lapc4  (ATCC)


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    LAPC 4
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    ATCC lapc4
    Busulfan effectively inhibited survival of prostate cancer cells. A. CCK-8 test probing influence of varied busulfan dose (0 mg/L, 5 mg/L, 20 mg/L, 50 mg/L and 100 mg/L) on prostate cancer cell 22RV1, <t>LAPC4</t> and LNCaP; B. CCK-8 test probing 60 mg/L busulfan

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    1) Product Images from "Molecular mechanism of prostate cancer cell apoptosis induced by busulfan via adjustment of androgen receptor phosphatization"

    Article Title: Molecular mechanism of prostate cancer cell apoptosis induced by busulfan via adjustment of androgen receptor phosphatization

    Journal: American Journal of Translational Research

    doi:

    Busulfan effectively inhibited survival of prostate cancer cells. A. CCK-8 test probing influence of varied busulfan dose (0 mg/L, 5 mg/L, 20 mg/L, 50 mg/L and 100 mg/L) on prostate cancer cell 22RV1, LAPC4 and LNCaP; B. CCK-8 test probing 60 mg/L busulfan
    Figure Legend Snippet: Busulfan effectively inhibited survival of prostate cancer cells. A. CCK-8 test probing influence of varied busulfan dose (0 mg/L, 5 mg/L, 20 mg/L, 50 mg/L and 100 mg/L) on prostate cancer cell 22RV1, LAPC4 and LNCaP; B. CCK-8 test probing 60 mg/L busulfan

    Techniques Used: CCK-8 Assay

    Busulfan effectively inhibited survival of prostate cancer cells by inducing cell apoptosis. A. Flow cytometry probing influence of varied busulfan dose (0 mg/L, 30 mg/L, 60 mg/L and 100 mg/L) on apoptosis of prostate cancer cell line LAPC4 at 24 h; B.
    Figure Legend Snippet: Busulfan effectively inhibited survival of prostate cancer cells by inducing cell apoptosis. A. Flow cytometry probing influence of varied busulfan dose (0 mg/L, 30 mg/L, 60 mg/L and 100 mg/L) on apoptosis of prostate cancer cell line LAPC4 at 24 h; B.

    Techniques Used: Flow Cytometry, Cytometry

    2) Product Images from "Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression"

    Article Title: Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression

    Journal: Genes & Development

    doi: 10.1101/gad.301564.117

    Rac1 is a novel mutant p53-binding protein in human cancer cells. ( A ) Rac1-Flag preferentially interacted with ectopic mutant p53 (R175H) compared with ectopic wild-type p53 in human p53-null H1299 cells. H1299 cells were transfected with Rac1-Flag expression vectors together with wild-type p53 or mutant p53 (R175H) expression vectors for co-IP assays. The antibodies used for immunoprecipitation assays were as follows: anti-Flag for Rac1-Flag and DO-1 for mutant p53 and wild-type p53. ( B ) Rac1-Flag interacted with different hot spot mutant p53s, including R175H, R248W, and R273H, in H1299 cells transfected with vectors as indicated. ( C ) Endogenous Rac1 interacted with endogenous mutant p53 in SK-BR-3 (R175H), MDA-MB468 (R273H), LAPC4 (R175H), SW480 (R273H), Huh7 (Y220C), and HT29 (R273H) cells. ( D ) Rac1 interacted with the mutant p53 (R175H) DBD. ( Left panel) The domain structure of mutant p53 (R175H) and mutant p53 fragments. ( Right panel) H1299 cells were transfected with expression vectors of HA-tagged mutant p53 R175H fragments together with Rac1-Flag expression vectors. The antibody used for immunoprecipitation assays was anti-Flag for Rac1-Flag. ( E ) Mutant p53 interacted with the central region of the Rac1 protein. ( Left panel) The domain structure of Rac1 and Rac1 fragments. ( Right panel) H1299 cells were transfected with vectors expressing Myc-tagged Rac1 fragments together with mutant p53 (R175H) expression vectors. The antibody used for immunoprecipitation assays was DO-1 for mutant p53.
    Figure Legend Snippet: Rac1 is a novel mutant p53-binding protein in human cancer cells. ( A ) Rac1-Flag preferentially interacted with ectopic mutant p53 (R175H) compared with ectopic wild-type p53 in human p53-null H1299 cells. H1299 cells were transfected with Rac1-Flag expression vectors together with wild-type p53 or mutant p53 (R175H) expression vectors for co-IP assays. The antibodies used for immunoprecipitation assays were as follows: anti-Flag for Rac1-Flag and DO-1 for mutant p53 and wild-type p53. ( B ) Rac1-Flag interacted with different hot spot mutant p53s, including R175H, R248W, and R273H, in H1299 cells transfected with vectors as indicated. ( C ) Endogenous Rac1 interacted with endogenous mutant p53 in SK-BR-3 (R175H), MDA-MB468 (R273H), LAPC4 (R175H), SW480 (R273H), Huh7 (Y220C), and HT29 (R273H) cells. ( D ) Rac1 interacted with the mutant p53 (R175H) DBD. ( Left panel) The domain structure of mutant p53 (R175H) and mutant p53 fragments. ( Right panel) H1299 cells were transfected with expression vectors of HA-tagged mutant p53 R175H fragments together with Rac1-Flag expression vectors. The antibody used for immunoprecipitation assays was anti-Flag for Rac1-Flag. ( E ) Mutant p53 interacted with the central region of the Rac1 protein. ( Left panel) The domain structure of Rac1 and Rac1 fragments. ( Right panel) H1299 cells were transfected with vectors expressing Myc-tagged Rac1 fragments together with mutant p53 (R175H) expression vectors. The antibody used for immunoprecipitation assays was DO-1 for mutant p53.

    Techniques Used: Mutagenesis, Binding Assay, Transfection, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Multiple Displacement Amplification

    Mutant p53 activates Rac1 in human cancer cells. ( A ) Mutant p53 increased Rac1 activity in H1299 cells, represented by increased levels of Rac1-GTP. H1299 cells with or without ectopic mutant p53 (R175H, R248W, or R273H) expression were used to perform the GST-p21-binding domain of PAK1 pull-down assays to specifically pull down Rac1-GTP in cells followed by Western blot assays to measure the Rac1-GTP levels. ( B ) Ectopic mutant p53 expression increased the levels of p-PAK1 Ser144 and p-PAK2 Ser141 (p-PAK1/2) in H1299 cells. ( C ) Knockdown of endogenous mutant p53 by shRNA vectors decreased the levels of Rac1-GTP and p-PAK1/2 in SK-BR-3, MDA-MB468, LAPC4, and SW480 cells. Two shRNA vectors against p53 were used for all cell lines, and very similar results were observed. For the sake of clarity, results of one shRNA vector are presented for SW480 and LAPC4 cells. ( D ) Wild-type p53 decreased Rac1 activity, represented by decreased Rac1-GTP levels in human cancer cells. H1299 cells with or without ectopic wild-type p53 expression, human colorectal cancer HCT116 cells with or without wild-type p53, and MCF7 cells with or without wild-type p53 knockdown were used. For A , C , and D , the top panels show representative Western blot images for Rac1 activity analysis, and the bottom panels show quantitation of relative Rac1-GTP/total Rac1/Actin levels. Data are presented as mean ± SD. n = 3. (*) P
    Figure Legend Snippet: Mutant p53 activates Rac1 in human cancer cells. ( A ) Mutant p53 increased Rac1 activity in H1299 cells, represented by increased levels of Rac1-GTP. H1299 cells with or without ectopic mutant p53 (R175H, R248W, or R273H) expression were used to perform the GST-p21-binding domain of PAK1 pull-down assays to specifically pull down Rac1-GTP in cells followed by Western blot assays to measure the Rac1-GTP levels. ( B ) Ectopic mutant p53 expression increased the levels of p-PAK1 Ser144 and p-PAK2 Ser141 (p-PAK1/2) in H1299 cells. ( C ) Knockdown of endogenous mutant p53 by shRNA vectors decreased the levels of Rac1-GTP and p-PAK1/2 in SK-BR-3, MDA-MB468, LAPC4, and SW480 cells. Two shRNA vectors against p53 were used for all cell lines, and very similar results were observed. For the sake of clarity, results of one shRNA vector are presented for SW480 and LAPC4 cells. ( D ) Wild-type p53 decreased Rac1 activity, represented by decreased Rac1-GTP levels in human cancer cells. H1299 cells with or without ectopic wild-type p53 expression, human colorectal cancer HCT116 cells with or without wild-type p53, and MCF7 cells with or without wild-type p53 knockdown were used. For A , C , and D , the top panels show representative Western blot images for Rac1 activity analysis, and the bottom panels show quantitation of relative Rac1-GTP/total Rac1/Actin levels. Data are presented as mean ± SD. n = 3. (*) P

    Techniques Used: Mutagenesis, Activity Assay, Expressing, Binding Assay, Western Blot, shRNA, Multiple Displacement Amplification, Plasmid Preparation, Quantitation Assay

    3) Product Images from "Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer"

    Article Title: Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer

    Journal: Genes & Development

    doi: 10.1101/gad.299958.117

    Cytoplasmic and nuclear mTOR activities are induced by androgen signaling. ( A ) Protein expression of components of the mTOR signaling pathway and AR in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 or vehicle. Tubulin is shown as a loading control. ( B ) Protein expression of components of the mTOR signaling pathway in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 and/or anti-androgens (bicalutamide [B] and enzalutamide [E]). Tubulin is shown as a loading control. ( C ) Western blot analysis of nuclear fractions of LNCaP cells treated with R1881 or vehicle for various amounts of time. Lamin B1 is shown as a loading control. ( D ) Western blot analysis of cytoplasmic and nuclear fractions of LNCaP cells transfected with control or anti-AR siRNA and treated with R1881 or vehicle for 48 h. Lamin B1 and tubulin are shown as controls for nuclear and cytoplasmic extracts, respectively. ( E ) Immunofluorescence showing increased mTOR (red) levels in the nucleus following a 48-h treatment with R1881 in LCNaP cells. Nuclei were stained with DAPI (blue). ( F ) Overlap between mTOR DNA-binding peaks in LNCaP cells treated for 48 h with R1881 or vehicle and heat maps of the signal intensity of mTOR genomic binding peaks in a window of ±2.5 kb. ( G ) Average ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) signal intensities normalized per reads for down-regulated, unregulated, and up-regulated mTOR peaks. ( H ) Examples of University of California at Santa Cruz Genome Browser graphical views of mTOR-binding peaks in LNCaP cells treated with R1881 or vehicle that are either unaffected (unregulated) or positively or negatively regulated by androgens. Genes and peaks (black boxes) are indicated below tag density graphs. ChIP-qPCR (ChIP combined with quantitative PCR) of mTOR in LNCaP ( I ) or LAPC4 ( J ) cells after 48 h of treatment with R1881 or vehicle. Relative fold enrichment was normalized over two negative regions and is shown relative to IgG (set at 1). Results are shown as the average of three independent experiments.
    Figure Legend Snippet: Cytoplasmic and nuclear mTOR activities are induced by androgen signaling. ( A ) Protein expression of components of the mTOR signaling pathway and AR in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 or vehicle. Tubulin is shown as a loading control. ( B ) Protein expression of components of the mTOR signaling pathway in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 and/or anti-androgens (bicalutamide [B] and enzalutamide [E]). Tubulin is shown as a loading control. ( C ) Western blot analysis of nuclear fractions of LNCaP cells treated with R1881 or vehicle for various amounts of time. Lamin B1 is shown as a loading control. ( D ) Western blot analysis of cytoplasmic and nuclear fractions of LNCaP cells transfected with control or anti-AR siRNA and treated with R1881 or vehicle for 48 h. Lamin B1 and tubulin are shown as controls for nuclear and cytoplasmic extracts, respectively. ( E ) Immunofluorescence showing increased mTOR (red) levels in the nucleus following a 48-h treatment with R1881 in LCNaP cells. Nuclei were stained with DAPI (blue). ( F ) Overlap between mTOR DNA-binding peaks in LNCaP cells treated for 48 h with R1881 or vehicle and heat maps of the signal intensity of mTOR genomic binding peaks in a window of ±2.5 kb. ( G ) Average ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) signal intensities normalized per reads for down-regulated, unregulated, and up-regulated mTOR peaks. ( H ) Examples of University of California at Santa Cruz Genome Browser graphical views of mTOR-binding peaks in LNCaP cells treated with R1881 or vehicle that are either unaffected (unregulated) or positively or negatively regulated by androgens. Genes and peaks (black boxes) are indicated below tag density graphs. ChIP-qPCR (ChIP combined with quantitative PCR) of mTOR in LNCaP ( I ) or LAPC4 ( J ) cells after 48 h of treatment with R1881 or vehicle. Relative fold enrichment was normalized over two negative regions and is shown relative to IgG (set at 1). Results are shown as the average of three independent experiments.

    Techniques Used: Expressing, Western Blot, Transfection, Immunofluorescence, Staining, Binding Assay, Chromatin Immunoprecipitation, Next-Generation Sequencing, Real-time Polymerase Chain Reaction

    4) Product Images from "AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion"

    Article Title: AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2013.05.008

    Effects of constitutively active AKT expression in different PCa cells Cells of LNCaP C4-2 (A) CWR22R v1 (B) , and LAPC4 (C) , infected with the pHAGE lentivirus expressing AKT-null or AKT-myr, were maintained in c.s.FBS medium for 48 hours. Total cell lysates from each experiment were analyzed by Western blotting for expression of the indicated proteins.
    Figure Legend Snippet: Effects of constitutively active AKT expression in different PCa cells Cells of LNCaP C4-2 (A) CWR22R v1 (B) , and LAPC4 (C) , infected with the pHAGE lentivirus expressing AKT-null or AKT-myr, were maintained in c.s.FBS medium for 48 hours. Total cell lysates from each experiment were analyzed by Western blotting for expression of the indicated proteins.

    Techniques Used: Expressing, Infection, Western Blot

    5) Product Images from "AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion"

    Article Title: AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2013.05.008

    Effects of constitutively active AKT expression in different PCa cells Cells of LNCaP C4-2 (A) CWR22R v1 (B) , and LAPC4 (C) , infected with the pHAGE lentivirus expressing AKT-null or AKT-myr, were maintained in c.s.FBS medium for 48 hours. Total cell lysates from each experiment were analyzed by Western blotting for expression of the indicated proteins.
    Figure Legend Snippet: Effects of constitutively active AKT expression in different PCa cells Cells of LNCaP C4-2 (A) CWR22R v1 (B) , and LAPC4 (C) , infected with the pHAGE lentivirus expressing AKT-null or AKT-myr, were maintained in c.s.FBS medium for 48 hours. Total cell lysates from each experiment were analyzed by Western blotting for expression of the indicated proteins.

    Techniques Used: Expressing, Infection, Western Blot

    6) Product Images from "Minoxidil may suppress androgen receptor-related functions"

    Article Title: Minoxidil may suppress androgen receptor-related functions

    Journal: Oncotarget

    doi:

    Minoxidil suppresses AR-related functions in skin cells (A-B) AR expression in cells and cell lines at the (A) mRNA and (B) protein levels. Lane 1, HHDPCs; lane 2, LNCaP cells; lane 3, PC-3 cells. (C) HHDPCs in 24-well plates were transfected with 1,000 ng of MMTV-Luc reporter plasmid. After 16 h, ethanol, 10 nM DHT, 10–100 μM minoxidil, and/or 1 mM tolbutamide was added and cells were incubated for an additional 16 h. Luciferase activity in cell lysates was normalized to protein concentration. Relative luciferase activity was calculated as described in Materials and methods. (D) After seeding HHDPCs in dishes and culturing for 24 h, 10 μg/ml cyclohexamide, 10 nM DHT, and different concentrations of minoxidil were added and cells were incubated for an additional 2 h. AR and β-actin (loading control) protein levels were determined by Western blotting. (E) Potassium channel subunit expression in different cells and cell lines. Prostate cancer cell lines: LAPC4, LNCaP, and PC-3; liver cancer cell line: HePG2; skin cells: HHDPCs. PC-3 cells expressed the same potassium channel subtype (SUR2B/Kir6.1) as HHDPCs. (F) Tolbutamide did not reverse minoxidil effects in AR transactivation reporter assays in PC-3 cell. The same procedure as in (C) was performed except with the addition of different concentrations of tolbutamide
    Figure Legend Snippet: Minoxidil suppresses AR-related functions in skin cells (A-B) AR expression in cells and cell lines at the (A) mRNA and (B) protein levels. Lane 1, HHDPCs; lane 2, LNCaP cells; lane 3, PC-3 cells. (C) HHDPCs in 24-well plates were transfected with 1,000 ng of MMTV-Luc reporter plasmid. After 16 h, ethanol, 10 nM DHT, 10–100 μM minoxidil, and/or 1 mM tolbutamide was added and cells were incubated for an additional 16 h. Luciferase activity in cell lysates was normalized to protein concentration. Relative luciferase activity was calculated as described in Materials and methods. (D) After seeding HHDPCs in dishes and culturing for 24 h, 10 μg/ml cyclohexamide, 10 nM DHT, and different concentrations of minoxidil were added and cells were incubated for an additional 2 h. AR and β-actin (loading control) protein levels were determined by Western blotting. (E) Potassium channel subunit expression in different cells and cell lines. Prostate cancer cell lines: LAPC4, LNCaP, and PC-3; liver cancer cell line: HePG2; skin cells: HHDPCs. PC-3 cells expressed the same potassium channel subtype (SUR2B/Kir6.1) as HHDPCs. (F) Tolbutamide did not reverse minoxidil effects in AR transactivation reporter assays in PC-3 cell. The same procedure as in (C) was performed except with the addition of different concentrations of tolbutamide

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Protein Concentration, Western Blot

    7) Product Images from "Crosstalk Between Nuclear MET and SOX9/β-Catenin Correlates with Castration-Resistant Prostate Cancer"

    Article Title: Crosstalk Between Nuclear MET and SOX9/β-Catenin Correlates with Castration-Resistant Prostate Cancer

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2014-1078

    Nuclear MET activates β-catenin pathway and a combined inhibition of MET and β-catenin increases the efficiency. A, The level of nuclear β-catenin was increased by nMET overexpression in LAPC4 cells. B, The level of AR was increased
    Figure Legend Snippet: Nuclear MET activates β-catenin pathway and a combined inhibition of MET and β-catenin increases the efficiency. A, The level of nuclear β-catenin was increased by nMET overexpression in LAPC4 cells. B, The level of AR was increased

    Techniques Used: Inhibition, Over Expression

    Androgen deprivation up-regulates endogenous nuclear MET and promotes stem-like cells self-renewal in androgen-responsive cells. A–C, Coup-regulation of endogenous nuclear MET and SOX9 in LAPC4 cells upon androgen depletion. LAPC4 cells were androgen
    Figure Legend Snippet: Androgen deprivation up-regulates endogenous nuclear MET and promotes stem-like cells self-renewal in androgen-responsive cells. A–C, Coup-regulation of endogenous nuclear MET and SOX9 in LAPC4 cells upon androgen depletion. LAPC4 cells were androgen

    Techniques Used:

    8) Product Images from "A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions"

    Article Title: A novel patient-derived intra-femoral xenograft model of bone metastatic prostate cancer that recapitulates mixed osteolytic and osteoblastic lesions

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-9-185

    PCSD1 xenograft tumors expressed human prostate PSA and human AR . A. RNA was extracted from secondary transplant sub-cutaneous xenograft tumors (Passage 1, P1) and used for RT-PCR analysis of human prostate specific antigen (PSA) and human androgen receptor (AR). Human PSA and human AR-specific primers were used for PCR amplification of cDNA synthesized with reverse transcriptase (RT+) or without (RT-) and confirmed by sequencing of correctly sized bands. Human GAPDH-specific primers were used as an internal control. Human PSA and AR were expressed in the PCSD1 xenograft tumor and the human prostate cancer cell line, LAPC4, but not in the human chronic myelogenous leukemia (CML) cell line, K562. RNA from mouse spleen, bone marrow and liver did not express human PSA or AR. B. Immunohistochemical analysis showed human PSA protein expression in PCSD1 xenograft tumors. Images show paraffin embedded (PPFE) sub-cutaneous PCSD1 secondary transplant xenograft sections stained with IgG isotype negative control or human PSA-specific antibody. (C) Upper panels show H and E stained intra-femoral PCSD1 secondary transplant xenograft cryosections at 10× and 20× magnification. Middle panels show cryosections from the left, un-injected, contralateral femurs immunostained with human PSA-specific antibody at 10× and 40×. Arrows show red blood cells in bone marrow. Lower panels show cryosections from secondary intra-femoral transplants of PCSD1 immunostained with anti-PSA. Arrows point to human PSA positive (+) prostate cancer cells.
    Figure Legend Snippet: PCSD1 xenograft tumors expressed human prostate PSA and human AR . A. RNA was extracted from secondary transplant sub-cutaneous xenograft tumors (Passage 1, P1) and used for RT-PCR analysis of human prostate specific antigen (PSA) and human androgen receptor (AR). Human PSA and human AR-specific primers were used for PCR amplification of cDNA synthesized with reverse transcriptase (RT+) or without (RT-) and confirmed by sequencing of correctly sized bands. Human GAPDH-specific primers were used as an internal control. Human PSA and AR were expressed in the PCSD1 xenograft tumor and the human prostate cancer cell line, LAPC4, but not in the human chronic myelogenous leukemia (CML) cell line, K562. RNA from mouse spleen, bone marrow and liver did not express human PSA or AR. B. Immunohistochemical analysis showed human PSA protein expression in PCSD1 xenograft tumors. Images show paraffin embedded (PPFE) sub-cutaneous PCSD1 secondary transplant xenograft sections stained with IgG isotype negative control or human PSA-specific antibody. (C) Upper panels show H and E stained intra-femoral PCSD1 secondary transplant xenograft cryosections at 10× and 20× magnification. Middle panels show cryosections from the left, un-injected, contralateral femurs immunostained with human PSA-specific antibody at 10× and 40×. Arrows show red blood cells in bone marrow. Lower panels show cryosections from secondary intra-femoral transplants of PCSD1 immunostained with anti-PSA. Arrows point to human PSA positive (+) prostate cancer cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Synthesized, Sequencing, Immunohistochemistry, Expressing, Staining, Negative Control, Injection

    PCSD1 xenograft tumors express luminal-type epithelial, advanced prostate cancer biomarkers . A. RT-PCR analysis was performed on cDNA synthesized with reverse transcriptase (RT+) or without (RT-) from RNA purified from PCSD1 sub-cutaneous xenograft tumors, cultures of LAPC4, a human prostate cancer cell line and K562, a human CML cell line culture, as well as murine spleen, bone marrow and liver or H2O alone and RNA from normal human prostate tissue. Human specific primers for keratins 5, 8, 14 and 18, AMACR and NKX3.1, GAPDH and mouse-specific GAPDH were used to detect expression of these genes. B. RT-PCR analysis was performed on intra-femoral xenograft PCSD1 tumor cells, cultured VCaP prostate cancer cell line as well as normal human prostate tissue. Primers were specific for RNA from full length human AR or the TMPRSS2-ERG fusion gene. GAPDH levels were comparable.
    Figure Legend Snippet: PCSD1 xenograft tumors express luminal-type epithelial, advanced prostate cancer biomarkers . A. RT-PCR analysis was performed on cDNA synthesized with reverse transcriptase (RT+) or without (RT-) from RNA purified from PCSD1 sub-cutaneous xenograft tumors, cultures of LAPC4, a human prostate cancer cell line and K562, a human CML cell line culture, as well as murine spleen, bone marrow and liver or H2O alone and RNA from normal human prostate tissue. Human specific primers for keratins 5, 8, 14 and 18, AMACR and NKX3.1, GAPDH and mouse-specific GAPDH were used to detect expression of these genes. B. RT-PCR analysis was performed on intra-femoral xenograft PCSD1 tumor cells, cultured VCaP prostate cancer cell line as well as normal human prostate tissue. Primers were specific for RNA from full length human AR or the TMPRSS2-ERG fusion gene. GAPDH levels were comparable.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Synthesized, Purification, Expressing, Cell Culture

    9) Product Images from "Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy"

    Article Title: Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07411-7

    Chronic-ADT in hypoxia induces resistance to targeted disruption of androgen/AR-axis. a Schematic representation of repeated-ADT treatment of LNCaP and LAPC4 cells in hypoxia (1% O 2 ) or normoxia (20% O 2 ). Cells were cultured in charcoal-stripped serum (CSS) media with or without 1 nM synthetic androgen R1881 (R1881 or ADT). Each round/cycle of treatment lasted 48 h, and the survived cells were recovered and re-plated for the same treatment in the following cycle. b Dose-dependent sensitivity of LNCaP clones to enzalutamide. Cells derived from a were cultured with CSS + R1881, and treated by increasing doses of enzalutamide in normoxia or hypoxia for 96 h. Afterwards, cell growth and viability were determined based on viable cells quantitated with SYTO 60 fluorescence staining and imaging. The AdtHs is represented by purple line. All values were mean ± s.d. relative to solvent-treated controls, n = 3. * P
    Figure Legend Snippet: Chronic-ADT in hypoxia induces resistance to targeted disruption of androgen/AR-axis. a Schematic representation of repeated-ADT treatment of LNCaP and LAPC4 cells in hypoxia (1% O 2 ) or normoxia (20% O 2 ). Cells were cultured in charcoal-stripped serum (CSS) media with or without 1 nM synthetic androgen R1881 (R1881 or ADT). Each round/cycle of treatment lasted 48 h, and the survived cells were recovered and re-plated for the same treatment in the following cycle. b Dose-dependent sensitivity of LNCaP clones to enzalutamide. Cells derived from a were cultured with CSS + R1881, and treated by increasing doses of enzalutamide in normoxia or hypoxia for 96 h. Afterwards, cell growth and viability were determined based on viable cells quantitated with SYTO 60 fluorescence staining and imaging. The AdtHs is represented by purple line. All values were mean ± s.d. relative to solvent-treated controls, n = 3. * P

    Techniques Used: Cell Culture, Clone Assay, Derivative Assay, Fluorescence, Staining, Imaging

    10) Product Images from "Identification of Therapeutic Vulnerabilities in Small Cell Neuroendocrine Prostate Cancer"

    Article Title: Identification of Therapeutic Vulnerabilities in Small Cell Neuroendocrine Prostate Cancer

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-19-0775

    BCL2 antagonists inhibit the growth of SCNPC. (A-D) Dose response curves of BCL2-family inhibitors across a panel of prostate cancer cell lines. NCI-H660 and MSKCC EF1 are SCNPC, and LNCaP, C4–2, LAPC4, and VCaP are ARPC. (E) Western Blot for cleaved caspase-3 following 24 hour treatment with ABT-263 across the indicated cell lines. (F) BCL2 expression in ARPC versus SCNPC PDX models determined by RNAseq. p = 0.011 by Student’s t-test. (G ) Western Blot of selected BCL2 family members across a panel of prostate cancer PDX models. (H) Quantification of BCL2 staining of LuCaP TMA. (I)Scatterplot depicting Pearson correlation between the NE signature score and BCL2 gene expression in PDX lines. (J) Normalized tumor volumes of PDX models treated with vehicle control or ABT-263. ***p
    Figure Legend Snippet: BCL2 antagonists inhibit the growth of SCNPC. (A-D) Dose response curves of BCL2-family inhibitors across a panel of prostate cancer cell lines. NCI-H660 and MSKCC EF1 are SCNPC, and LNCaP, C4–2, LAPC4, and VCaP are ARPC. (E) Western Blot for cleaved caspase-3 following 24 hour treatment with ABT-263 across the indicated cell lines. (F) BCL2 expression in ARPC versus SCNPC PDX models determined by RNAseq. p = 0.011 by Student’s t-test. (G ) Western Blot of selected BCL2 family members across a panel of prostate cancer PDX models. (H) Quantification of BCL2 staining of LuCaP TMA. (I)Scatterplot depicting Pearson correlation between the NE signature score and BCL2 gene expression in PDX lines. (J) Normalized tumor volumes of PDX models treated with vehicle control or ABT-263. ***p

    Techniques Used: Western Blot, Expressing, Staining

    11) Product Images from "Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy"

    Article Title: Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07411-7

    Chronic-ADT in hypoxia induces resistance to targeted disruption of androgen/AR-axis. a Schematic representation of repeated-ADT treatment of LNCaP and LAPC4 cells in hypoxia (1% O 2 ) or normoxia (20% O 2 ). Cells were cultured in charcoal-stripped serum (CSS) media with or without 1 nM synthetic androgen R1881 (R1881 or ADT). Each round/cycle of treatment lasted 48 h, and the survived cells were recovered and re-plated for the same treatment in the following cycle. b Dose-dependent sensitivity of LNCaP clones to enzalutamide. Cells derived from a were cultured with CSS + R1881, and treated by increasing doses of enzalutamide in normoxia or hypoxia for 96 h. Afterwards, cell growth and viability were determined based on viable cells quantitated with SYTO 60 fluorescence staining and imaging. The AdtHs is represented by purple line. All values were mean ± s.d. relative to solvent-treated controls, n = 3. * P
    Figure Legend Snippet: Chronic-ADT in hypoxia induces resistance to targeted disruption of androgen/AR-axis. a Schematic representation of repeated-ADT treatment of LNCaP and LAPC4 cells in hypoxia (1% O 2 ) or normoxia (20% O 2 ). Cells were cultured in charcoal-stripped serum (CSS) media with or without 1 nM synthetic androgen R1881 (R1881 or ADT). Each round/cycle of treatment lasted 48 h, and the survived cells were recovered and re-plated for the same treatment in the following cycle. b Dose-dependent sensitivity of LNCaP clones to enzalutamide. Cells derived from a were cultured with CSS + R1881, and treated by increasing doses of enzalutamide in normoxia or hypoxia for 96 h. Afterwards, cell growth and viability were determined based on viable cells quantitated with SYTO 60 fluorescence staining and imaging. The AdtHs is represented by purple line. All values were mean ± s.d. relative to solvent-treated controls, n = 3. * P

    Techniques Used: Cell Culture, Clone Assay, Derivative Assay, Fluorescence, Staining, Imaging

    12) Product Images from "Inhibition of base excision repair by natamycin suppresses prostate cancer cell proliferation"

    Article Title: Inhibition of base excision repair by natamycin suppresses prostate cancer cell proliferation

    Journal: Biochimie

    doi: 10.1016/j.biochi.2019.11.008

    Natamycin suppresses cellular proliferation and adhesion in androgen dependent prostate cancer cell lines. A-B. LNCaP cells were plated onto xCelligence E-plates at 10 4 cells per well in medium supplemented with either 10% FBS (A) or 10% CSS (B) serum. Cells attached overnight and were treated with vehicle (DMSO) or 10 μM of the indicated compounds. C-D. LAPC4 cells were plated at 5x10 4 cells per well onto xCelligence E-plates in media supplemented with FBS (C) or CSS (D) and allowed to attach overnight. Treatment was conducted as in (A-B) and impedance measured continuously in 30 min intervals. Impedance in all graphs was normalized to the time of treatment, which was assigned a value of 1.
    Figure Legend Snippet: Natamycin suppresses cellular proliferation and adhesion in androgen dependent prostate cancer cell lines. A-B. LNCaP cells were plated onto xCelligence E-plates at 10 4 cells per well in medium supplemented with either 10% FBS (A) or 10% CSS (B) serum. Cells attached overnight and were treated with vehicle (DMSO) or 10 μM of the indicated compounds. C-D. LAPC4 cells were plated at 5x10 4 cells per well onto xCelligence E-plates in media supplemented with FBS (C) or CSS (D) and allowed to attach overnight. Treatment was conducted as in (A-B) and impedance measured continuously in 30 min intervals. Impedance in all graphs was normalized to the time of treatment, which was assigned a value of 1.

    Techniques Used:

    Cellular toxicity of the lead compounds in androgen-dependent prostate cancer cell lines. A. LNCaP cells were plated in a 96-well plate at 1x10 4 cells/well in the presence of FBS or CSS. Cells were allowed to attach overnight and were treated with either vehicle (DMSO) or 10 μM of indicated compound. At 24 and 48 h cellular viability was compared using the Cell Titer Glo Luminescent Cell Viability kit. B. LAPC4 cells were plated in a 96-well plate at a density of 1x10 4 cells/well in the presence of FBS or CSS. Cells were allowed to attach overnight and treated with 10 μM of indicated compound. At 24 h and 48 h cellular viability was compared as in A. (* signifies a difference between vehicle and treatment with *p≤0.05, ** p≤0.01, *** p≤0.001 **** p≤0.0001).
    Figure Legend Snippet: Cellular toxicity of the lead compounds in androgen-dependent prostate cancer cell lines. A. LNCaP cells were plated in a 96-well plate at 1x10 4 cells/well in the presence of FBS or CSS. Cells were allowed to attach overnight and were treated with either vehicle (DMSO) or 10 μM of indicated compound. At 24 and 48 h cellular viability was compared using the Cell Titer Glo Luminescent Cell Viability kit. B. LAPC4 cells were plated in a 96-well plate at a density of 1x10 4 cells/well in the presence of FBS or CSS. Cells were allowed to attach overnight and treated with 10 μM of indicated compound. At 24 h and 48 h cellular viability was compared as in A. (* signifies a difference between vehicle and treatment with *p≤0.05, ** p≤0.01, *** p≤0.001 **** p≤0.0001).

    Techniques Used:

    Related Articles

    Multiple Displacement Amplification:

    Article Title: Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression
    Article Snippet: .. H1299, SK-BR-3, MDA-MB468, LAPC4, SW480, HT29, and Huh7 cell lines were obtained from American Type Culture Collection. .. H1299 cells with stable ectopic mutant p53 (R175H, R248W, and R273H) overexpression were established as described previously ( ).

    Cell Culture:

    Article Title: Arctigenin inhibits prostate tumor growth in high-fat diet fed mice through dual actions on adipose tissue and tumor
    Article Snippet: .. Cell culture and adipocyte differentiation Androgen-sensitive LNCaP and LAPC-4 human prostate cancer cell lines were purchased from ATCC (Chicago, IL, USA), maintained in RPMI 1640 medium supplemented with 10% (v:v) of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 µg/mL of streptomycin at 37 °C in a 5% CO2 incubator. .. 3T3-L1 mouse embryonic fibroblasts (ATCC) were cultured in Dulbecco’s modified Eagle’s medium supplanted with 10% FBS, 100 IU/mL of penicillin and 100 µg/mL of streptomycin.

    Article Title: Aberrant Activation of the Androgen Receptor by NF-?B2/p52 in Prostate Cancer Cells
    Article Snippet: .. LAPC-4, LNCaP, C4-2, and PC-3 prostate cancer cells were obtained from the American Type Culture Collection and cultured in RPMI 1640 containing either 10% complete fetal bovine serum (FBS) or 10% charcoal-dextran–stripped FBS (CS-FBS) and penicillin/streptomycin. ..

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    ATCC lapc4
    Busulfan effectively inhibited survival of prostate cancer cells. A. CCK-8 test probing influence of varied busulfan dose (0 mg/L, 5 mg/L, 20 mg/L, 50 mg/L and 100 mg/L) on prostate cancer cell 22RV1, <t>LAPC4</t> and LNCaP; B. CCK-8 test probing 60 mg/L busulfan
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    Busulfan effectively inhibited survival of prostate cancer cells. A. CCK-8 test probing influence of varied busulfan dose (0 mg/L, 5 mg/L, 20 mg/L, 50 mg/L and 100 mg/L) on prostate cancer cell 22RV1, LAPC4 and LNCaP; B. CCK-8 test probing 60 mg/L busulfan

    Journal: American Journal of Translational Research

    Article Title: Molecular mechanism of prostate cancer cell apoptosis induced by busulfan via adjustment of androgen receptor phosphatization

    doi:

    Figure Lengend Snippet: Busulfan effectively inhibited survival of prostate cancer cells. A. CCK-8 test probing influence of varied busulfan dose (0 mg/L, 5 mg/L, 20 mg/L, 50 mg/L and 100 mg/L) on prostate cancer cell 22RV1, LAPC4 and LNCaP; B. CCK-8 test probing 60 mg/L busulfan

    Article Snippet: Prostate cancer cell line 22RV1, LAPC4 and LNCaP were from ATCC.

    Techniques: CCK-8 Assay

    Busulfan effectively inhibited survival of prostate cancer cells by inducing cell apoptosis. A. Flow cytometry probing influence of varied busulfan dose (0 mg/L, 30 mg/L, 60 mg/L and 100 mg/L) on apoptosis of prostate cancer cell line LAPC4 at 24 h; B.

    Journal: American Journal of Translational Research

    Article Title: Molecular mechanism of prostate cancer cell apoptosis induced by busulfan via adjustment of androgen receptor phosphatization

    doi:

    Figure Lengend Snippet: Busulfan effectively inhibited survival of prostate cancer cells by inducing cell apoptosis. A. Flow cytometry probing influence of varied busulfan dose (0 mg/L, 30 mg/L, 60 mg/L and 100 mg/L) on apoptosis of prostate cancer cell line LAPC4 at 24 h; B.

    Article Snippet: Prostate cancer cell line 22RV1, LAPC4 and LNCaP were from ATCC.

    Techniques: Flow Cytometry, Cytometry

    Rac1 is a novel mutant p53-binding protein in human cancer cells. ( A ) Rac1-Flag preferentially interacted with ectopic mutant p53 (R175H) compared with ectopic wild-type p53 in human p53-null H1299 cells. H1299 cells were transfected with Rac1-Flag expression vectors together with wild-type p53 or mutant p53 (R175H) expression vectors for co-IP assays. The antibodies used for immunoprecipitation assays were as follows: anti-Flag for Rac1-Flag and DO-1 for mutant p53 and wild-type p53. ( B ) Rac1-Flag interacted with different hot spot mutant p53s, including R175H, R248W, and R273H, in H1299 cells transfected with vectors as indicated. ( C ) Endogenous Rac1 interacted with endogenous mutant p53 in SK-BR-3 (R175H), MDA-MB468 (R273H), LAPC4 (R175H), SW480 (R273H), Huh7 (Y220C), and HT29 (R273H) cells. ( D ) Rac1 interacted with the mutant p53 (R175H) DBD. ( Left panel) The domain structure of mutant p53 (R175H) and mutant p53 fragments. ( Right panel) H1299 cells were transfected with expression vectors of HA-tagged mutant p53 R175H fragments together with Rac1-Flag expression vectors. The antibody used for immunoprecipitation assays was anti-Flag for Rac1-Flag. ( E ) Mutant p53 interacted with the central region of the Rac1 protein. ( Left panel) The domain structure of Rac1 and Rac1 fragments. ( Right panel) H1299 cells were transfected with vectors expressing Myc-tagged Rac1 fragments together with mutant p53 (R175H) expression vectors. The antibody used for immunoprecipitation assays was DO-1 for mutant p53.

    Journal: Genes & Development

    Article Title: Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression

    doi: 10.1101/gad.301564.117

    Figure Lengend Snippet: Rac1 is a novel mutant p53-binding protein in human cancer cells. ( A ) Rac1-Flag preferentially interacted with ectopic mutant p53 (R175H) compared with ectopic wild-type p53 in human p53-null H1299 cells. H1299 cells were transfected with Rac1-Flag expression vectors together with wild-type p53 or mutant p53 (R175H) expression vectors for co-IP assays. The antibodies used for immunoprecipitation assays were as follows: anti-Flag for Rac1-Flag and DO-1 for mutant p53 and wild-type p53. ( B ) Rac1-Flag interacted with different hot spot mutant p53s, including R175H, R248W, and R273H, in H1299 cells transfected with vectors as indicated. ( C ) Endogenous Rac1 interacted with endogenous mutant p53 in SK-BR-3 (R175H), MDA-MB468 (R273H), LAPC4 (R175H), SW480 (R273H), Huh7 (Y220C), and HT29 (R273H) cells. ( D ) Rac1 interacted with the mutant p53 (R175H) DBD. ( Left panel) The domain structure of mutant p53 (R175H) and mutant p53 fragments. ( Right panel) H1299 cells were transfected with expression vectors of HA-tagged mutant p53 R175H fragments together with Rac1-Flag expression vectors. The antibody used for immunoprecipitation assays was anti-Flag for Rac1-Flag. ( E ) Mutant p53 interacted with the central region of the Rac1 protein. ( Left panel) The domain structure of Rac1 and Rac1 fragments. ( Right panel) H1299 cells were transfected with vectors expressing Myc-tagged Rac1 fragments together with mutant p53 (R175H) expression vectors. The antibody used for immunoprecipitation assays was DO-1 for mutant p53.

    Article Snippet: H1299, SK-BR-3, MDA-MB468, LAPC4, SW480, HT29, and Huh7 cell lines were obtained from American Type Culture Collection.

    Techniques: Mutagenesis, Binding Assay, Transfection, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Multiple Displacement Amplification

    Mutant p53 activates Rac1 in human cancer cells. ( A ) Mutant p53 increased Rac1 activity in H1299 cells, represented by increased levels of Rac1-GTP. H1299 cells with or without ectopic mutant p53 (R175H, R248W, or R273H) expression were used to perform the GST-p21-binding domain of PAK1 pull-down assays to specifically pull down Rac1-GTP in cells followed by Western blot assays to measure the Rac1-GTP levels. ( B ) Ectopic mutant p53 expression increased the levels of p-PAK1 Ser144 and p-PAK2 Ser141 (p-PAK1/2) in H1299 cells. ( C ) Knockdown of endogenous mutant p53 by shRNA vectors decreased the levels of Rac1-GTP and p-PAK1/2 in SK-BR-3, MDA-MB468, LAPC4, and SW480 cells. Two shRNA vectors against p53 were used for all cell lines, and very similar results were observed. For the sake of clarity, results of one shRNA vector are presented for SW480 and LAPC4 cells. ( D ) Wild-type p53 decreased Rac1 activity, represented by decreased Rac1-GTP levels in human cancer cells. H1299 cells with or without ectopic wild-type p53 expression, human colorectal cancer HCT116 cells with or without wild-type p53, and MCF7 cells with or without wild-type p53 knockdown were used. For A , C , and D , the top panels show representative Western blot images for Rac1 activity analysis, and the bottom panels show quantitation of relative Rac1-GTP/total Rac1/Actin levels. Data are presented as mean ± SD. n = 3. (*) P

    Journal: Genes & Development

    Article Title: Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression

    doi: 10.1101/gad.301564.117

    Figure Lengend Snippet: Mutant p53 activates Rac1 in human cancer cells. ( A ) Mutant p53 increased Rac1 activity in H1299 cells, represented by increased levels of Rac1-GTP. H1299 cells with or without ectopic mutant p53 (R175H, R248W, or R273H) expression were used to perform the GST-p21-binding domain of PAK1 pull-down assays to specifically pull down Rac1-GTP in cells followed by Western blot assays to measure the Rac1-GTP levels. ( B ) Ectopic mutant p53 expression increased the levels of p-PAK1 Ser144 and p-PAK2 Ser141 (p-PAK1/2) in H1299 cells. ( C ) Knockdown of endogenous mutant p53 by shRNA vectors decreased the levels of Rac1-GTP and p-PAK1/2 in SK-BR-3, MDA-MB468, LAPC4, and SW480 cells. Two shRNA vectors against p53 were used for all cell lines, and very similar results were observed. For the sake of clarity, results of one shRNA vector are presented for SW480 and LAPC4 cells. ( D ) Wild-type p53 decreased Rac1 activity, represented by decreased Rac1-GTP levels in human cancer cells. H1299 cells with or without ectopic wild-type p53 expression, human colorectal cancer HCT116 cells with or without wild-type p53, and MCF7 cells with or without wild-type p53 knockdown were used. For A , C , and D , the top panels show representative Western blot images for Rac1 activity analysis, and the bottom panels show quantitation of relative Rac1-GTP/total Rac1/Actin levels. Data are presented as mean ± SD. n = 3. (*) P

    Article Snippet: H1299, SK-BR-3, MDA-MB468, LAPC4, SW480, HT29, and Huh7 cell lines were obtained from American Type Culture Collection.

    Techniques: Mutagenesis, Activity Assay, Expressing, Binding Assay, Western Blot, shRNA, Multiple Displacement Amplification, Plasmid Preparation, Quantitation Assay

    Cytoplasmic and nuclear mTOR activities are induced by androgen signaling. ( A ) Protein expression of components of the mTOR signaling pathway and AR in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 or vehicle. Tubulin is shown as a loading control. ( B ) Protein expression of components of the mTOR signaling pathway in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 and/or anti-androgens (bicalutamide [B] and enzalutamide [E]). Tubulin is shown as a loading control. ( C ) Western blot analysis of nuclear fractions of LNCaP cells treated with R1881 or vehicle for various amounts of time. Lamin B1 is shown as a loading control. ( D ) Western blot analysis of cytoplasmic and nuclear fractions of LNCaP cells transfected with control or anti-AR siRNA and treated with R1881 or vehicle for 48 h. Lamin B1 and tubulin are shown as controls for nuclear and cytoplasmic extracts, respectively. ( E ) Immunofluorescence showing increased mTOR (red) levels in the nucleus following a 48-h treatment with R1881 in LCNaP cells. Nuclei were stained with DAPI (blue). ( F ) Overlap between mTOR DNA-binding peaks in LNCaP cells treated for 48 h with R1881 or vehicle and heat maps of the signal intensity of mTOR genomic binding peaks in a window of ±2.5 kb. ( G ) Average ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) signal intensities normalized per reads for down-regulated, unregulated, and up-regulated mTOR peaks. ( H ) Examples of University of California at Santa Cruz Genome Browser graphical views of mTOR-binding peaks in LNCaP cells treated with R1881 or vehicle that are either unaffected (unregulated) or positively or negatively regulated by androgens. Genes and peaks (black boxes) are indicated below tag density graphs. ChIP-qPCR (ChIP combined with quantitative PCR) of mTOR in LNCaP ( I ) or LAPC4 ( J ) cells after 48 h of treatment with R1881 or vehicle. Relative fold enrichment was normalized over two negative regions and is shown relative to IgG (set at 1). Results are shown as the average of three independent experiments.

    Journal: Genes & Development

    Article Title: Nuclear mTOR acts as a transcriptional integrator of the androgen signaling pathway in prostate cancer

    doi: 10.1101/gad.299958.117

    Figure Lengend Snippet: Cytoplasmic and nuclear mTOR activities are induced by androgen signaling. ( A ) Protein expression of components of the mTOR signaling pathway and AR in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 or vehicle. Tubulin is shown as a loading control. ( B ) Protein expression of components of the mTOR signaling pathway in whole-cell lysates from LNCaP cells after a 48-h treatment with R1881 and/or anti-androgens (bicalutamide [B] and enzalutamide [E]). Tubulin is shown as a loading control. ( C ) Western blot analysis of nuclear fractions of LNCaP cells treated with R1881 or vehicle for various amounts of time. Lamin B1 is shown as a loading control. ( D ) Western blot analysis of cytoplasmic and nuclear fractions of LNCaP cells transfected with control or anti-AR siRNA and treated with R1881 or vehicle for 48 h. Lamin B1 and tubulin are shown as controls for nuclear and cytoplasmic extracts, respectively. ( E ) Immunofluorescence showing increased mTOR (red) levels in the nucleus following a 48-h treatment with R1881 in LCNaP cells. Nuclei were stained with DAPI (blue). ( F ) Overlap between mTOR DNA-binding peaks in LNCaP cells treated for 48 h with R1881 or vehicle and heat maps of the signal intensity of mTOR genomic binding peaks in a window of ±2.5 kb. ( G ) Average ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) signal intensities normalized per reads for down-regulated, unregulated, and up-regulated mTOR peaks. ( H ) Examples of University of California at Santa Cruz Genome Browser graphical views of mTOR-binding peaks in LNCaP cells treated with R1881 or vehicle that are either unaffected (unregulated) or positively or negatively regulated by androgens. Genes and peaks (black boxes) are indicated below tag density graphs. ChIP-qPCR (ChIP combined with quantitative PCR) of mTOR in LNCaP ( I ) or LAPC4 ( J ) cells after 48 h of treatment with R1881 or vehicle. Relative fold enrichment was normalized over two negative regions and is shown relative to IgG (set at 1). Results are shown as the average of three independent experiments.

    Article Snippet: LNCaP, LAPC4, PC3, DU145, and 22rv1 were originally obtained from American Type Culture Collection (ATCC).

    Techniques: Expressing, Western Blot, Transfection, Immunofluorescence, Staining, Binding Assay, Chromatin Immunoprecipitation, Next-Generation Sequencing, Real-time Polymerase Chain Reaction

    Effects of constitutively active AKT expression in different PCa cells Cells of LNCaP C4-2 (A) CWR22R v1 (B) , and LAPC4 (C) , infected with the pHAGE lentivirus expressing AKT-null or AKT-myr, were maintained in c.s.FBS medium for 48 hours. Total cell lysates from each experiment were analyzed by Western blotting for expression of the indicated proteins.

    Journal: Experimental cell research

    Article Title: AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

    doi: 10.1016/j.yexcr.2013.05.008

    Figure Lengend Snippet: Effects of constitutively active AKT expression in different PCa cells Cells of LNCaP C4-2 (A) CWR22R v1 (B) , and LAPC4 (C) , infected with the pHAGE lentivirus expressing AKT-null or AKT-myr, were maintained in c.s.FBS medium for 48 hours. Total cell lysates from each experiment were analyzed by Western blotting for expression of the indicated proteins.

    Article Snippet: LAPC4 (ATCC) was grown in Iscove's medium with 10% FBS.

    Techniques: Expressing, Infection, Western Blot