but not castrate-resistant prostate cancer cells"

    Article Title: Nrdp1-mediated regulation of ErbB3 expression by the androgen receptor in androgen-dependent but not castrate-resistant prostate cancer cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-09-4440

    ErbB3 levels increase with prostate cancer progression (A) Tissue microarrays representing (i) benign prostate (n=36) (BENIGN), (ii) high grade PIN (n=21) (HGPIN) and (iii) localized tumors (n=65) (TUMOR) obtained by prostatectomy and (iv) prostatic tissues (n=68) (CRPC/MET) from warm autopsies of men who died of CRPC were immunostained with anti-ErbB3 antibody (brown staining) and counterstained with hematoxylin (blue staining). (Upper): sections stained with anti-ErbB3. Note the strong ErbB3 stain in the tumor containing regions whereas the benign tissue alongside stained only weakly. The antibody used did not stain the nuclei in these tissues (20X magnification). (Lower): box plots representing range of ErbB3 expression in benign prostate, HGPIN, localized tumors and metastatic and localized tissues from warm autopsies of men who died of CRPC. (B) Increased ErbB3 expression in prostate cancer cells compared to lines derived from normal prostate- (upper): Normal prostate derived RWPE-1 cells, androgen-dependent LNCaP and its CRPC subline LNCaP-AI cells compared to stable LNCaP sublines overexpressing ErbB3 (LNCaP-ErbB3-1 and -2), (lower): ErbB3 expression in pRNS-1-1 cells derived from a normal prostate which upon culture lost the expression of the AR, and AR-null PC-3 and DU-145 cells. pRNS-1-1 cells were transfected with vector only, or mutant AR(T877A) or AR(K580R).
    Figure Legend Snippet: ErbB3 levels increase with prostate cancer progression (A) Tissue microarrays representing (i) benign prostate (n=36) (BENIGN), (ii) high grade PIN (n=21) (HGPIN) and (iii) localized tumors (n=65) (TUMOR) obtained by prostatectomy and (iv) prostatic tissues (n=68) (CRPC/MET) from warm autopsies of men who died of CRPC were immunostained with anti-ErbB3 antibody (brown staining) and counterstained with hematoxylin (blue staining). (Upper): sections stained with anti-ErbB3. Note the strong ErbB3 stain in the tumor containing regions whereas the benign tissue alongside stained only weakly. The antibody used did not stain the nuclei in these tissues (20X magnification). (Lower): box plots representing range of ErbB3 expression in benign prostate, HGPIN, localized tumors and metastatic and localized tissues from warm autopsies of men who died of CRPC. (B) Increased ErbB3 expression in prostate cancer cells compared to lines derived from normal prostate- (upper): Normal prostate derived RWPE-1 cells, androgen-dependent LNCaP and its CRPC subline LNCaP-AI cells compared to stable LNCaP sublines overexpressing ErbB3 (LNCaP-ErbB3-1 and -2), (lower): ErbB3 expression in pRNS-1-1 cells derived from a normal prostate which upon culture lost the expression of the AR, and AR-null PC-3 and DU-145 cells. pRNS-1-1 cells were transfected with vector only, or mutant AR(T877A) or AR(K580R).

    Techniques Used: Staining, Expressing, Derivative Assay, Transfection, Plasmid Preparation, Mutagenesis

    18) Product Images from "Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway"

    Article Title: Notch1 suppresses prostate cancer cell invasion via the metastasis-associated 1-KiSS-1 metastasis-suppressor pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.6761

    Notch1 and cleaved-Notch1 expression in the human prostatic carcinoma cell lines LNCaP, PC-3 and DU 145 and the immortalized human prostatic epithelial RWPE-1 cell line. (A) Western blot analysis. Total cell lysates were collected from LNCaP, PC-3, DU
    Figure Legend Snippet: Notch1 and cleaved-Notch1 expression in the human prostatic carcinoma cell lines LNCaP, PC-3 and DU 145 and the immortalized human prostatic epithelial RWPE-1 cell line. (A) Western blot analysis. Total cell lysates were collected from LNCaP, PC-3, DU

    Techniques Used: Expressing, Western Blot

    19) Product Images from "Downregulation of C-Terminal Tensin-Like Protein (CTEN) Suppresses Prostate Cell Proliferation and Contributes to Acinar Morphogenesis"

    Article Title: Downregulation of C-Terminal Tensin-Like Protein (CTEN) Suppresses Prostate Cell Proliferation and Contributes to Acinar Morphogenesis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19103190

    CTEN acts as a binding partner of integrin β1 and increases the activity of FAK and RhoA. ( a ) Whole cell lysate of RWPE-1 were coimmunoprecipitated (CoIP) with anti-CTEN antibody or normal rabbit IgG. The precipitates were examined by immunoblotting (IB) with anti-integrin β1 and anti-CTEN antibodies. ( b ) Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 6 days in the presence of doxycycline (0.2 μg/mL). The cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. The densities of the protein bands from the Western blot were quantified. The levels of the phosphorylated FAK Y397 (pFAK Y397 ) was normalized to those of the total FAK. The ratio of pFAKY397/FAK in each group was presented. ( c ) RWPE-1 cells were transfected with the indicated siRNA. The GTP-RhoA in RWPE-1 lysates was purified using GTP-RhoA pull-down assays as described in Materials and Methods and examined by Western blot analyses with an anti-RhoA antibody. The protein levels in whole cell lysates were also examined by Western analyses using the indicated antibodies. Total RhoA was used as loading control. The densities of the protein bands from the Western blot were quantified. The levels of Rho-GTP was normalized to those of total RhoA. The ratio of Rho-GTP/total RhoA was presented.
    Figure Legend Snippet: CTEN acts as a binding partner of integrin β1 and increases the activity of FAK and RhoA. ( a ) Whole cell lysate of RWPE-1 were coimmunoprecipitated (CoIP) with anti-CTEN antibody or normal rabbit IgG. The precipitates were examined by immunoblotting (IB) with anti-integrin β1 and anti-CTEN antibodies. ( b ) Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 6 days in the presence of doxycycline (0.2 μg/mL). The cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. The densities of the protein bands from the Western blot were quantified. The levels of the phosphorylated FAK Y397 (pFAK Y397 ) was normalized to those of the total FAK. The ratio of pFAKY397/FAK in each group was presented. ( c ) RWPE-1 cells were transfected with the indicated siRNA. The GTP-RhoA in RWPE-1 lysates was purified using GTP-RhoA pull-down assays as described in Materials and Methods and examined by Western blot analyses with an anti-RhoA antibody. The protein levels in whole cell lysates were also examined by Western analyses using the indicated antibodies. Total RhoA was used as loading control. The densities of the protein bands from the Western blot were quantified. The levels of Rho-GTP was normalized to those of total RhoA. The ratio of Rho-GTP/total RhoA was presented.

    Techniques Used: Binding Assay, Activity Assay, Co-Immunoprecipitation Assay, Expressing, Incubation, Western Blot, Transfection, Purification

    Inducible EGFP-expressing and CTEN-expressing RWPE-1 cells perform similar phenotypes in the absence of doxycycline in 3D culture. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days without doxycycline induction. ( a ) The appearance of acinar structures was observed and classified to three types (normal, notched and deformed). The numbers of the three-type acini in each group were counted and presented as a percentage of the whole (upper panel). Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; no significant difference between aON-EGFP and aON-CTEN in each phenotype). The representative images of acinar structure were also shown in the lower panel. Scale bar: 100 μm. ( b ) aON-EGFP and aON-CTEN RWPE-1 cells grown in 3D culture were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control.
    Figure Legend Snippet: Inducible EGFP-expressing and CTEN-expressing RWPE-1 cells perform similar phenotypes in the absence of doxycycline in 3D culture. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days without doxycycline induction. ( a ) The appearance of acinar structures was observed and classified to three types (normal, notched and deformed). The numbers of the three-type acini in each group were counted and presented as a percentage of the whole (upper panel). Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; no significant difference between aON-EGFP and aON-CTEN in each phenotype). The representative images of acinar structure were also shown in the lower panel. Scale bar: 100 μm. ( b ) aON-EGFP and aON-CTEN RWPE-1 cells grown in 3D culture were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control.

    Techniques Used: Expressing, Incubation, Western Blot

    Forced CTEN expression disrupts acinar morphogenesis. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days in the presence of doxycycline (0.2 μg/mL). ( a ) Cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. ( b ) The numbers of the three-type acini in each group were counted at Day 9 and presented as a percentage of the whole. Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; * p
    Figure Legend Snippet: Forced CTEN expression disrupts acinar morphogenesis. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days in the presence of doxycycline (0.2 μg/mL). ( a ) Cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. ( b ) The numbers of the three-type acini in each group were counted at Day 9 and presented as a percentage of the whole. Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; * p

    Techniques Used: Expressing, Incubation, Western Blot

    Knockdown of CTEN suppresses cell proliferation and downregulation of CTEN occurs during cell differentiation. ( a ) RWPE-1 cells were transfected with CTEN-specific siRNA (siCTEN) or nontargeting siRNA (siCtrl). Cell proliferation was evaluated by WST-1 based assay as described in Materials and Methods. Data were expressed as mean ± standard deviation (SD) of three independent experiments analyzed in triplicate (Student’s t test; ** p
    Figure Legend Snippet: Knockdown of CTEN suppresses cell proliferation and downregulation of CTEN occurs during cell differentiation. ( a ) RWPE-1 cells were transfected with CTEN-specific siRNA (siCTEN) or nontargeting siRNA (siCtrl). Cell proliferation was evaluated by WST-1 based assay as described in Materials and Methods. Data were expressed as mean ± standard deviation (SD) of three independent experiments analyzed in triplicate (Student’s t test; ** p

    Techniques Used: Cell Differentiation, Transfection, WST-1 Assay, Standard Deviation

    20) Product Images from "Unleashing the Diagnostic, Prognostic and Therapeutic Potential of the Neuronostatin/GPR107 System in Prostate Cancer"

    Article Title: Unleashing the Diagnostic, Prognostic and Therapeutic Potential of the Neuronostatin/GPR107 System in Prostate Cancer

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm9061703

    Functional effects after neuronostatin (NST) treatment and after the combination of NST and G protein-coupled receptor 107 (GPR107)-silencing treatment in prostate cell lines. ( a ) Proliferation rate of normal prostate (RWPE-1) and Prostate Cancer (PCa) (LNCaP, 22Rv1 and PC-3) cells in response to NST treatment (10 –7 M; after 24, 48 and 72 h). ( b ) Migration rate of normal prostate (RWPE-1) and PCa (PC-3) cells after 24 h of NST treatment (10 –7 M). Proliferation ( c ) and migration ( d ) rate of androgen-independent PCa cells in response to NST and GPR107-silencing alone or in combination. Data were represented as percent of vehicle-treated cells (set at 100%). Asterisks (* p
    Figure Legend Snippet: Functional effects after neuronostatin (NST) treatment and after the combination of NST and G protein-coupled receptor 107 (GPR107)-silencing treatment in prostate cell lines. ( a ) Proliferation rate of normal prostate (RWPE-1) and Prostate Cancer (PCa) (LNCaP, 22Rv1 and PC-3) cells in response to NST treatment (10 –7 M; after 24, 48 and 72 h). ( b ) Migration rate of normal prostate (RWPE-1) and PCa (PC-3) cells after 24 h of NST treatment (10 –7 M). Proliferation ( c ) and migration ( d ) rate of androgen-independent PCa cells in response to NST and GPR107-silencing alone or in combination. Data were represented as percent of vehicle-treated cells (set at 100%). Asterisks (* p

    Techniques Used: Functional Assay, Migration

    Screening of G protein-coupled receptor 107 (GPR107) expression level and functional effects of its silencing in normal and tumor prostate cell lines. ( a ) Comparison of GPR107 expression levels between a non-tumor prostate cell line (RWPE-1) and different Prostate Cancer (PCa) cell lines (LNCaP, 22Rv1 and PC-3). Messenger RNA (mRNA) levels were determined by quantitative Polymerase Chain Reaction (qPCR) and adjusted by a normalization factor (NF) generated by the combination of Actin Beta (ACTB) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) expression levels. ( b) –( c ) Proliferation rate of 22Rv1 ( b ) and PC-3 ( c ) cells after 24, 48 and 72 h of GPR107-silencing. ( d ) Migration rate of PC-3 cells after 24 h of GPR107-silencing. In ( b )–( d ), data were represented as percent of scrambled cells (set at 100%). Asterisks (* p
    Figure Legend Snippet: Screening of G protein-coupled receptor 107 (GPR107) expression level and functional effects of its silencing in normal and tumor prostate cell lines. ( a ) Comparison of GPR107 expression levels between a non-tumor prostate cell line (RWPE-1) and different Prostate Cancer (PCa) cell lines (LNCaP, 22Rv1 and PC-3). Messenger RNA (mRNA) levels were determined by quantitative Polymerase Chain Reaction (qPCR) and adjusted by a normalization factor (NF) generated by the combination of Actin Beta (ACTB) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) expression levels. ( b) –( c ) Proliferation rate of 22Rv1 ( b ) and PC-3 ( c ) cells after 24, 48 and 72 h of GPR107-silencing. ( d ) Migration rate of PC-3 cells after 24 h of GPR107-silencing. In ( b )–( d ), data were represented as percent of scrambled cells (set at 100%). Asterisks (* p

    Techniques Used: Expressing, Functional Assay, Real-time Polymerase Chain Reaction, Generated, Migration

    21) Product Images from "Profiling and bioinformatics analyses of differential circular RNA expression in prostate cancer cells"

    Article Title: Profiling and bioinformatics analyses of differential circular RNA expression in prostate cancer cells

    Journal: Future Science OA

    doi: 10.4155/fsoa-2018-0046

    Gene ontology term analysis and KEGG pathway analysis of differentially expressed circular RNA host genes. (A–F) Main GO term analysis of 22RV1 versus RWPE-1, PC3 versus RWPE-1 and PC3 versus 22RV1 with p-value
    Figure Legend Snippet: Gene ontology term analysis and KEGG pathway analysis of differentially expressed circular RNA host genes. (A–F) Main GO term analysis of 22RV1 versus RWPE-1, PC3 versus RWPE-1 and PC3 versus 22RV1 with p-value

    Techniques Used:

    Circular RNAs expression profiles in prostate cancer cell lines. (A) The number of circRNAs identified in RWPE-1, 22RV1, PC3 cell lines. (B) The distribution of circRNAs from different database resources. (C) The distribution of circRNAs from different classifications based on the genomic origin. (D) The distribution of circRNAs in different lengths. (E) The distribution of circRNAs in different chromosomes. circRNA: Circular RNA.
    Figure Legend Snippet: Circular RNAs expression profiles in prostate cancer cell lines. (A) The number of circRNAs identified in RWPE-1, 22RV1, PC3 cell lines. (B) The distribution of circRNAs from different database resources. (C) The distribution of circRNAs from different classifications based on the genomic origin. (D) The distribution of circRNAs in different lengths. (E) The distribution of circRNAs in different chromosomes. circRNA: Circular RNA.

    Techniques Used: Expressing

    Validation of differentially expressed circular RNAs in prostate cancer cell lines. (A) The relative expression levels of significantly up- and down-regulated circRNAs in RWPE-1, 22RV1 and PC3 cell lines by real-time qPCR. (B) The Sanger sequencing results of 12 circRNAs PCR amplified products selected randomly. (C) Real-time qPCR results of circRNAs in PCa cells treated with RNase R. The amount of circRNAs was normalized to the value measured in the mock treatment. circRNA: Circular RNA.
    Figure Legend Snippet: Validation of differentially expressed circular RNAs in prostate cancer cell lines. (A) The relative expression levels of significantly up- and down-regulated circRNAs in RWPE-1, 22RV1 and PC3 cell lines by real-time qPCR. (B) The Sanger sequencing results of 12 circRNAs PCR amplified products selected randomly. (C) Real-time qPCR results of circRNAs in PCa cells treated with RNase R. The amount of circRNAs was normalized to the value measured in the mock treatment. circRNA: Circular RNA.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Amplification

    22) Product Images from ""

    Article Title:

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M111.014845

    Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different androgen independent and androgen sensitive prostate cancer cell lines including PC3, DU145, VCaP, LNCaP and C4–2 versus benign epithelial prostate cell line RWPE-1. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–100 nm size were identified as being exosomes.
    Figure Legend Snippet: Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different androgen independent and androgen sensitive prostate cancer cell lines including PC3, DU145, VCaP, LNCaP and C4–2 versus benign epithelial prostate cell line RWPE-1. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–100 nm size were identified as being exosomes.

    Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Derivative Assay, Staining

    Exosome lipidomic data. The lipid content of four major lipid classes was measured in PC3, DU145 and VCaP, LNCaP, C4–2 and RWPE-1 cells and compared with their derived exosomes, using LC-MS. The bar diagrams are representative of A , glycerolipid, B , glycerophospholipid, C , sphingolipid, D , glycosphingolipid in cell lysates and exosomes. Relative amounts of each lipid group were calculated as (sum of all AUC's for a particular lipid group) ÷ (sum of AUC's for all lipid groups) ×100%. * denotes a significant difference ( p
    Figure Legend Snippet: Exosome lipidomic data. The lipid content of four major lipid classes was measured in PC3, DU145 and VCaP, LNCaP, C4–2 and RWPE-1 cells and compared with their derived exosomes, using LC-MS. The bar diagrams are representative of A , glycerolipid, B , glycerophospholipid, C , sphingolipid, D , glycosphingolipid in cell lysates and exosomes. Relative amounts of each lipid group were calculated as (sum of all AUC's for a particular lipid group) ÷ (sum of AUC's for all lipid groups) ×100%. * denotes a significant difference ( p

    Techniques Used: Derivative Assay, Liquid Chromatography with Mass Spectroscopy

    Cholesterol concentration. The bar diagrams show the cholesterol concentration of A , lysates of PC3, DU145 and VCaP, LNCaP, C4–2, and RWPE-1 cells B , exosomes derived from the six different prostate cell lines. Cholesterol results were normalized to protein concentration of each sample and expressed as μg Cholesterol/μg Protein. * indicate significantly difference ( p
    Figure Legend Snippet: Cholesterol concentration. The bar diagrams show the cholesterol concentration of A , lysates of PC3, DU145 and VCaP, LNCaP, C4–2, and RWPE-1 cells B , exosomes derived from the six different prostate cell lines. Cholesterol results were normalized to protein concentration of each sample and expressed as μg Cholesterol/μg Protein. * indicate significantly difference ( p

    Techniques Used: Concentration Assay, Derivative Assay, Protein Concentration

    23) Product Images from "ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor"

    Article Title: ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor

    Journal: eLife

    doi: 10.7554/eLife.13964

    AR and ERG expression in RWPE-1, and mutation of AR LBD. ( B ) RWPE-1 control cells and stably expressing AR wild type (AR WT) were either left untreated ( C ) or treated with 100ng/ml doxycycline ( D ) to induce ERG expression, in the absence ( D ) or presence (R/D) of 1nM R1881 for 24 hr. Western blot analysis shows expression levels of AR and ERG. ( B ) qRT-PCR of the luminal genes PSA (blue) and NKX3-1 (red) from RWPE-1 cells treated as in ( A ). Data represent normalized expression of PSA and NKX3-1 mRNA relative to the B2M transcript. Error bars represent + SEM of three replicates. ( C ) Western blot analysis of AR, ERG and GAPDH expression levels from RWPE-1 parental cells or cells stably expressing either AR wild type (WT), R711K, R727K, R753K, R761K, R775K, R780K, R787K, R789K, R787/789K, R832K, R841K, R847K, R855K, R856K, R855/856K or R872K AR mutant. ( D ) RWPE-1 cells stably expressing either AR R711K, R727K, R753K, R775K, R780K, R787K, R789K, R787/789K, R832K, R841K, R847K, R855K, R856K, R855/856K or R872K mutant were used for AR IP followed by western blot analysis for MMA, SDMA and total AR levels. DOI: http://dx.doi.org/10.7554/eLife.13964.012
    Figure Legend Snippet: AR and ERG expression in RWPE-1, and mutation of AR LBD. ( B ) RWPE-1 control cells and stably expressing AR wild type (AR WT) were either left untreated ( C ) or treated with 100ng/ml doxycycline ( D ) to induce ERG expression, in the absence ( D ) or presence (R/D) of 1nM R1881 for 24 hr. Western blot analysis shows expression levels of AR and ERG. ( B ) qRT-PCR of the luminal genes PSA (blue) and NKX3-1 (red) from RWPE-1 cells treated as in ( A ). Data represent normalized expression of PSA and NKX3-1 mRNA relative to the B2M transcript. Error bars represent + SEM of three replicates. ( C ) Western blot analysis of AR, ERG and GAPDH expression levels from RWPE-1 parental cells or cells stably expressing either AR wild type (WT), R711K, R727K, R753K, R761K, R775K, R780K, R787K, R789K, R787/789K, R832K, R841K, R847K, R855K, R856K, R855/856K or R872K AR mutant. ( D ) RWPE-1 cells stably expressing either AR R711K, R727K, R753K, R775K, R780K, R787K, R789K, R787/789K, R832K, R841K, R847K, R855K, R856K, R855/856K or R872K mutant were used for AR IP followed by western blot analysis for MMA, SDMA and total AR levels. DOI: http://dx.doi.org/10.7554/eLife.13964.012

    Techniques Used: Expressing, Mutagenesis, Stable Transfection, Western Blot, Quantitative RT-PCR

    AR R761K mutation effects in RWPE-1. ( A ) Western blot of RWPE-1 parental cells and cells stably expressing AR wild type (AR WT) or AR R761K mutant. Cells were either untreated ( C ) or treated with 100ng/ml doxycycline ( D ) to induce ERG expression, in the absence or presence of 1 nM R1881 for 24 hr (R, R/D). Blots for AR, ERG, and GAPDH are shown. ( B ) Representative immunofluorescence images of AR from RWPE-1 cells stably expressing either AR WT or R761K mutant. Cells were either untreated (-) or treated with 0.1 or 1nM R1881 for 24 hr. Cells were also stained with DAPI to visualize nuclei. Cells were fixed and stained as described in Materials and methods. Scale bar, 50 µm. Data shown is a representative example of three biological replicates. ( C ) ChIP qPCR of ERG (top row) and PRMT5 (bottom row) at the PSA promoter and enhancer regions in RWPE-1 cells expressing either wild type AR (AR WT, left column) or AR R761K mutant (right column) in the absence and presence of ERG (Dox) or R1881 (R; 1nM for 24 hrs). All recruitments are normalized to the IgG control ChIP. Error bars represent + SEM of three biological replicates, each with three technical repeats. DOI: http://dx.doi.org/10.7554/eLife.13964.014
    Figure Legend Snippet: AR R761K mutation effects in RWPE-1. ( A ) Western blot of RWPE-1 parental cells and cells stably expressing AR wild type (AR WT) or AR R761K mutant. Cells were either untreated ( C ) or treated with 100ng/ml doxycycline ( D ) to induce ERG expression, in the absence or presence of 1 nM R1881 for 24 hr (R, R/D). Blots for AR, ERG, and GAPDH are shown. ( B ) Representative immunofluorescence images of AR from RWPE-1 cells stably expressing either AR WT or R761K mutant. Cells were either untreated (-) or treated with 0.1 or 1nM R1881 for 24 hr. Cells were also stained with DAPI to visualize nuclei. Cells were fixed and stained as described in Materials and methods. Scale bar, 50 µm. Data shown is a representative example of three biological replicates. ( C ) ChIP qPCR of ERG (top row) and PRMT5 (bottom row) at the PSA promoter and enhancer regions in RWPE-1 cells expressing either wild type AR (AR WT, left column) or AR R761K mutant (right column) in the absence and presence of ERG (Dox) or R1881 (R; 1nM for 24 hrs). All recruitments are normalized to the IgG control ChIP. Error bars represent + SEM of three biological replicates, each with three technical repeats. DOI: http://dx.doi.org/10.7554/eLife.13964.014

    Techniques Used: Mutagenesis, Western Blot, Stable Transfection, Expressing, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    24) Product Images from "ERG Deregulation Induces PIM1 Over-Expression and Aneuploidy in Prostate Epithelial Cells"

    Article Title: ERG Deregulation Induces PIM1 Over-Expression and Aneuploidy in Prostate Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028162

    Sensitivity of RWPE-1_tERG cells to chemotherapeutic drugs. a ) Proliferative potential of tERG over-expressing and control (empty) cells measured by thymidine uptake assay up to 72 hrs growth in standard conditions. b–c ) 72 hours treatment of RWPE-1 transfectants with taxol or idarubicin-hydrochloride. Proliferative potential was measured by thymidine uptake assay. d–e ) Re-growth of RWPE-1 transfectants after 10 nM taxol ( d ) or 0.5 µM idarubcicin-hydrochloride ( e ): following 24-hrs treatment, drugs were removed and the cells allowed to grow for additional 72 hours. Proliferation of cells collected at the time of wash-out is set as 100%. * = p
    Figure Legend Snippet: Sensitivity of RWPE-1_tERG cells to chemotherapeutic drugs. a ) Proliferative potential of tERG over-expressing and control (empty) cells measured by thymidine uptake assay up to 72 hrs growth in standard conditions. b–c ) 72 hours treatment of RWPE-1 transfectants with taxol or idarubicin-hydrochloride. Proliferative potential was measured by thymidine uptake assay. d–e ) Re-growth of RWPE-1 transfectants after 10 nM taxol ( d ) or 0.5 µM idarubcicin-hydrochloride ( e ): following 24-hrs treatment, drugs were removed and the cells allowed to grow for additional 72 hours. Proliferation of cells collected at the time of wash-out is set as 100%. * = p

    Techniques Used: Expressing

    The tERG/PIM1 axis favours aneuploidy. a ) Immunoblot assay demonstrates Cyclin B1 increase in RWPE-1_tERG cells. siRNA knockdown of PIM1 (siPIM1) reduces this effect compared to non-targeting siRNA (siNT). The same amount of whole cell lysates were loaded in two different polyacrilamide gels. The tERG_siPIM1 sample for the HA immunoblot was loaded in a different position compared to the gel for PIM1, Actin and Cyclin B1 blot b ) Flow cytometric analysis of non-synchronized propidium iodide stained cells. Treatment with taxol for 15/24/42 hours induces an important increase in the > 4n fraction of RWPE-1_tERG compared to empty cells. c ) Percentage of > 4n cells after taxol treatment obtained from the average of at least two independent experiments. ** = p
    Figure Legend Snippet: The tERG/PIM1 axis favours aneuploidy. a ) Immunoblot assay demonstrates Cyclin B1 increase in RWPE-1_tERG cells. siRNA knockdown of PIM1 (siPIM1) reduces this effect compared to non-targeting siRNA (siNT). The same amount of whole cell lysates were loaded in two different polyacrilamide gels. The tERG_siPIM1 sample for the HA immunoblot was loaded in a different position compared to the gel for PIM1, Actin and Cyclin B1 blot b ) Flow cytometric analysis of non-synchronized propidium iodide stained cells. Treatment with taxol for 15/24/42 hours induces an important increase in the > 4n fraction of RWPE-1_tERG compared to empty cells. c ) Percentage of > 4n cells after taxol treatment obtained from the average of at least two independent experiments. ** = p

    Techniques Used: Flow Cytometry, Staining

    ERG dependent PIM1 induction in RWPE-1 cells. a–b ) PIM1 over-expression in RWPE-1 transfected with HA-tagged TMPRSS2/ERG coding region (RWPE-1_tERG) as evidenced by immunoblot ( a ) and Real-Time quantitative PCR ( b ). Actin is shown as a loading control. ERG and PIM1 fold changes were normalized against empty-vector cells. c ) siRNA knockdown of ERG (siERG) in RWPE-1_tERG induces a decrease in PIM1 levels compared to non-targeting siRNA (siNT). d ) Sequence of the 194 bp-long PIM1 promoter region amplified in ChIP analysis. The 7 bp putative ERG binding site identified by Transcription Element Search System (TESS) is shown in italic. The distances from the ATG starting site are presented. e–f ) Chromatin Immunoprecipitation (ChIP) showed a significant enrichment of HA-tERG binding to PIM1 promoter compared to IgG control in RWPE-1_tERG. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was used as a negative control. ** = p
    Figure Legend Snippet: ERG dependent PIM1 induction in RWPE-1 cells. a–b ) PIM1 over-expression in RWPE-1 transfected with HA-tagged TMPRSS2/ERG coding region (RWPE-1_tERG) as evidenced by immunoblot ( a ) and Real-Time quantitative PCR ( b ). Actin is shown as a loading control. ERG and PIM1 fold changes were normalized against empty-vector cells. c ) siRNA knockdown of ERG (siERG) in RWPE-1_tERG induces a decrease in PIM1 levels compared to non-targeting siRNA (siNT). d ) Sequence of the 194 bp-long PIM1 promoter region amplified in ChIP analysis. The 7 bp putative ERG binding site identified by Transcription Element Search System (TESS) is shown in italic. The distances from the ATG starting site are presented. e–f ) Chromatin Immunoprecipitation (ChIP) showed a significant enrichment of HA-tERG binding to PIM1 promoter compared to IgG control in RWPE-1_tERG. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was used as a negative control. ** = p

    Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Amplification, Chromatin Immunoprecipitation, Binding Assay, Negative Control

    25) Product Images from "Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells"

    Article Title: Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S105725

    RO inhibits the viability of prostate cancer cells, but not normal prostate cells, in vitro. Notes: ( A ) RO reduces viability of hormone-dependent (LNCaP) and castration-resistant (PC-3) prostate cancer cell lines. LNCaP cells (7×10 3 /well) in 20% FBS RPMI-1640 medium and PC-3 cells (4×10 3 /well) and DU145 cells (4×10 3 /well) in 10% FBS RPMI-1640 medium were seeded in 96-well plates overnight. Cells were then treated with different concentrations of RO in 5% FBS RPMI-1640 medium (PC-3 and DU145) or 10% FBS RPMI-1640 medium (LNCaP) for 24 or 48 hours. Cell viability was evaluated by SRB assay. ( B ) RO does not reduce the viability of normal human prostate RWPE-1 cells. RWPE-1 cells (5×10 3 /well) in complete growth medium and PC-3 prostate cancer cells (4×10 3 /well) in 10% FBS RPMI-1640 medium were seeded in 96-well plates overnight. Cells were washed once with medium without FBS and then treated with different concentrations of RO in 5% FBS RPMI-1640 medium (PC-3 cells) or complete growth medium (RWPE-1 cells) for 24 hours. Cell viability was evaluated by SRB assay. * Significantly different from controls ( P
    Figure Legend Snippet: RO inhibits the viability of prostate cancer cells, but not normal prostate cells, in vitro. Notes: ( A ) RO reduces viability of hormone-dependent (LNCaP) and castration-resistant (PC-3) prostate cancer cell lines. LNCaP cells (7×10 3 /well) in 20% FBS RPMI-1640 medium and PC-3 cells (4×10 3 /well) and DU145 cells (4×10 3 /well) in 10% FBS RPMI-1640 medium were seeded in 96-well plates overnight. Cells were then treated with different concentrations of RO in 5% FBS RPMI-1640 medium (PC-3 and DU145) or 10% FBS RPMI-1640 medium (LNCaP) for 24 or 48 hours. Cell viability was evaluated by SRB assay. ( B ) RO does not reduce the viability of normal human prostate RWPE-1 cells. RWPE-1 cells (5×10 3 /well) in complete growth medium and PC-3 prostate cancer cells (4×10 3 /well) in 10% FBS RPMI-1640 medium were seeded in 96-well plates overnight. Cells were washed once with medium without FBS and then treated with different concentrations of RO in 5% FBS RPMI-1640 medium (PC-3 cells) or complete growth medium (RWPE-1 cells) for 24 hours. Cell viability was evaluated by SRB assay. * Significantly different from controls ( P

    Techniques Used: In Vitro, Sulforhodamine B Assay

    26) Product Images from "Beta-Defensin 124 Is Required for Efficient Innate Immune Responses in Prostate Epithelial RWPE-1 Cells"

    Article Title: Beta-Defensin 124 Is Required for Efficient Innate Immune Responses in Prostate Epithelial RWPE-1 Cells

    Journal: Korean Journal of Urology

    doi: 10.4111/kju.2014.55.6.417

    Beta-Defensin 124 (DEFB124) upregulation induces increased production of cytokines and chemokines. DEFB124 promotes mRNA expression for cytokines (A) and chemokines (B) in DEFB124-induced RWPE-1 cells. The mRNA expression of these genes was determined by using quantitative real-time polymerase chain reaction. Relative expression levels of each gene were calculated from cycle threshold values and were normalized with ACTB, and the expression ratio was calculated against the expression of each gene in the empty vector-transfected RWPE-1 cells. Experiments were repeated at least three times, and data are expressed as the mean±standard error of the mean (SEM). Asterisks, * and ** , represent statistical significance at p
    Figure Legend Snippet: Beta-Defensin 124 (DEFB124) upregulation induces increased production of cytokines and chemokines. DEFB124 promotes mRNA expression for cytokines (A) and chemokines (B) in DEFB124-induced RWPE-1 cells. The mRNA expression of these genes was determined by using quantitative real-time polymerase chain reaction. Relative expression levels of each gene were calculated from cycle threshold values and were normalized with ACTB, and the expression ratio was calculated against the expression of each gene in the empty vector-transfected RWPE-1 cells. Experiments were repeated at least three times, and data are expressed as the mean±standard error of the mean (SEM). Asterisks, * and ** , represent statistical significance at p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection

    Overexpression of β-defensin 124 (DEFB124) in RWPE-1 cells. (A) DEFB124 mRNA overexpression. The RWPE-1 cells were transfected with DEFB124-DDK-Myc vector or empty vector, and DEFB124 mRNA expression was determined by reverse transcription-polymerase chain reaction (left) and quantitative real-time polymerase chain reaction (right). ACTB was used as an internal control. Asterisk represents statistical significance at p
    Figure Legend Snippet: Overexpression of β-defensin 124 (DEFB124) in RWPE-1 cells. (A) DEFB124 mRNA overexpression. The RWPE-1 cells were transfected with DEFB124-DDK-Myc vector or empty vector, and DEFB124 mRNA expression was determined by reverse transcription-polymerase chain reaction (left) and quantitative real-time polymerase chain reaction (right). ACTB was used as an internal control. Asterisk represents statistical significance at p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Peptidoglycan (PGN) induces gene expression of β-defensin 124 (DEFB124) through nuclear factor-kappa B (NF-κB) activation in RWPE-1 cells. (A) PGN induces DEFB124 mRNA expression. The RWPE-1 cells were treated with PGN (10 µg/mL) for the indicated times and DEFB124 expression was determined by reverse transcription-polymerase chain reaction. (B) PGN leads to NF-κB activation in RWPE-1 cells. The cells were stimulated with 10 µg/mL of PGN, and protein was extracted at the indicated time points. The phosphorylation and ubiquitination of IκBα were examined by western blot. ACTB was used as an internal control. (C) The activation of NF-κB is required for the upregulation of PGN-induced DEFB124. The RWPE-1 cells were treated with PGN or NF-κB inhibitor Bay11-7082 (10 µM), and the concentration of secreted DEFB124 protein was measured in the culture supernatant by enzyme-linked immunosorbent assay. Concentrations are picograms of protein per mL, and data are the mean results of three distinct experiments. Different letters indicate significant differences at p
    Figure Legend Snippet: Peptidoglycan (PGN) induces gene expression of β-defensin 124 (DEFB124) through nuclear factor-kappa B (NF-κB) activation in RWPE-1 cells. (A) PGN induces DEFB124 mRNA expression. The RWPE-1 cells were treated with PGN (10 µg/mL) for the indicated times and DEFB124 expression was determined by reverse transcription-polymerase chain reaction. (B) PGN leads to NF-κB activation in RWPE-1 cells. The cells were stimulated with 10 µg/mL of PGN, and protein was extracted at the indicated time points. The phosphorylation and ubiquitination of IκBα were examined by western blot. ACTB was used as an internal control. (C) The activation of NF-κB is required for the upregulation of PGN-induced DEFB124. The RWPE-1 cells were treated with PGN or NF-κB inhibitor Bay11-7082 (10 µM), and the concentration of secreted DEFB124 protein was measured in the culture supernatant by enzyme-linked immunosorbent assay. Concentrations are picograms of protein per mL, and data are the mean results of three distinct experiments. Different letters indicate significant differences at p

    Techniques Used: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Beta-Defensin 124 (DEFB124)-mediated upregulation of cytokines and chemokines promote chemotactic response of THP-1 monocytes. DEFB124- or DEFB124-mediated cytokines and chemokines induce chemotaxis for THP-1 monocytes. THP-1 cells (1×10 6 cells) were added to the upper chamber, and the lower chamber contained supernatants from either the empty vector- or DEFB124-DDK-Myc-transfected RWPE-1 cells. The results are presented as a migration index denoting the fold increase of cell migration over the empty vector. Monocyte chemoattractant protein-1 (MCP-1) (100 ng/mL) was used as a positive control. Results are representative of three independent experiments. Asterisk represents statistical significance at p
    Figure Legend Snippet: Beta-Defensin 124 (DEFB124)-mediated upregulation of cytokines and chemokines promote chemotactic response of THP-1 monocytes. DEFB124- or DEFB124-mediated cytokines and chemokines induce chemotaxis for THP-1 monocytes. THP-1 cells (1×10 6 cells) were added to the upper chamber, and the lower chamber contained supernatants from either the empty vector- or DEFB124-DDK-Myc-transfected RWPE-1 cells. The results are presented as a migration index denoting the fold increase of cell migration over the empty vector. Monocyte chemoattractant protein-1 (MCP-1) (100 ng/mL) was used as a positive control. Results are representative of three independent experiments. Asterisk represents statistical significance at p

    Techniques Used: Chemotaxis Assay, Plasmid Preparation, Transfection, Migration, Positive Control

    27) Product Images from "Epigenetic Suppression of SERPINB1 Promotes Inflammation-Mediated Prostate Cancer Progression"

    Article Title: Epigenetic Suppression of SERPINB1 Promotes Inflammation-Mediated Prostate Cancer Progression

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-18-0638

    SERPINB1 expression is induced via ERK1/2 signaling in normal prostate but not prostate cancer cells. A. RWPE-1 cells were serum starved and treated with epidermal growth factor (EGF; 20 ng/mL) for 4 hours. mRNA expression of SERPINB1 , PI3 , and SLPI were determined using quantitative PCR and normalized to GAPDH . Data were normalized to untreated samples, and differences were assessed using Student’s t-test (n = 3; ** p
    Figure Legend Snippet: SERPINB1 expression is induced via ERK1/2 signaling in normal prostate but not prostate cancer cells. A. RWPE-1 cells were serum starved and treated with epidermal growth factor (EGF; 20 ng/mL) for 4 hours. mRNA expression of SERPINB1 , PI3 , and SLPI were determined using quantitative PCR and normalized to GAPDH . Data were normalized to untreated samples, and differences were assessed using Student’s t-test (n = 3; ** p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    SERPINB1 is epigenetically repressed by EZH2-mediated histone methylation and DNMT-mediated DNA methylation in prostate cancer. A. Chromatin immunoprecipitation for H3K27me3 on the SERPINB1 promoter was performed in RWPE-1 and C4-2 cells. Data are presented as fold enrichment over IgG controls, and the average of two experiments is shown. B. C4-2 cells were treated with EZH2 inhibitor GSK343 (10 μM) for 72 hours. mRNA expression of SERPINB1 , PI3 , and SLPI were determined using quantitative PCR and normalized to GAPDH . Data were normalized to untreated samples, and differences were assessed using Student’s t-test (n = 3; * p
    Figure Legend Snippet: SERPINB1 is epigenetically repressed by EZH2-mediated histone methylation and DNMT-mediated DNA methylation in prostate cancer. A. Chromatin immunoprecipitation for H3K27me3 on the SERPINB1 promoter was performed in RWPE-1 and C4-2 cells. Data are presented as fold enrichment over IgG controls, and the average of two experiments is shown. B. C4-2 cells were treated with EZH2 inhibitor GSK343 (10 μM) for 72 hours. mRNA expression of SERPINB1 , PI3 , and SLPI were determined using quantitative PCR and normalized to GAPDH . Data were normalized to untreated samples, and differences were assessed using Student’s t-test (n = 3; * p

    Techniques Used: Methylation, DNA Methylation Assay, Chromatin Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction

    SERPINB1 loss stimulates EMT and a proliferative phenotype in normal prostate cells. A. RWPE-1 cells were transfected with non-specific (NSP) and SERPINB1-specific siRNA, and knockdown was verified using Western blot. GAPDH was used as a loading control. A representative blot is shown. WCL=whole cell lystate, CM=complete media (secreted SERPBINB1). B. mRNA expression of SERPINB1 and EMT markers MMP9 , TWIST1 , SNAI1 were determined in RWPE-1 cells after SERPINB1 knockdown using quantitative PCR and normalized to GAPDH . Data were normalized to NSP treated samples, and differences were assessed using Student’s t-test (n = 7; * p
    Figure Legend Snippet: SERPINB1 loss stimulates EMT and a proliferative phenotype in normal prostate cells. A. RWPE-1 cells were transfected with non-specific (NSP) and SERPINB1-specific siRNA, and knockdown was verified using Western blot. GAPDH was used as a loading control. A representative blot is shown. WCL=whole cell lystate, CM=complete media (secreted SERPBINB1). B. mRNA expression of SERPINB1 and EMT markers MMP9 , TWIST1 , SNAI1 were determined in RWPE-1 cells after SERPINB1 knockdown using quantitative PCR and normalized to GAPDH . Data were normalized to NSP treated samples, and differences were assessed using Student’s t-test (n = 7; * p

    Techniques Used: Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction

    28) Product Images from "A HUMAN-DERIVED PROSTATE CO-CULTURE MICROTISSUE MODEL USING EPITHELIAL (RWPE-1) AND STROMAL (WPMY-1) CELL LINES"

    Article Title: A HUMAN-DERIVED PROSTATE CO-CULTURE MICROTISSUE MODEL USING EPITHELIAL (RWPE-1) AND STROMAL (WPMY-1) CELL LINES

    Journal: Toxicology in vitro : an international journal published in association with BIBRA

    doi: 10.1016/j.tiv.2019.05.023

    Confocal images of prostate co-culture microtissues. The co-cultures of epithelial RWPE-1 cells (yellow) and stromal WPMY-1 cells (green) were grown in medium containing charcoal-stripped serumfor 3 (A) or 5 (B) days and undergo a spontaneous re-arrangement so that by day 5 most microtissues consist of acore of WPMY-1 cells surrounded by RWPE-1 cells. Scale bar = 2 mm.
    Figure Legend Snippet: Confocal images of prostate co-culture microtissues. The co-cultures of epithelial RWPE-1 cells (yellow) and stromal WPMY-1 cells (green) were grown in medium containing charcoal-stripped serumfor 3 (A) or 5 (B) days and undergo a spontaneous re-arrangement so that by day 5 most microtissues consist of acore of WPMY-1 cells surrounded by RWPE-1 cells. Scale bar = 2 mm.

    Techniques Used: Co-Culture Assay

    Immunohistochemical staining for CK5/6 (A, C, E, G) and confocal images (B, D, F, H) of prostate microtissue co-cultures. Prostate microtissues were exposed for 4 days to flutamide (C, D), DHT (E, F), or DHT+flutamide (G, H). Flutamide causes an increased incidence of fragmented cores of WPMY-1 cells (green in confocal images) compared with control and DHT, whilst DHT+flutamide causes fewer microtissues with clearly defined WPMY-1 cores, and in many cases the core consists of RWPE-1 cells (yellow in confocal images).
    Figure Legend Snippet: Immunohistochemical staining for CK5/6 (A, C, E, G) and confocal images (B, D, F, H) of prostate microtissue co-cultures. Prostate microtissues were exposed for 4 days to flutamide (C, D), DHT (E, F), or DHT+flutamide (G, H). Flutamide causes an increased incidence of fragmented cores of WPMY-1 cells (green in confocal images) compared with control and DHT, whilst DHT+flutamide causes fewer microtissues with clearly defined WPMY-1 cores, and in many cases the core consists of RWPE-1 cells (yellow in confocal images).

    Techniques Used: Immunohistochemistry, Staining

    29) Product Images from "Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer"

    Article Title: Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-77

    Native electroblot activity staining and anti-OPH reveal OPH bands. LNCaP and RWPE-1 cell lysates each containing 120 μg of protein were subjected to 6% n-PAGE followed by electroblot transfer to a nitrocellulose membrane. A) The blot was stained with 800 μM S-ANAA and Fast Blue RR salt. A parallel unstained blot was probed with anti-OPH as stated in the Materials and methods section. B) The esterase activity and C) anti-OPH bands were measured by densitometry and expressed in arbitrary units (A.U.). Letters that are not the same are significantly different at P
    Figure Legend Snippet: Native electroblot activity staining and anti-OPH reveal OPH bands. LNCaP and RWPE-1 cell lysates each containing 120 μg of protein were subjected to 6% n-PAGE followed by electroblot transfer to a nitrocellulose membrane. A) The blot was stained with 800 μM S-ANAA and Fast Blue RR salt. A parallel unstained blot was probed with anti-OPH as stated in the Materials and methods section. B) The esterase activity and C) anti-OPH bands were measured by densitometry and expressed in arbitrary units (A.U.). Letters that are not the same are significantly different at P

    Techniques Used: Activity Assay, Staining, Polyacrylamide Gel Electrophoresis

    LNCaP prostate cancer cells contain higher esterase activity than non-tumorigenic RWPE-1 cells. A) LNCaP and RWPE-1 cell cultures were incubated with 800 μM α-naphthyl acetate, R-ANAA, or S-ANAA substrate and Fast Blue RR salt for 20 min. The dark color indicates in situ esterase activity. B) Microscopy images were analyzed with ImageJ to measure the relative staining between cell lines. Percent area threshold was defined as the percent area stained that exceeded background staining. Letters that are not the same are significantly different at P
    Figure Legend Snippet: LNCaP prostate cancer cells contain higher esterase activity than non-tumorigenic RWPE-1 cells. A) LNCaP and RWPE-1 cell cultures were incubated with 800 μM α-naphthyl acetate, R-ANAA, or S-ANAA substrate and Fast Blue RR salt for 20 min. The dark color indicates in situ esterase activity. B) Microscopy images were analyzed with ImageJ to measure the relative staining between cell lines. Percent area threshold was defined as the percent area stained that exceeded background staining. Letters that are not the same are significantly different at P

    Techniques Used: Activity Assay, Incubation, In Situ, Microscopy, Staining

    OPH protein levels are differentially expressed in prostate cell lysates. A) RWPE-1, LNCaP, DU 145, and PC3 lysates containing 90 μg of protein were separated by 6% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-OPH antibody. Anti-OPH bands were measured by densitometry and expressed in arbitrary units (A.U.). B) RWPE-1, LNCaP, DU 145, and PC3 lysates containing 90 μg of protein were incubated with 4 mM AcApNA and the amount of p-nitroaniline released after 10 minutes were calculated. PLE (0.5 units) and semi-purified rat liver OPH (250 ng) were used as negative and positive controls respectively. Letters that are not the same are significantly different at P
    Figure Legend Snippet: OPH protein levels are differentially expressed in prostate cell lysates. A) RWPE-1, LNCaP, DU 145, and PC3 lysates containing 90 μg of protein were separated by 6% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-OPH antibody. Anti-OPH bands were measured by densitometry and expressed in arbitrary units (A.U.). B) RWPE-1, LNCaP, DU 145, and PC3 lysates containing 90 μg of protein were incubated with 4 mM AcApNA and the amount of p-nitroaniline released after 10 minutes were calculated. PLE (0.5 units) and semi-purified rat liver OPH (250 ng) were used as negative and positive controls respectively. Letters that are not the same are significantly different at P

    Techniques Used: SDS Page, Incubation, Purification

    Non-tumorigenic RWPE-1 and tumorigenic LNCaP prostate cell lysates display differential esterase activity and substrate specificity. A) Non-tumorigenic RWPE-1 and tumorigenic LNCaP cell lysates containing 120 μg of protein were separated on a native 10-20% polyacrylamide gel. The gels were stained with 800 μM α-naphthyl acetate, R-ANAA, or S-ANAA substrate and Fast Blue RR salt. Native molecular weight markers (left side) were used to estimate the relative migration of some esterase bands (right side). B) The 359 kDa n-PAGE LNCaP and RWPE-1 esterase bands stained with R-ANAA or S-ANAA were measured by densitometry. C) The 432 kDa n-PAGE LNCaP and RWPE-1 esterase bands were also measured by densitometry. Letters that are not the same are significantly different at P
    Figure Legend Snippet: Non-tumorigenic RWPE-1 and tumorigenic LNCaP prostate cell lysates display differential esterase activity and substrate specificity. A) Non-tumorigenic RWPE-1 and tumorigenic LNCaP cell lysates containing 120 μg of protein were separated on a native 10-20% polyacrylamide gel. The gels were stained with 800 μM α-naphthyl acetate, R-ANAA, or S-ANAA substrate and Fast Blue RR salt. Native molecular weight markers (left side) were used to estimate the relative migration of some esterase bands (right side). B) The 359 kDa n-PAGE LNCaP and RWPE-1 esterase bands stained with R-ANAA or S-ANAA were measured by densitometry. C) The 432 kDa n-PAGE LNCaP and RWPE-1 esterase bands were also measured by densitometry. Letters that are not the same are significantly different at P

    Techniques Used: Activity Assay, Staining, Molecular Weight, Migration, Polyacrylamide Gel Electrophoresis

    Prostate cell lysate esterases form distinct bands when separated by 6% n-PAGE. A) RWPE-1, RWPE-2, LNCaP, DU 145, and PC3 cell lysates containing 120 μg of protein were separated by 6% n-PAGE followed by staining with either 800 μM α-naphthyl acetate, S-ANAA, or R-ANAA. Native molecular weight markers (left side) were used to calculate the relative migration of some esterase bands (right side). The B) 216 kDa and C) 198 kDa esterase bands visualized with S-ANAA were measured by densitometry. Letters that are not the same are significantly different at P
    Figure Legend Snippet: Prostate cell lysate esterases form distinct bands when separated by 6% n-PAGE. A) RWPE-1, RWPE-2, LNCaP, DU 145, and PC3 cell lysates containing 120 μg of protein were separated by 6% n-PAGE followed by staining with either 800 μM α-naphthyl acetate, S-ANAA, or R-ANAA. Native molecular weight markers (left side) were used to calculate the relative migration of some esterase bands (right side). The B) 216 kDa and C) 198 kDa esterase bands visualized with S-ANAA were measured by densitometry. Letters that are not the same are significantly different at P

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Migration

    Pre-clearance of OPH or inhibition by DFP ablates OPH activity bands. A) LNCaP and RWPE-1 lysates were separated by 6% n-PAGE. The gel was pre-incubated in phosphate buffer or phosphate buffer containing 50 μM DFP for 30 min. Esterase activity bands were visualized with S-ANAA and Fast Blue RR salt. B) LNCaP lysates containing 120 μg of protein were pre-cleared with protein A beads or anti-OPH antibody bound to protein A beads. The collected lysates were separated by 6% n-PAGE and the esterase activity visualized with S-ANAA and Fast Blue RR salt.
    Figure Legend Snippet: Pre-clearance of OPH or inhibition by DFP ablates OPH activity bands. A) LNCaP and RWPE-1 lysates were separated by 6% n-PAGE. The gel was pre-incubated in phosphate buffer or phosphate buffer containing 50 μM DFP for 30 min. Esterase activity bands were visualized with S-ANAA and Fast Blue RR salt. B) LNCaP lysates containing 120 μg of protein were pre-cleared with protein A beads or anti-OPH antibody bound to protein A beads. The collected lysates were separated by 6% n-PAGE and the esterase activity visualized with S-ANAA and Fast Blue RR salt.

    Techniques Used: Inhibition, Activity Assay, Polyacrylamide Gel Electrophoresis, Incubation

    30) Product Images from "Oncolytic targeting of androgen-sensitive prostate tumor by the respiratory syncytial virus (RSV): consequences of deficient interferon-dependent antiviral defense"

    Article Title: Oncolytic targeting of androgen-sensitive prostate tumor by the respiratory syncytial virus (RSV): consequences of deficient interferon-dependent antiviral defense

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-11-43

    Interferon response of prostate cancer cells . (a) IFN-β production from mock-infected and RSV-infected RWPE-1 and LNCaP cells was measured at 12 h and 24 h post-infection. Amount of IFN-β deduced from the ELISA assay was expressed as pg/ml and each value represents mean ± standard deviation for three determinations. (b) RSV infectivity in untreated (UT) and IFN pre-treated cells was measured by plaque assay at 24 h post-infection. The values are fold reductions in the RSV titer following IFN pre-treatment compared to untreated cells. Mean ± standard deviation for three determinations are shown. (c) Plaque assay showing RSV infectivity in untreated (UT) and IFN pre-treated RWPE-1 and LNCaP cells. Culture supernatant collected from RSV infected cells were added to CV-1 cells at various dilutions (1 × 10 4 - 1 × 10 6 dilutions). (d) Plaque assay showing RSV infectivity in untreated (UT) and IFN pre-treated RWPE-1 and PC-3 cells at 24 h post-infection. Culture supernatant collected from RSV infected cells was added to CV-1 cells at various dilutions (1 × 10 5 and 1 × 10 8 dilutions). For figures (c) and (d) the plaques were observed on methyl-cellulose after crystal-violet staining. Please note that figures (c) and (d) are two separate experiments.
    Figure Legend Snippet: Interferon response of prostate cancer cells . (a) IFN-β production from mock-infected and RSV-infected RWPE-1 and LNCaP cells was measured at 12 h and 24 h post-infection. Amount of IFN-β deduced from the ELISA assay was expressed as pg/ml and each value represents mean ± standard deviation for three determinations. (b) RSV infectivity in untreated (UT) and IFN pre-treated cells was measured by plaque assay at 24 h post-infection. The values are fold reductions in the RSV titer following IFN pre-treatment compared to untreated cells. Mean ± standard deviation for three determinations are shown. (c) Plaque assay showing RSV infectivity in untreated (UT) and IFN pre-treated RWPE-1 and LNCaP cells. Culture supernatant collected from RSV infected cells were added to CV-1 cells at various dilutions (1 × 10 4 - 1 × 10 6 dilutions). (d) Plaque assay showing RSV infectivity in untreated (UT) and IFN pre-treated RWPE-1 and PC-3 cells at 24 h post-infection. Culture supernatant collected from RSV infected cells was added to CV-1 cells at various dilutions (1 × 10 5 and 1 × 10 8 dilutions). For figures (c) and (d) the plaques were observed on methyl-cellulose after crystal-violet staining. Please note that figures (c) and (d) are two separate experiments.

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Plaque Assay, Staining

    Impaired IFNα- and RSV-mediated STAT-1 activation in LNCaP cells . STAT-1-specific EMSA of nuclear extracts prepared from mock-infected and RSV-infected LNCaP and RWPE-1 cells (a) PC-3 cells (b) . The STAT-1/DNA EMSA complex is indicated with arrowheads. (c) STAT-specific EMSA of nuclear extracts prepared from cells treated with (+) or without (-) IFNα for 4 hours. Asterisks represent STAT-1/DNA complex. NS: non specific band.
    Figure Legend Snippet: Impaired IFNα- and RSV-mediated STAT-1 activation in LNCaP cells . STAT-1-specific EMSA of nuclear extracts prepared from mock-infected and RSV-infected LNCaP and RWPE-1 cells (a) PC-3 cells (b) . The STAT-1/DNA EMSA complex is indicated with arrowheads. (c) STAT-specific EMSA of nuclear extracts prepared from cells treated with (+) or without (-) IFNα for 4 hours. Asterisks represent STAT-1/DNA complex. NS: non specific band.

    Techniques Used: Activation Assay, Infection

    Interferon response is essential for limiting RSV infection in prostate cells . (a) RSV infection of RWPE-1 in the absence (untreated, UT) or in the presence of either control (Con Ab) or IFN neutralizing (IFN Ab) antibody. Infection was measured by plaque assay at 36 h post-infection. Plaque assay values expressed as pfu/ml represent mean ± standard deviation for three independent determinations. Standard deviations are shown by error bars. (b) Plaque assay showing RSV infectivity in Con Ab or IFN Ab treated cells. Culture supernatant from RSV infected RWPE-1 cells (+/- Con Ab or IFN Ab) was added to CV-1 cells at a dilution of 1 × 10 6 . Plaques were observed on methyl-cellulose after crystal-violet staining.
    Figure Legend Snippet: Interferon response is essential for limiting RSV infection in prostate cells . (a) RSV infection of RWPE-1 in the absence (untreated, UT) or in the presence of either control (Con Ab) or IFN neutralizing (IFN Ab) antibody. Infection was measured by plaque assay at 36 h post-infection. Plaque assay values expressed as pfu/ml represent mean ± standard deviation for three independent determinations. Standard deviations are shown by error bars. (b) Plaque assay showing RSV infectivity in Con Ab or IFN Ab treated cells. Culture supernatant from RSV infected RWPE-1 cells (+/- Con Ab or IFN Ab) was added to CV-1 cells at a dilution of 1 × 10 6 . Plaques were observed on methyl-cellulose after crystal-violet staining.

    Techniques Used: Infection, Plaque Assay, Standard Deviation, Staining

    Oncolytic activity of RSV . (a) RSV infection of RWPE-1, LNCaP and RM1 cells were measured by plaque assay at 36 h post-infection. Plaque assay values expressed as pfu/ml represent mean ± standard deviation for three independent determinations. Standard deviations are shown by the error bars. (b) Morphology of mock-infected or RSV-infected cells at the indicated post-infection time periods. (c) MTT cell viability assay of cells infected with RSV for 36 h. MTT assay values are mean ± standard deviation of 6 wells and triplicate experiments. Uninfected (-) cells indicate 100% cell viability. (d) RSV mediated apoptosis at 24 h post-infection. % Apoptosis represents the % of cells that are undergoing apoptosis (i.e. positive for annexin V staining). The values represent mean ± SD for three determinations.
    Figure Legend Snippet: Oncolytic activity of RSV . (a) RSV infection of RWPE-1, LNCaP and RM1 cells were measured by plaque assay at 36 h post-infection. Plaque assay values expressed as pfu/ml represent mean ± standard deviation for three independent determinations. Standard deviations are shown by the error bars. (b) Morphology of mock-infected or RSV-infected cells at the indicated post-infection time periods. (c) MTT cell viability assay of cells infected with RSV for 36 h. MTT assay values are mean ± standard deviation of 6 wells and triplicate experiments. Uninfected (-) cells indicate 100% cell viability. (d) RSV mediated apoptosis at 24 h post-infection. % Apoptosis represents the % of cells that are undergoing apoptosis (i.e. positive for annexin V staining). The values represent mean ± SD for three determinations.

    Techniques Used: Activity Assay, Infection, Plaque Assay, Standard Deviation, MTT Assay, Viability Assay, Staining

    NF-κB activation in RSV infected prostate cancer cells . NF-κB specific EMSA of nuclear extracts prepared from mock-infected and RSV-infected LNCaP ( a ) RWPE-1 ( b ) and PC-3 (c) cells. Duration of infection (in hours) with RSV is indicated. The NF-κB/DNA EMSA complex is indicated with arrowheads. Competition of the EMSA complex with the cold NF-κB duplex oligonucleotide, but not with cold STAT1/2 oligo duplex in panel (a) ensures the specific nature of the EMSA complex.
    Figure Legend Snippet: NF-κB activation in RSV infected prostate cancer cells . NF-κB specific EMSA of nuclear extracts prepared from mock-infected and RSV-infected LNCaP ( a ) RWPE-1 ( b ) and PC-3 (c) cells. Duration of infection (in hours) with RSV is indicated. The NF-κB/DNA EMSA complex is indicated with arrowheads. Competition of the EMSA complex with the cold NF-κB duplex oligonucleotide, but not with cold STAT1/2 oligo duplex in panel (a) ensures the specific nature of the EMSA complex.

    Techniques Used: Activation Assay, Infection

    31) Product Images from "MiR-491-5p negatively regulates cell proliferation and motility by targeting PDGFRA in prostate cancer"

    Article Title: MiR-491-5p negatively regulates cell proliferation and motility by targeting PDGFRA in prostate cancer

    Journal: American Journal of Cancer Research

    doi:

    miR-491-5p expression was downregulated in PCa cell lines and tissues. A. Decreased miR-491-5p expression was detected in all three PCa cell lines (LNCaP, DU145, and PC-3) compared to in the normal human prostate epithelial cell line RWPE-1. B. qRT-PCR analysis of miR-491-5p expression in 18 pairs of human PCa tissues and their adjacent normal prostate tissues. The error bars represent the mean ± S.D. of three independent experiments. * P
    Figure Legend Snippet: miR-491-5p expression was downregulated in PCa cell lines and tissues. A. Decreased miR-491-5p expression was detected in all three PCa cell lines (LNCaP, DU145, and PC-3) compared to in the normal human prostate epithelial cell line RWPE-1. B. qRT-PCR analysis of miR-491-5p expression in 18 pairs of human PCa tissues and their adjacent normal prostate tissues. The error bars represent the mean ± S.D. of three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    32) Product Images from "Downregulation of C-Terminal Tensin-Like Protein (CTEN) Suppresses Prostate Cell Proliferation and Contributes to Acinar Morphogenesis"

    Article Title: Downregulation of C-Terminal Tensin-Like Protein (CTEN) Suppresses Prostate Cell Proliferation and Contributes to Acinar Morphogenesis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19103190

    CTEN acts as a binding partner of integrin β1 and increases the activity of FAK and RhoA. ( a ) Whole cell lysate of RWPE-1 were coimmunoprecipitated (CoIP) with anti-CTEN antibody or normal rabbit IgG. The precipitates were examined by immunoblotting (IB) with anti-integrin β1 and anti-CTEN antibodies. ( b ) Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 6 days in the presence of doxycycline (0.2 μg/mL). The cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. The densities of the protein bands from the Western blot were quantified. The levels of the phosphorylated FAK Y397 (pFAK Y397 ) was normalized to those of the total FAK. The ratio of pFAKY397/FAK in each group was presented. ( c ) RWPE-1 cells were transfected with the indicated siRNA. The GTP-RhoA in RWPE-1 lysates was purified using GTP-RhoA pull-down assays as described in Materials and Methods and examined by Western blot analyses with an anti-RhoA antibody. The protein levels in whole cell lysates were also examined by Western analyses using the indicated antibodies. Total RhoA was used as loading control. The densities of the protein bands from the Western blot were quantified. The levels of Rho-GTP was normalized to those of total RhoA. The ratio of Rho-GTP/total RhoA was presented.
    Figure Legend Snippet: CTEN acts as a binding partner of integrin β1 and increases the activity of FAK and RhoA. ( a ) Whole cell lysate of RWPE-1 were coimmunoprecipitated (CoIP) with anti-CTEN antibody or normal rabbit IgG. The precipitates were examined by immunoblotting (IB) with anti-integrin β1 and anti-CTEN antibodies. ( b ) Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 6 days in the presence of doxycycline (0.2 μg/mL). The cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. The densities of the protein bands from the Western blot were quantified. The levels of the phosphorylated FAK Y397 (pFAK Y397 ) was normalized to those of the total FAK. The ratio of pFAKY397/FAK in each group was presented. ( c ) RWPE-1 cells were transfected with the indicated siRNA. The GTP-RhoA in RWPE-1 lysates was purified using GTP-RhoA pull-down assays as described in Materials and Methods and examined by Western blot analyses with an anti-RhoA antibody. The protein levels in whole cell lysates were also examined by Western analyses using the indicated antibodies. Total RhoA was used as loading control. The densities of the protein bands from the Western blot were quantified. The levels of Rho-GTP was normalized to those of total RhoA. The ratio of Rho-GTP/total RhoA was presented.

    Techniques Used: Binding Assay, Activity Assay, Co-Immunoprecipitation Assay, Expressing, Incubation, Western Blot, Transfection, Purification

    Inducible EGFP-expressing and CTEN-expressing RWPE-1 cells perform similar phenotypes in the absence of doxycycline in 3D culture. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days without doxycycline induction. ( a ) The appearance of acinar structures was observed and classified to three types (normal, notched and deformed). The numbers of the three-type acini in each group were counted and presented as a percentage of the whole (upper panel). Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; no significant difference between aON-EGFP and aON-CTEN in each phenotype). The representative images of acinar structure were also shown in the lower panel. Scale bar: 100 μm. ( b ) aON-EGFP and aON-CTEN RWPE-1 cells grown in 3D culture were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control.
    Figure Legend Snippet: Inducible EGFP-expressing and CTEN-expressing RWPE-1 cells perform similar phenotypes in the absence of doxycycline in 3D culture. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days without doxycycline induction. ( a ) The appearance of acinar structures was observed and classified to three types (normal, notched and deformed). The numbers of the three-type acini in each group were counted and presented as a percentage of the whole (upper panel). Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; no significant difference between aON-EGFP and aON-CTEN in each phenotype). The representative images of acinar structure were also shown in the lower panel. Scale bar: 100 μm. ( b ) aON-EGFP and aON-CTEN RWPE-1 cells grown in 3D culture were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control.

    Techniques Used: Expressing, Incubation, Western Blot

    Forced CTEN expression disrupts acinar morphogenesis. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days in the presence of doxycycline (0.2 μg/mL). ( a ) Cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. ( b ) The numbers of the three-type acini in each group were counted at Day 9 and presented as a percentage of the whole. Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; * p
    Figure Legend Snippet: Forced CTEN expression disrupts acinar morphogenesis. Inducible EGFP-expressing (aON-EGFP) or CTEN-expressing (aON-CTEN) RWPE-1 cells were incubated in 3D culture system for 9 days in the presence of doxycycline (0.2 μg/mL). ( a ) Cells were collected at the indicated time and the protein levels were examined by Western analyses using the indicated antibodies. α-tubulin was used as a loading control. ( b ) The numbers of the three-type acini in each group were counted at Day 9 and presented as a percentage of the whole. Data were expressed as mean ± SD of three independent experiments. More than 50 acini were evaluated within each sample (Student’s t test; * p

    Techniques Used: Expressing, Incubation, Western Blot

    Knockdown of CTEN suppresses cell proliferation and downregulation of CTEN occurs during cell differentiation. ( a ) RWPE-1 cells were transfected with CTEN-specific siRNA (siCTEN) or nontargeting siRNA (siCtrl). Cell proliferation was evaluated by WST-1 based assay as described in Materials and Methods. Data were expressed as mean ± standard deviation (SD) of three independent experiments analyzed in triplicate (Student’s t test; ** p
    Figure Legend Snippet: Knockdown of CTEN suppresses cell proliferation and downregulation of CTEN occurs during cell differentiation. ( a ) RWPE-1 cells were transfected with CTEN-specific siRNA (siCTEN) or nontargeting siRNA (siCtrl). Cell proliferation was evaluated by WST-1 based assay as described in Materials and Methods. Data were expressed as mean ± standard deviation (SD) of three independent experiments analyzed in triplicate (Student’s t test; ** p

    Techniques Used: Cell Differentiation, Transfection, WST-1 Assay, Standard Deviation

    33) Product Images from "Spatial enrichment of phosphoinositide metabolism is a molecular switch to promote metastasis"

    Article Title: Spatial enrichment of phosphoinositide metabolism is a molecular switch to promote metastasis

    Journal: bioRxiv

    doi: 10.1101/851410

    Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression from q-PCR. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 biological replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.
    Figure Legend Snippet: Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression from q-PCR. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 biological replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Techniques Used: Expressing, Polymerase Chain Reaction, Transformation Assay, Activation Assay, Generated, Western Blot

    34) Product Images from "Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis"

    Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

    Journal: bioRxiv

    doi: 10.1101/851410

    Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.
    Figure Legend Snippet: Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

    Techniques Used: Expressing, Transformation Assay, Activation Assay, Generated, Western Blot, Quantitation Assay

    35) Product Images from "Identification of PLX4032-resistance mechanisms and implications for novel RAF inhibitors"

    Article Title: Identification of PLX4032-resistance mechanisms and implications for novel RAF inhibitors

    Journal: Pigment Cell & Melanoma Research

    doi: 10.1111/pcmr.12197

    BRAF L505H mutation induces oncogenic MAPK signaling. (A) Changes in pMEK and pERK in 293T cells expressing the indicated BRAF variant. (B) In vitro kinase activity of WT (wild-type) BRAF, BRAF V600E , BRAF L505H , and BRAF V600E/L505H , employing MEK as a substrate. The figure shows the total immunoprecipitated BRAF, the phosphorylated levels of the substrate MEK (pMEK), and total MEK. (C) Changes in pMEK and pERK in 293T cells expressing the indicated BRAF variant ( BRAF V600E , BRAF L505H , or BRAF V600E/L505H ) after 18 h treatment with DMSO or PLX4032 (1 μ M, or 3 μ M). (D) IP kinase assay performed on lysates from 293T cells expressing the indicated BRAF variant, treated with DMSO, or increasing concentration of PLX4032 (1, 3, or 10 μ M) employing MEK as a substrate. (E) Changes in pERK in response to MEK inhibitor (AZD6244). 293T cells expressing the indicated BRAF variant were treated with DMSO, or AZD6244 (0.03, 0.1, 0.3, 1, or 3 μ M) for 18 h. (F) Changes in pERK and pMEK in RWPE-1 prostate cells expressing the indicated doxycycline inducible BRAF variant. (G) Soft agar colonies of RWPE-1 prostate cells expressing the indicated doxycycline-inducible BRAF variant; numbers are averages of triplicate cultures after 3 weeks incubation. *indicates a P-value
    Figure Legend Snippet: BRAF L505H mutation induces oncogenic MAPK signaling. (A) Changes in pMEK and pERK in 293T cells expressing the indicated BRAF variant. (B) In vitro kinase activity of WT (wild-type) BRAF, BRAF V600E , BRAF L505H , and BRAF V600E/L505H , employing MEK as a substrate. The figure shows the total immunoprecipitated BRAF, the phosphorylated levels of the substrate MEK (pMEK), and total MEK. (C) Changes in pMEK and pERK in 293T cells expressing the indicated BRAF variant ( BRAF V600E , BRAF L505H , or BRAF V600E/L505H ) after 18 h treatment with DMSO or PLX4032 (1 μ M, or 3 μ M). (D) IP kinase assay performed on lysates from 293T cells expressing the indicated BRAF variant, treated with DMSO, or increasing concentration of PLX4032 (1, 3, or 10 μ M) employing MEK as a substrate. (E) Changes in pERK in response to MEK inhibitor (AZD6244). 293T cells expressing the indicated BRAF variant were treated with DMSO, or AZD6244 (0.03, 0.1, 0.3, 1, or 3 μ M) for 18 h. (F) Changes in pERK and pMEK in RWPE-1 prostate cells expressing the indicated doxycycline inducible BRAF variant. (G) Soft agar colonies of RWPE-1 prostate cells expressing the indicated doxycycline-inducible BRAF variant; numbers are averages of triplicate cultures after 3 weeks incubation. *indicates a P-value

    Techniques Used: Mutagenesis, Expressing, Variant Assay, In Vitro, Activity Assay, Immunoprecipitation, IP-Kinase Assay, Concentration Assay, Incubation

    36) Product Images from "Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin"

    Article Title: Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-11-70

    Real-time quantitative RT-PCR assays. Assays were performed to determine the relative mRNA expression levels of (A) GOAT ( MBOAT4 ), (B) PC1/3 ( PCSK1 ), (C) PC2 ( PCSK2 ), and (D) furin ( FURIN ) in the RWPE-1 and RWPE-2 normal-prostate derived cell lines and the DU145, LNCaP and PC3 prostate cancer-derived cell lines. Data are expressed relative to the RWPE-1 cell line mRNA levels (set as 1) and are presented as means ± S.E.M. (n = 2, assays performed in duplicate) and compared by one-way ANOVA, followed by Tukey’s post-hoc analysis.* = P
    Figure Legend Snippet: Real-time quantitative RT-PCR assays. Assays were performed to determine the relative mRNA expression levels of (A) GOAT ( MBOAT4 ), (B) PC1/3 ( PCSK1 ), (C) PC2 ( PCSK2 ), and (D) furin ( FURIN ) in the RWPE-1 and RWPE-2 normal-prostate derived cell lines and the DU145, LNCaP and PC3 prostate cancer-derived cell lines. Data are expressed relative to the RWPE-1 cell line mRNA levels (set as 1) and are presented as means ± S.E.M. (n = 2, assays performed in duplicate) and compared by one-way ANOVA, followed by Tukey’s post-hoc analysis.* = P

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay

    Real-time quantitative RT-PCR analysis of GOAT gene expression in response to ghrelin and desacyl ghrelin treatments for 6 hours. GOAT mRNA expression in ghrelin treated (A) RWPE-1 normal prostate derived cell line, and the (C) DU145, (E) LNCaP and (G) PC3 prostate cancer cell lines and desacyl ghrelin-treated (B) RWPE-1, (D) DU145 and (F) LNCaP and (H) PC3 cell lines. Data is represented as means and standard error of two technical replicates from two independent replicate experiments (n = 2). * P
    Figure Legend Snippet: Real-time quantitative RT-PCR analysis of GOAT gene expression in response to ghrelin and desacyl ghrelin treatments for 6 hours. GOAT mRNA expression in ghrelin treated (A) RWPE-1 normal prostate derived cell line, and the (C) DU145, (E) LNCaP and (G) PC3 prostate cancer cell lines and desacyl ghrelin-treated (B) RWPE-1, (D) DU145 and (F) LNCaP and (H) PC3 cell lines. Data is represented as means and standard error of two technical replicates from two independent replicate experiments (n = 2). * P

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay

    37) Product Images from "CD82 Suppresses ADAM17-Dependent E-Cadherin Cleavage and Cell Migration in Prostate Cancer"

    Article Title: CD82 Suppresses ADAM17-Dependent E-Cadherin Cleavage and Cell Migration in Prostate Cancer

    Journal: Disease Markers

    doi: 10.1155/2020/8899924

    The expression of CD82 in PCa tissues and cell lines. (a) Immunohistochemical assay was applied to detect the expressions of CD82 in normal prostate tissues, prostate cancers, and matched adjacent normal tissues. Quantification of CD82 staining was shown in cancer and matched adjacent tissue and in cancers with different Gleason scores (N: matched adjacent normal tissue, T: prostate cancer). (b) Real-time PCR (left) and Western blot (right) showed the expressions of CD82 in prostate normal epithelia cells RWPE-1, benign prostatic hyperplasia cell BPH-1, and various PCa cell lines. Error bars indicate s.d. from at least two technical replicates.
    Figure Legend Snippet: The expression of CD82 in PCa tissues and cell lines. (a) Immunohistochemical assay was applied to detect the expressions of CD82 in normal prostate tissues, prostate cancers, and matched adjacent normal tissues. Quantification of CD82 staining was shown in cancer and matched adjacent tissue and in cancers with different Gleason scores (N: matched adjacent normal tissue, T: prostate cancer). (b) Real-time PCR (left) and Western blot (right) showed the expressions of CD82 in prostate normal epithelia cells RWPE-1, benign prostatic hyperplasia cell BPH-1, and various PCa cell lines. Error bars indicate s.d. from at least two technical replicates.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Western Blot

    38) Product Images from "Exploiting the Metabolic Consequences of PTEN Loss and Akt/Hexokinase 2 Hyperactivation in Prostate Cancer: A New Role for δ-Tocotrienol"

    Article Title: Exploiting the Metabolic Consequences of PTEN Loss and Akt/Hexokinase 2 Hyperactivation in Prostate Cancer: A New Role for δ-Tocotrienol

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23095269

    δ-TT downregulates HK2 in LNCaP PCa cells via inhibition of Akt. ( a ) PTEN expression was evaluated in RWPE-1 and LNCaP cells by Western blot analysis. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown. ( b ) Akt hyperactivation was evaluated in RWPE-1 and LNCaP cells by Western blot analysis. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown. ( c ) After δ-TT treatment (15 μg/mL, 1–24 h), Western blot analysis was performed to investigate the expression levels of p-Akt in LNCaP cells. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown.
    Figure Legend Snippet: δ-TT downregulates HK2 in LNCaP PCa cells via inhibition of Akt. ( a ) PTEN expression was evaluated in RWPE-1 and LNCaP cells by Western blot analysis. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown. ( b ) Akt hyperactivation was evaluated in RWPE-1 and LNCaP cells by Western blot analysis. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown. ( c ) After δ-TT treatment (15 μg/mL, 1–24 h), Western blot analysis was performed to investigate the expression levels of p-Akt in LNCaP cells. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown.

    Techniques Used: Inhibition, Expressing, Western Blot

    δ-TT cytotoxicity correlates with the suppression of the HK2-driven Warburg effect in LNCaP PCa cells. ( a ) HK2 expression was evaluated in RWPE-1 and LNCaP cells by Western blot analysis. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown. ( b ) After δ-TT treatment (15 μg/mL, 1–24 h), Western blot analysis was performed to investigate the expression levels of HK2 in LNCaP cells. Tubulin expression was evaluated as a loading control. Data represent mean values ± SEM and were analyzed by Dunnett’s test after one-way analysis of variance. * p
    Figure Legend Snippet: δ-TT cytotoxicity correlates with the suppression of the HK2-driven Warburg effect in LNCaP PCa cells. ( a ) HK2 expression was evaluated in RWPE-1 and LNCaP cells by Western blot analysis. Tubulin expression was evaluated as a loading control. One representative of three experiments performed is shown. ( b ) After δ-TT treatment (15 μg/mL, 1–24 h), Western blot analysis was performed to investigate the expression levels of HK2 in LNCaP cells. Tubulin expression was evaluated as a loading control. Data represent mean values ± SEM and were analyzed by Dunnett’s test after one-way analysis of variance. * p

    Techniques Used: Expressing, Western Blot

    39) Product Images from "Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner"

    Article Title: Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167894

    Human mammary and prostate epithelial cells simultaneously activate matriptase and prostasin in response to transient exposure to a pH 6.0 buffer. Human prostate epithelial cells RWPE-1 ( A .), mammary epithelial cells 184 A1N4 ( B .), MTSV 1.1B ( C .) and MTSV 1.7 ( D .) were exposed to PBS as a non-activation control (N, lanes 1 and 3) or phosphate buffer pH 6.0 for 20 min (A, lanes 2 and 4). Cell lysates were prepared and analyzed by Western blot for species containing matriptase (MTP, lanes 1 and 2, bands a and c ) or prostasin (Pros., lanes 3 and 4, bands b , d , and e ). This experiment was conducted at least twice with the same profile observed. Representative data are shown.
    Figure Legend Snippet: Human mammary and prostate epithelial cells simultaneously activate matriptase and prostasin in response to transient exposure to a pH 6.0 buffer. Human prostate epithelial cells RWPE-1 ( A .), mammary epithelial cells 184 A1N4 ( B .), MTSV 1.1B ( C .) and MTSV 1.7 ( D .) were exposed to PBS as a non-activation control (N, lanes 1 and 3) or phosphate buffer pH 6.0 for 20 min (A, lanes 2 and 4). Cell lysates were prepared and analyzed by Western blot for species containing matriptase (MTP, lanes 1 and 2, bands a and c ) or prostasin (Pros., lanes 3 and 4, bands b , d , and e ). This experiment was conducted at least twice with the same profile observed. Representative data are shown.

    Techniques Used: Activation Assay, Western Blot

    40) Product Images from "CTRP3 Stimulates Proliferation and Anti-Apoptosis of Prostate Cells through PKC Signaling Pathways"

    Article Title: CTRP3 Stimulates Proliferation and Anti-Apoptosis of Prostate Cells through PKC Signaling Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134006

    Growth curve demonstrating the effects of CTRP3 on the proliferation of prostate cells. Cells were treated with various concentrations of CTRP3: (A) 0 μg/mL, (B) 3 μg/mL, (C) 10 μg/mL, (D) 30 μg/mL. The optimum concentration of CTRP3 for the growth of RWPE-1 was 10 μg/mL.
    Figure Legend Snippet: Growth curve demonstrating the effects of CTRP3 on the proliferation of prostate cells. Cells were treated with various concentrations of CTRP3: (A) 0 μg/mL, (B) 3 μg/mL, (C) 10 μg/mL, (D) 30 μg/mL. The optimum concentration of CTRP3 for the growth of RWPE-1 was 10 μg/mL.

    Techniques Used: Concentration Assay

    Effect of CTRP3 on cell cycle was mediated through the PKC signaling pathway. RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. The percent of RWPE-1 cells in the G1 phase decreased significantly upon CTRP3 treatment whereas the percentage of cells in the S and G2 phase increased. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated effect. Mean±SD. * P
    Figure Legend Snippet: Effect of CTRP3 on cell cycle was mediated through the PKC signaling pathway. RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. The percent of RWPE-1 cells in the G1 phase decreased significantly upon CTRP3 treatment whereas the percentage of cells in the S and G2 phase increased. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated effect. Mean±SD. * P

    Techniques Used: Flow Cytometry, Cytometry

    Effect of CTRP3 on proliferation and apoptosis was mediated through the PKC signaling pathway. (A) Phosphorylation of intracellular PKC substrates increased in CTRP3 treated RWPE-1 cells and went back to normal when pretreated with PKC inhibitor. (B) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by a MTT assay. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated proliferation in RWPE-1 cells. (C) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated anti-apoptosis effect. Mean±SD. * P
    Figure Legend Snippet: Effect of CTRP3 on proliferation and apoptosis was mediated through the PKC signaling pathway. (A) Phosphorylation of intracellular PKC substrates increased in CTRP3 treated RWPE-1 cells and went back to normal when pretreated with PKC inhibitor. (B) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by a MTT assay. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated proliferation in RWPE-1 cells. (C) RWPE-1 cells were treated with 10 μg/mL of CTRP3 or staurosporine and then analyzed by flow cytometry. PKC inhibitor staurosporine completely abolished the CTRP3-stimulated anti-apoptosis effect. Mean±SD. * P

    Techniques Used: MTT Assay, Flow Cytometry, Cytometry

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  • rwpe 1  (ATCC)
    97
    ATCC rwpe 1
    Western blotting of mAbs 2-7A50 and 2-5C11 against cell extracts from tumoral and non-tumoral cell lines. MW: molecular weight marker; RW: <t>RWPE-1</t> cell line extract; PC-3: PC-3 cell line extract; β-A RW: β-actin antibody against RWPE-1 cell line extract; β-A PC-3: β-actin antibody against PC-3 cell line extract. Both mAbs recognized bands present only at the PC-3 cell line. No reactivity is shown against the RWPE-1 cell line. mAb 2-7A50 specifically detected a 45kDa band while mAb 2-5C11 specifically detected a 62 kDa band. Detection was performed using chemiluminescent method.
    Rwpe 1, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Western blotting of mAbs 2-7A50 and 2-5C11 against cell extracts from tumoral and non-tumoral cell lines. MW: molecular weight marker; RW: RWPE-1 cell line extract; PC-3: PC-3 cell line extract; β-A RW: β-actin antibody against RWPE-1 cell line extract; β-A PC-3: β-actin antibody against PC-3 cell line extract. Both mAbs recognized bands present only at the PC-3 cell line. No reactivity is shown against the RWPE-1 cell line. mAb 2-7A50 specifically detected a 45kDa band while mAb 2-5C11 specifically detected a 62 kDa band. Detection was performed using chemiluminescent method.

    Journal: Frontiers in Immunology

    Article Title: Multiple Tolerization Subtractive Immunization (MTSI) Protocol: Effects on Mice and Monoclonal Antibody Specificity

    doi: 10.3389/fimmu.2021.760817

    Figure Lengend Snippet: Western blotting of mAbs 2-7A50 and 2-5C11 against cell extracts from tumoral and non-tumoral cell lines. MW: molecular weight marker; RW: RWPE-1 cell line extract; PC-3: PC-3 cell line extract; β-A RW: β-actin antibody against RWPE-1 cell line extract; β-A PC-3: β-actin antibody against PC-3 cell line extract. Both mAbs recognized bands present only at the PC-3 cell line. No reactivity is shown against the RWPE-1 cell line. mAb 2-7A50 specifically detected a 45kDa band while mAb 2-5C11 specifically detected a 62 kDa band. Detection was performed using chemiluminescent method.

    Article Snippet: RWPE-1 and PC-3 cell extracts were obtained by cultivating these cells, separately, in 6-well plates.

    Techniques: Western Blot, Molecular Weight, Marker

    Hydroxytyrosol (HT) specifically inhibits the growth of prostate cancer cells. (A) Chemical structure of HT/3,4-dihydroxyphenylethanol (HT). (B) Normal prostate epithelium cells, RWPE1 and RWPE2, and prostate cancer cells, C4–2 and LNCaP, grown in 96-well plates, were treated with increasing dose of HT. Percent viability of these cells was measured using the WST-1 assay after 48 or 72 h of HT treatment. (C) Dose dependent HT treatment of LNCaP and C4–2 cell lines significantly altered cell morphology and the cells became round and shrunken in a dose-dependent manner, as examined under phase-contrast microscope. Representative images are shown.

    Journal: Nutrition and cancer

    Article Title: Hydroxytyrosol Induces Apoptosis, Cell Cycle Arrest and Suppresses Multiple Oncogenic Signaling Pathways in Prostate Cancer Cells

    doi: 10.1080/01635581.2017.1339818

    Figure Lengend Snippet: Hydroxytyrosol (HT) specifically inhibits the growth of prostate cancer cells. (A) Chemical structure of HT/3,4-dihydroxyphenylethanol (HT). (B) Normal prostate epithelium cells, RWPE1 and RWPE2, and prostate cancer cells, C4–2 and LNCaP, grown in 96-well plates, were treated with increasing dose of HT. Percent viability of these cells was measured using the WST-1 assay after 48 or 72 h of HT treatment. (C) Dose dependent HT treatment of LNCaP and C4–2 cell lines significantly altered cell morphology and the cells became round and shrunken in a dose-dependent manner, as examined under phase-contrast microscope. Representative images are shown.

    Article Snippet: The human normal prostate epithelial cell lines RWPE1 and RWPE2 (ATCC) were cultured in keratinocyte serum free medium containing 50 mg/ml gentamycin, 0.05 mg/ml bovine pituitary extract (BPE), and 5 ng/mL epidermal growth factor.

    Techniques: WST-1 Assay, Microscopy

    Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different androgen independent and androgen sensitive prostate cancer cell lines including PC3, DU145, VCaP, LNCaP and C4–2 versus benign epithelial prostate cell line RWPE-1. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–100 nm size were identified as being exosomes.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.014845

    Figure Lengend Snippet: Transmission Electron Microscopy (TEM). TEM images of exosomes derived from different androgen independent and androgen sensitive prostate cancer cell lines including PC3, DU145, VCaP, LNCaP and C4–2 versus benign epithelial prostate cell line RWPE-1. Exosomes were negatively stained with 2% uracyl acetate after removing the extra moisture. Cup-shaped structures, with 30–100 nm size were identified as being exosomes.

    Article Snippet: Proteomic profiles of two AR null PCa cell lines (DU145 and PC3) were compared with AR +ve cells including; VCaP, LNCaP, and C4–2 (PCa Cells), and RWPE-1 (benign epithelial prostate cell line) cells.).

    Techniques: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Derivative Assay, Staining

    Exosome lipidomic data. The lipid content of four major lipid classes was measured in PC3, DU145 and VCaP, LNCaP, C4–2 and RWPE-1 cells and compared with their derived exosomes, using LC-MS. The bar diagrams are representative of A , glycerolipid, B , glycerophospholipid, C , sphingolipid, D , glycosphingolipid in cell lysates and exosomes. Relative amounts of each lipid group were calculated as (sum of all AUC's for a particular lipid group) ÷ (sum of AUC's for all lipid groups) ×100%. * denotes a significant difference ( p

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.014845

    Figure Lengend Snippet: Exosome lipidomic data. The lipid content of four major lipid classes was measured in PC3, DU145 and VCaP, LNCaP, C4–2 and RWPE-1 cells and compared with their derived exosomes, using LC-MS. The bar diagrams are representative of A , glycerolipid, B , glycerophospholipid, C , sphingolipid, D , glycosphingolipid in cell lysates and exosomes. Relative amounts of each lipid group were calculated as (sum of all AUC's for a particular lipid group) ÷ (sum of AUC's for all lipid groups) ×100%. * denotes a significant difference ( p

    Article Snippet: Proteomic profiles of two AR null PCa cell lines (DU145 and PC3) were compared with AR +ve cells including; VCaP, LNCaP, and C4–2 (PCa Cells), and RWPE-1 (benign epithelial prostate cell line) cells.).

    Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy

    Cholesterol concentration. The bar diagrams show the cholesterol concentration of A , lysates of PC3, DU145 and VCaP, LNCaP, C4–2, and RWPE-1 cells B , exosomes derived from the six different prostate cell lines. Cholesterol results were normalized to protein concentration of each sample and expressed as μg Cholesterol/μg Protein. * indicate significantly difference ( p

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.014845

    Figure Lengend Snippet: Cholesterol concentration. The bar diagrams show the cholesterol concentration of A , lysates of PC3, DU145 and VCaP, LNCaP, C4–2, and RWPE-1 cells B , exosomes derived from the six different prostate cell lines. Cholesterol results were normalized to protein concentration of each sample and expressed as μg Cholesterol/μg Protein. * indicate significantly difference ( p

    Article Snippet: Proteomic profiles of two AR null PCa cell lines (DU145 and PC3) were compared with AR +ve cells including; VCaP, LNCaP, and C4–2 (PCa Cells), and RWPE-1 (benign epithelial prostate cell line) cells.).

    Techniques: Concentration Assay, Derivative Assay, Protein Concentration

    DLC1 suppresses NF-κB activation. (A) Relative luciferase activity of NF-κB reporter in RWPE-1, C4-2-B2 and PC-3 cells, (B) Western blot showing the level of DLC1, pNF-κB (p65), NF-κB (p65), RhoAGTP, and total RhoA in RWPE-1, C4-2-B2 and PC-3 cells and level of DLC1 in RWPE-1 and Ad-DLC1 transduced (50MOI) C4-2-B2 and PC-3cells. (C) Western blot showing level of phosphorylated p65 subunit of NF-κB in the Ad-Lac Z and Ad-DLC1 transduced C4-2-B2 and PC-3 cells. (D) Relative luciferase activity of NF-κB reporter in AD-LacZ and DLC1 transduced C4-2-B2 and PC-3 cells. All values are means ± SD of three independent counts. (E) Western blot showing the level of phosphorylated p65 subunit of NF-κB in DLC1- and α-catenin-knock down RWPE-1 cells.

    Journal: SpringerPlus

    Article Title: DLC1 suppresses NF-?B activity in prostate cancer cells due to its stabilizing effect on adherens junctions

    doi: 10.1186/2193-1801-3-27

    Figure Lengend Snippet: DLC1 suppresses NF-κB activation. (A) Relative luciferase activity of NF-κB reporter in RWPE-1, C4-2-B2 and PC-3 cells, (B) Western blot showing the level of DLC1, pNF-κB (p65), NF-κB (p65), RhoAGTP, and total RhoA in RWPE-1, C4-2-B2 and PC-3 cells and level of DLC1 in RWPE-1 and Ad-DLC1 transduced (50MOI) C4-2-B2 and PC-3cells. (C) Western blot showing level of phosphorylated p65 subunit of NF-κB in the Ad-Lac Z and Ad-DLC1 transduced C4-2-B2 and PC-3 cells. (D) Relative luciferase activity of NF-κB reporter in AD-LacZ and DLC1 transduced C4-2-B2 and PC-3 cells. All values are means ± SD of three independent counts. (E) Western blot showing the level of phosphorylated p65 subunit of NF-κB in DLC1- and α-catenin-knock down RWPE-1 cells.

    Article Snippet: PC-3 and RWPE-1 cells lines were purchased from American Type Culture Collection (Rockville, MD).

    Techniques: Activation Assay, Luciferase, Activity Assay, Western Blot