panc-1 cells Search Results


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  • 99
    ATCC pancreatic panc 1 cells
    Comparison of MCF-7 and <t>Panc-1</t> cell proliferation before and after transfection with UGT2B4, 2B7, and 2B15. Cell viability was determined in triplicate by the trypan blue exclusion assay 24 and 48 hours after transfection with the indicated UGT2B expression plasmid. Untransfected and Lipofectamine 2000 (sham) transfected cells were used as controls. Bars represent mean percent of viable cells at each time point and vertical lines indicate SEM, n=3. All treatment conditions were compared to the sham (Lipofectamine 2000 only) transfection samples for statistical significance; ** = p
    Pancreatic Panc 1 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore experimental protocol panc 1 cells
    The effect of DHA on monosialotetrahexosylganglioside (GM1) and EGFR levels in the lipid rafts of <t>PANC-1</t> cells. Cells were treated for 4 h with 150 µM DHA ( A ) or the indicated concentrations of DHA ( B ). ( A ) Confocal microscope images. Fixed cells were stained with Alexa-fluor 555-conjugated cholera toxin subunit B (CTB-555) to identify the GM1 of lipid rafts, and with 4′,6-diamidino-2-phenylindole (DAPI) to locate the nucleus. The row of images labeled “None” corresponds to the extract from untreated cells and that labeled “DHA” to the extracts of cells treated with 150 µM DHA. ( B ) Western blot detection of EGFR associated with the soluble vs. insoluble fractions of PANC-1 cellular membranes generated using the detergent Triton X-100. Caveolin-1 was used as a marker for the plasma membrane. The column labeled “None” corresponds to the extract from untreated cells, and the columns labeled “50”, “100”, and “150” correspond to the extracts of cells treated with 50, 100, and 150 µM DHA, respectively.
    Experimental Protocol Panc 1 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    National Centre for Cell Science panc 1 cells
    Effect of MFX and CFX on apoptotic and survival pathway proteins. Western blot analysis of apoptotic and survival pathway protein in MIA PaCa-2 ( a ), and <t>panc-1</t> cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p
    Panc 1 Cells, supplied by National Centre for Cell Science, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa panc 1 cells
    Chmp1A knockdown abolished ATRA mediated growth inhibition and the increase of protein levels of Chmp1A, CRBP, P53 and phospho-P53 . (A) Chmp1A shRNA was cloned into shRNA RNAintro™ pSM2 retroviral vector to generate stable <t>PanC-1</t> clones expressing Chmp1A shRNA. Western blot analysis demonstrated the knockdown efficiency of two colonies (KD1 and KD2). Non-silencing shRNA was used as control. (B) Chmp1A expression was reduced in Chmp1A silenced PanC-1 cells more than 50% on Day 1 and 40% on Day 2 regardless of ATRA treatment, compared with non-silencing shRNA expressing cells that were treated with vehicle DMSO. ATRA treatment increased Chmp1A expression slightly. (C) Silencing of Chmp1A induced growth promotion in the presence or absence of ATRA, compared with control cells that were treated with either DMSO or ATRA. ATRA did not have an effect on growth upon stable expression of Chmp1A shRNA compared to control shRNA. (D) Control shRNA expressing cells showed an increase in P53 and phospho-P53 at serine 15 and 37 without exhibiting an increase in CRBP-1. However, Chmp1A shRNA expressing cells exhibited a decline in CRBP-1 and phospho-P53 expression at serine 15 and 37 when compared with control shRNA expressing cells that were treated with DMSO. ATRA increased P53 expression when Chmp1A was silenced, however, Chmp1A depletion did not decrease P53 expression. D: DMSO, R: ATRA
    Panc 1 Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript panc 1 cells genscript
    Chmp1A knockdown abolished ATRA mediated growth inhibition and the increase of protein levels of Chmp1A, CRBP, P53 and phospho-P53 . (A) Chmp1A shRNA was cloned into shRNA RNAintro™ pSM2 retroviral vector to generate stable <t>PanC-1</t> clones expressing Chmp1A shRNA. Western blot analysis demonstrated the knockdown efficiency of two colonies (KD1 and KD2). Non-silencing shRNA was used as control. (B) Chmp1A expression was reduced in Chmp1A silenced PanC-1 cells more than 50% on Day 1 and 40% on Day 2 regardless of ATRA treatment, compared with non-silencing shRNA expressing cells that were treated with vehicle DMSO. ATRA treatment increased Chmp1A expression slightly. (C) Silencing of Chmp1A induced growth promotion in the presence or absence of ATRA, compared with control cells that were treated with either DMSO or ATRA. ATRA did not have an effect on growth upon stable expression of Chmp1A shRNA compared to control shRNA. (D) Control shRNA expressing cells showed an increase in P53 and phospho-P53 at serine 15 and 37 without exhibiting an increase in CRBP-1. However, Chmp1A shRNA expressing cells exhibited a decline in CRBP-1 and phospho-P53 expression at serine 15 and 37 when compared with control shRNA expressing cells that were treated with DMSO. ATRA increased P53 expression when Chmp1A was silenced, however, Chmp1A depletion did not decrease P53 expression. D: DMSO, R: ATRA
    Panc 1 Cells Genscript, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    LC Sciences panc 1 cells
    Selinexor treatment or miR-145 mimic transfection suppressed the migration activity of PDAC cells Selinexor treated or miR-145 mimic transfected MiaPaCa-2 (A) , AsPC-1 (B) and <t>PANC-1</t> (C) PDAC cells were seeded in 6 well plate and subjected to wound healing assay for 3 days as described in the section of “Materials and Methods”. The cells were photographed in each day.
    Panc 1 Cells, supplied by LC Sciences, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson panc 1 cells
    <t>Panc-1</t> cells treated with Ni NWs which potentiates apoptosis. Cells were treated with 200 μL Ni NWs for 8, 16, and 24 hours and stained with ethidium bromide (EB) and acridine orange (AO) and were visualized and photographed immediately with fluorescence microscope. ( A ) Control; ( B ) 8 hours after treatment – blue arrow indicates initial membrane blebbing of the apoptosed cells; ( C ) 16 hours after treatment – the white arrow indicates late membrane blebbing; ( D ) 24 hours after treatment. Cells were stained with AO and EB where viable cells were green nuclear fluorescence and apoptotic cells exhibit a red nucleus.
    Panc 1 Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mirus Bio panc 1 cells
    Ectopic expression of RhoB, not RhoA, inhibits Ras/PI3K/Akt-mediated transformation and migration. (a) Parental NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-PI3K, or CA-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The cells were split, and an aliquot of the cells was evaluated for expression of transfected RhoA and RhoB by Western blotting. The rest of the cells were then cultured for another 4 weeks. The plates were examined for focus formation by staining. H-Ras/NIH 3T3 (b) and <t>PANC-1</t> (c) cells were transiently transfected with pcDNA3 vector control, CA-Akt, or WT-Akt in the presence or absence of RhoA or RhoB or pcDNA3 for 36 h, internal ribosome entry site-EGFP was cotransfected as a transfection indicator. The cells were then split and analyzed for their migration capabilities through collagen type I-coated transfilters as described in Materials and Methods. (d and e) NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-Akt, or WT-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The monolayers were then scratched with a yellow tip and microphotographed at the time points indicated to analyze their capability to migrate into and fill the wounded area.
    Panc 1 Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    3-D Matrix panc 1 cells
    Ectopic expression of RhoB, not RhoA, inhibits Ras/PI3K/Akt-mediated transformation and migration. (a) Parental NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-PI3K, or CA-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The cells were split, and an aliquot of the cells was evaluated for expression of transfected RhoA and RhoB by Western blotting. The rest of the cells were then cultured for another 4 weeks. The plates were examined for focus formation by staining. H-Ras/NIH 3T3 (b) and <t>PANC-1</t> (c) cells were transiently transfected with pcDNA3 vector control, CA-Akt, or WT-Akt in the presence or absence of RhoA or RhoB or pcDNA3 for 36 h, internal ribosome entry site-EGFP was cotransfected as a transfection indicator. The cells were then split and analyzed for their migration capabilities through collagen type I-coated transfilters as described in Materials and Methods. (d and e) NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-Akt, or WT-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The monolayers were then scratched with a yellow tip and microphotographed at the time points indicated to analyze their capability to migrate into and fill the wounded area.
    Panc 1 Cells, supplied by 3-D Matrix, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Corning Life Sciences panc 1 cells
    Tracking individual cells in heterogeneous populations. ( a ) Comparison of ROC curves based on data from Harmony AUC = 0.93, CI 95% = [0.87,0.98] (blue), CellProfiler, AUC = 0.92, CI 95% = [0.84, 0.99] (yellow) and Imaris, AUC = 0.89 95%CI = [0.81–0.97] (green). ( b ) Cell not rejected due to TrAM outlined in green (τ = 2.79). Below outlined in red, cell excluded from evaluation due to TrAM value > 4.81 threshold (τ = 36.33). Nuclear area and roundness are plotted over time. Smoothed curves represent typical fluctuation between adjacent time points. Cell segmentation images correspond to highlighted data points. ( c ) Adjustment of PC3 cell motility to high density imaging areas (R2 = 0.66). Plot correlates number of cells per imaging field with current speed. Each dot represents an imaging well of a 96-well plate. ( d ) Speed distribution of PC3, HeLa and <t>Panc-1</t> cells before (blue) and after (yellow) TrAM filtering. PC3 data points indicate average speed of examples of high and low τ shown in b . ( e ) Speed distribution of HeLa (n = 26 fields, SEM = 0.005) vs. Panc-1 (6 fields, SEM = 0.006) cells post-filtering, p
    Panc 1 Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Scientific Cell Company panc 1 cells
    α -Mangostin modulates the expression of EMT-related genes in pancreatic cancer cells cells. (a) <t>Panc-1</t> and BxPC-3 cells were treated with α -mangostin 16 μ M at the indicated concentrations for 24 h. Protein (a) and mRNA (b) levels of MMP-2, MMP-9, E-cadherin, vimentin, and N-cadherin were measured by Western blotting and qRT-PCR, respectively. * P
    Panc 1 Cells, supplied by Scientific Cell Company, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Ted Pella panc 1 cells
    Activation of RhoG GTPase in hTERT-HPNE, BxPC3 and <t>PANC-1</t> pancreatic cell lines. Cells were starved 6 hours and confronted with rhEGF for the period of 0, 2 and 10 minutes (Marked as 0, 2 and 10). Fluorescence microscopic staining of RhoG (green) was carried out in hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoG activity was performed using RhoG pulldown assay. Immunoblots for RhoG, Rac-1 and GAPDH proteins for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) are shown. To quantify the amount of RhoG-GTP and bound-Rac-1 through the temporal course, densitometric analysis was performed using Image Lab software, hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I). For comparison of RhoG activity, the total amount of RhoG in cell lysates was normalized to total RhoG. GAPDH was used as a protein loading control. ELMO1-GST beads coomassie are shown as beads loading control. Arrowheads denote the localization of RhoG into the peripheral membrane; boxes with number represent the number of cells with this phenotype. Scale bar 100 μm.
    Panc 1 Cells, supplied by Ted Pella, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Inserm Transfert panc 1 cells
    AXL knockdown in the <t>Panc-1</t> pancreatic cancer cell line affects cell viability, colony formation, migration and impairs tumor growth. Analysis of cell viability after five days in culture by MTS (A), colony formation after 14 days by Giemsa staining (B),
    Panc 1 Cells, supplied by Inserm Transfert, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher panc 1 cells
    Cell size specific delivery. A) Diameters of the three different cell types (T-cells, BxPc3, <t>PANC-1</t> cells). B) Percentage delivery and viability of T-cell (left), BxPc3 (center), and PANC-1 (right) when passed through chips with different constriction widths at 100 psi. The average and standard deviation of 3 to 5 independent experiments are shown. C) Delivery efficacy of PANC-1, where delivery efficacy is the product of percent viability and percent delivery at each condition (constriction width and pressure). D) Heat map of delivery efficacy when a suspension of one cell type (x-axis) is passed through chips with different constriction widths (y-axis) at 50 and 100 psi. Darker colors indicate higher delivery efficacy and lighter colors indicates lower delivery efficacy. E) Flow cytometry plot of a mixture of T-cells (low FITC region) and PANC-1 (high FITC region) cells incubated with the dextran blue dye (Pacific Blue), but not processed through the microfluidic delivery platform (left panel) demonstrating negative controls to establish the basal levels of surface receptor-mediated binding and nonspecific uptake of dye. Flow cytometry plot of a mixture of T-cell and PANC-1 cells passed through a chip with constrictions that were 6 µm wide (and 10 µm long) at 100 psi (right panel). F) Percentage delivery of a mixture of T-cell and GFP-labeled PANC-1 cells when passed through chips with different constriction widths at 100 psi. Each condition represents the average and standard deviation of three independent experiments.
    Panc 1 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of MCF-7 and Panc-1 cell proliferation before and after transfection with UGT2B4, 2B7, and 2B15. Cell viability was determined in triplicate by the trypan blue exclusion assay 24 and 48 hours after transfection with the indicated UGT2B expression plasmid. Untransfected and Lipofectamine 2000 (sham) transfected cells were used as controls. Bars represent mean percent of viable cells at each time point and vertical lines indicate SEM, n=3. All treatment conditions were compared to the sham (Lipofectamine 2000 only) transfection samples for statistical significance; ** = p

    Journal: Cancer Biology & Therapy

    Article Title: Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines

    doi: 10.1080/15384047.2015.1026480

    Figure Lengend Snippet: Comparison of MCF-7 and Panc-1 cell proliferation before and after transfection with UGT2B4, 2B7, and 2B15. Cell viability was determined in triplicate by the trypan blue exclusion assay 24 and 48 hours after transfection with the indicated UGT2B expression plasmid. Untransfected and Lipofectamine 2000 (sham) transfected cells were used as controls. Bars represent mean percent of viable cells at each time point and vertical lines indicate SEM, n=3. All treatment conditions were compared to the sham (Lipofectamine 2000 only) transfection samples for statistical significance; ** = p

    Article Snippet: Human breast cancer MCF-7 cells (ATCC, Catalog# HTB-22), human pancreatic carcinoma Panc-1 cells (ATCC, Catalog# CRL-1469), Capan-2 cells (ATCC, Catalog# HTB-80), BxPC-3 cells (ATCC, Catalog# CRL-1687), and MIA PaCa-2 cells (ATCC, Catalog# CRL-1420) were grown at 5% CO2 /95% air in a humidified atmosphere at 37°C in Dulbecco's Modified Eagle's Medium (DMEM; ATCC, Catalog# 30-2002) supplemented with 10% fetal bovine serum (Fisher Scientific, Catalog#SH3091003).

    Techniques: Transfection, Trypan Blue Exclusion Assay, Expressing, Plasmid Preparation

    Comparison of steady-state mRNA levels for UGT2B isoforms in breast and pancreatic cancer cells lines measured using qPCR. Steady-state levels of UGT2B family mRNAs were quantified by qPCR in breast and pancreatic cancer cell lines. UGT2B mRNA expression is depicted normalized to 18s RNA. Breast: MCF-10A, a non-tumorigenic immortalized mammary epithelial cell line; MCF-7, a well-differentiated, non-metastatic cell line; MDA-231 and MDA-468, poorly-differentiated, metastatic cell lines. Pancreas: Capan-2, a well-differentiated, non-metastatic cell line; BxPC-3, a moderately-differentiated, non-metastatic cell line, Panc-1 and MIA PaCa-2, poorly-differentiated cell lines, PK-1, a metastatic cell line.

    Journal: Cancer Biology & Therapy

    Article Title: Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines

    doi: 10.1080/15384047.2015.1026480

    Figure Lengend Snippet: Comparison of steady-state mRNA levels for UGT2B isoforms in breast and pancreatic cancer cells lines measured using qPCR. Steady-state levels of UGT2B family mRNAs were quantified by qPCR in breast and pancreatic cancer cell lines. UGT2B mRNA expression is depicted normalized to 18s RNA. Breast: MCF-10A, a non-tumorigenic immortalized mammary epithelial cell line; MCF-7, a well-differentiated, non-metastatic cell line; MDA-231 and MDA-468, poorly-differentiated, metastatic cell lines. Pancreas: Capan-2, a well-differentiated, non-metastatic cell line; BxPC-3, a moderately-differentiated, non-metastatic cell line, Panc-1 and MIA PaCa-2, poorly-differentiated cell lines, PK-1, a metastatic cell line.

    Article Snippet: Human breast cancer MCF-7 cells (ATCC, Catalog# HTB-22), human pancreatic carcinoma Panc-1 cells (ATCC, Catalog# CRL-1469), Capan-2 cells (ATCC, Catalog# HTB-80), BxPC-3 cells (ATCC, Catalog# CRL-1687), and MIA PaCa-2 cells (ATCC, Catalog# CRL-1420) were grown at 5% CO2 /95% air in a humidified atmosphere at 37°C in Dulbecco's Modified Eagle's Medium (DMEM; ATCC, Catalog# 30-2002) supplemented with 10% fetal bovine serum (Fisher Scientific, Catalog#SH3091003).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Multiple Displacement Amplification

    Flow cytometry analysis of MCF-7 and Panc-1 cells transfected with UGT2B4, 2B7, and 2B15. MCF-7 and Panc-1 cells were stained with annexin V/PI at 48 and 72 h after transfection. The data shown represent the average percentage of gated cells in each quadrant collected from 3 experiments. The error bars represent standard error of the mean (SEM).

    Journal: Cancer Biology & Therapy

    Article Title: Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines

    doi: 10.1080/15384047.2015.1026480

    Figure Lengend Snippet: Flow cytometry analysis of MCF-7 and Panc-1 cells transfected with UGT2B4, 2B7, and 2B15. MCF-7 and Panc-1 cells were stained with annexin V/PI at 48 and 72 h after transfection. The data shown represent the average percentage of gated cells in each quadrant collected from 3 experiments. The error bars represent standard error of the mean (SEM).

    Article Snippet: Human breast cancer MCF-7 cells (ATCC, Catalog# HTB-22), human pancreatic carcinoma Panc-1 cells (ATCC, Catalog# CRL-1469), Capan-2 cells (ATCC, Catalog# HTB-80), BxPC-3 cells (ATCC, Catalog# CRL-1687), and MIA PaCa-2 cells (ATCC, Catalog# CRL-1420) were grown at 5% CO2 /95% air in a humidified atmosphere at 37°C in Dulbecco's Modified Eagle's Medium (DMEM; ATCC, Catalog# 30-2002) supplemented with 10% fetal bovine serum (Fisher Scientific, Catalog#SH3091003).

    Techniques: Flow Cytometry, Cytometry, Transfection, Staining

    TUNEL and caspase-3 immunohistochemistry in Panc-1 cells transfected with UGT2B4, 2B7, and 2B15. Cells were transfected with the indicated UGT2B expression plasmid or Lipofectamine 2000 alone as a sham control. Forty-eight hours after transfection, TUNEL and activated caspase-3 assays were performed as described in Methods and visualized by fluorescence microscopy using 200 x magnification. Nuclei were visualized by DAPI stain. For quantification, 10 independent fields were analyzed. Bar graphs depict the average quantification of imaged fluorescence signals from TUNEL and caspase-3 positive cells and vertical lines indicate SEM, n=4. *= p

    Journal: Cancer Biology & Therapy

    Article Title: Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines

    doi: 10.1080/15384047.2015.1026480

    Figure Lengend Snippet: TUNEL and caspase-3 immunohistochemistry in Panc-1 cells transfected with UGT2B4, 2B7, and 2B15. Cells were transfected with the indicated UGT2B expression plasmid or Lipofectamine 2000 alone as a sham control. Forty-eight hours after transfection, TUNEL and activated caspase-3 assays were performed as described in Methods and visualized by fluorescence microscopy using 200 x magnification. Nuclei were visualized by DAPI stain. For quantification, 10 independent fields were analyzed. Bar graphs depict the average quantification of imaged fluorescence signals from TUNEL and caspase-3 positive cells and vertical lines indicate SEM, n=4. *= p

    Article Snippet: Human breast cancer MCF-7 cells (ATCC, Catalog# HTB-22), human pancreatic carcinoma Panc-1 cells (ATCC, Catalog# CRL-1469), Capan-2 cells (ATCC, Catalog# HTB-80), BxPC-3 cells (ATCC, Catalog# CRL-1687), and MIA PaCa-2 cells (ATCC, Catalog# CRL-1420) were grown at 5% CO2 /95% air in a humidified atmosphere at 37°C in Dulbecco's Modified Eagle's Medium (DMEM; ATCC, Catalog# 30-2002) supplemented with 10% fetal bovine serum (Fisher Scientific, Catalog#SH3091003).

    Techniques: TUNEL Assay, Immunohistochemistry, Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Staining

    Expression of markers of pancreatic differentiation in cells derived from human control and HNF1A MODY reprogrammed cells. (A). Expression of markers in cells subjected to direct differentiation method. (B). Expression of markers in cells subjected to embryoid bodies-mediated method. Agarose gel electrophoresis of representative RT-PCR products. In case of direct differentiation RT-PCR was performed for cells from each patients and in case of embryoid bodies-mediated differentiation RT-PCR was performed in duplicates. EF2 was used as a housekeeping control for each performed reaction. (C). The efficiency of detection of pancreatic differentiation markers (both differentiation methods are combined, n = 3–5). Percentage of positive RT-PCR results in relation to expression of EF2 housekeeping gene which was detected in all analysed samples. (D). Production of glucagon in differentiated Panc-1 cells as well as control and HNF-1A MODY reprogrammed cells differentiated with embryoid bodies-mediated method (EB) and direct method (direct). Medium – media used to perform the last step of differentiation (in case of Panc-1 cells one type of medium was utilized); cells – media collected from differentiated cells in the last day of differentiation (n = 2).

    Journal: Scientific Reports

    Article Title: Induced pluripotent stem cells as a model for diabetes investigation

    doi: 10.1038/srep08597

    Figure Lengend Snippet: Expression of markers of pancreatic differentiation in cells derived from human control and HNF1A MODY reprogrammed cells. (A). Expression of markers in cells subjected to direct differentiation method. (B). Expression of markers in cells subjected to embryoid bodies-mediated method. Agarose gel electrophoresis of representative RT-PCR products. In case of direct differentiation RT-PCR was performed for cells from each patients and in case of embryoid bodies-mediated differentiation RT-PCR was performed in duplicates. EF2 was used as a housekeeping control for each performed reaction. (C). The efficiency of detection of pancreatic differentiation markers (both differentiation methods are combined, n = 3–5). Percentage of positive RT-PCR results in relation to expression of EF2 housekeeping gene which was detected in all analysed samples. (D). Production of glucagon in differentiated Panc-1 cells as well as control and HNF-1A MODY reprogrammed cells differentiated with embryoid bodies-mediated method (EB) and direct method (direct). Medium – media used to perform the last step of differentiation (in case of Panc-1 cells one type of medium was utilized); cells – media collected from differentiated cells in the last day of differentiation (n = 2).

    Article Snippet: Differentiation of Panc-1 pancreatic cancer cell line Panc-1 cells (ATCC, CRL-1469) were cultured in DMEM medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) and differentiated toward insulin- and glucagon-expressing cells according to Suzuki J et al. protocol .

    Techniques: Expressing, Derivative Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    EIF2α phosphorylation in NUPR1 deficient Panc-1 and matched control (Panc-1) in response to Thapsigargin induced ER-stress. A) The whole cell lysates were analysed by Western Blotting using phosphorylated eIF2α antibody. B) mRNA expression of CHOP and GADD34 genes in NUPR1 deficient cells and matched control cells was analyzed overtime by qPCR after 1 μM incubation with thapsigargin. Significant results are reported in the graph (****p

    Journal: bioRxiv

    Article Title: The stress-induced protein NUPR1 orchestrates protein translation during ER-stress by interacting with eIF2α

    doi: 10.1101/2020.02.18.954115

    Figure Lengend Snippet: EIF2α phosphorylation in NUPR1 deficient Panc-1 and matched control (Panc-1) in response to Thapsigargin induced ER-stress. A) The whole cell lysates were analysed by Western Blotting using phosphorylated eIF2α antibody. B) mRNA expression of CHOP and GADD34 genes in NUPR1 deficient cells and matched control cells was analyzed overtime by qPCR after 1 μM incubation with thapsigargin. Significant results are reported in the graph (****p

    Article Snippet: Cell culture MiaPaCa-2 and Panc-1 cells were purchased from ATCC and cultures in DMEM medium (Dulbecco’s modified Eagle’s medium, Gibco, Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland) and cultured at 37°C and 5% CO2 .

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

    Imaging nascent proteins in pancreatic cultured cells with OP-puro. A) Cultured Panc-1 WT or Crisper/Cas9 edited for NUPR1 (KO-Clones 1 and 2, B and C) cells were incubated for 1h or 3h with 1 μM of TPS. The cells were subsequently incubated for 1h with OP-puromycin. PFA fixed cells were stained by CuAAC with FITC-azide, and imaged by Confocal microscopy. Arbitrary Fluorescent intensity (AFU) was measured with imageJ is reported in graph D. Two-way ANOVA with a post hoc Sidak test were used to determine statistical significance (**p = 0.001, ****p

    Journal: bioRxiv

    Article Title: The stress-induced protein NUPR1 orchestrates protein translation during ER-stress by interacting with eIF2α

    doi: 10.1101/2020.02.18.954115

    Figure Lengend Snippet: Imaging nascent proteins in pancreatic cultured cells with OP-puro. A) Cultured Panc-1 WT or Crisper/Cas9 edited for NUPR1 (KO-Clones 1 and 2, B and C) cells were incubated for 1h or 3h with 1 μM of TPS. The cells were subsequently incubated for 1h with OP-puromycin. PFA fixed cells were stained by CuAAC with FITC-azide, and imaged by Confocal microscopy. Arbitrary Fluorescent intensity (AFU) was measured with imageJ is reported in graph D. Two-way ANOVA with a post hoc Sidak test were used to determine statistical significance (**p = 0.001, ****p

    Article Snippet: Cell culture MiaPaCa-2 and Panc-1 cells were purchased from ATCC and cultures in DMEM medium (Dulbecco’s modified Eagle’s medium, Gibco, Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland) and cultured at 37°C and 5% CO2 .

    Techniques: Imaging, Cell Culture, Incubation, Staining, Confocal Microscopy

    GLV-1h153 infection and killing in cell culture and in vivo . a. PANC-1 cells were infected by various GLV-1h153at MOIs of 0.01, 0.1, and 1.0. Cell viability was determined via lactate dehydrogenase assays, and was set at 100% before infection. GLV-1h153 infected and was cytotxic at various MOIs, with less than 20% survival of cells as compared to control at an MOI of 1.0 by day 9. The values are the mean of triplicate samples, and bars indicate SD. b. GFP expression is shown to be time-dependent, with abundant GFP expression by day 3. Phase overlay pictures shows gradual cell death and thus decline of GFP expression by day 7. Closer examination of infected cells reveals loss of normal morphology and cell progressive cell detachment. c. 2 × 10 6 PFUs of GLV-1h153 or GLV-1h68, or PBS were injected IVly or ITly into nude mice bearing s.c. PANC-1 tumors on the hindleg (~100 mm 3 ). GLV-1h153 was able to regress pancreatic tumor xenograft both ITly and IVly starting at day 13. The values are a mean of 4-5 mice, with bars indicating SEM. d. GLV-1h153 infection of pancreatic tumor xenografts did not have adverse effects on body weight at 5 weeks post injection, with the IT group even gaining weight compared to control.

    Journal: Journal of Translational Medicine

    Article Title: Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    doi: 10.1186/1479-5876-9-36

    Figure Lengend Snippet: GLV-1h153 infection and killing in cell culture and in vivo . a. PANC-1 cells were infected by various GLV-1h153at MOIs of 0.01, 0.1, and 1.0. Cell viability was determined via lactate dehydrogenase assays, and was set at 100% before infection. GLV-1h153 infected and was cytotxic at various MOIs, with less than 20% survival of cells as compared to control at an MOI of 1.0 by day 9. The values are the mean of triplicate samples, and bars indicate SD. b. GFP expression is shown to be time-dependent, with abundant GFP expression by day 3. Phase overlay pictures shows gradual cell death and thus decline of GFP expression by day 7. Closer examination of infected cells reveals loss of normal morphology and cell progressive cell detachment. c. 2 × 10 6 PFUs of GLV-1h153 or GLV-1h68, or PBS were injected IVly or ITly into nude mice bearing s.c. PANC-1 tumors on the hindleg (~100 mm 3 ). GLV-1h153 was able to regress pancreatic tumor xenograft both ITly and IVly starting at day 13. The values are a mean of 4-5 mice, with bars indicating SEM. d. GLV-1h153 infection of pancreatic tumor xenografts did not have adverse effects on body weight at 5 weeks post injection, with the IT group even gaining weight compared to control.

    Article Snippet: Virus and cell culture African green monkey kidney fibroblast CV-1 cells and human pancreatic ductal carcinoma PANC-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% antibiotic-antimycotic solution (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS) (Mediatech, Inc.) at 37°C under 5% CO2 .

    Techniques: Infection, Cell Culture, In Vivo, Expressing, Injection, Mouse Assay

    Viral proliferation assay of GLV-1h153-in PANC-1 cells . a. PANC-1 cells were grown in 6-well plates and infected with GLV-1h153 or GLV-1h68 at an MOI of 0.01 and 1.0. Three wells of each virus were harvested at 1, 24, 48, and 72 hours postinfection. GLV-1h153 replicated in a similar manner to GLV-1h68, with a 4-log increase in viral load at an MOI of 0.01 by 72 hours, reaching similar levels as that in cells infected with an MOI of 1.0. This demonstrates that GLV-1h153 is able to replicate efficiently within PANC-1 cells in vitro as well as parental virus GLV-1h68. b. GFP expression was quantified via flow cytometry in PANC-1 cells infected with GLV-1h153 at MOIs of 1.0 and 0.01 and was shown to be MOI dependent. GFP expression mimicked the viral replication growth curve, with GFP expression in the MOI 0.01 infected cells reaching similar levels as the MOI of 1.0 by 72 hours after infection. c. GFP expression was quantified via flow cytometry in PANC-1 cells infected with an MOI of 0.01, 0.1, 0.5, 1.0 2.0, and 5.0 at 24 hours after infection, and was shown to be MOI-dependent.

    Journal: Journal of Translational Medicine

    Article Title: Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    doi: 10.1186/1479-5876-9-36

    Figure Lengend Snippet: Viral proliferation assay of GLV-1h153-in PANC-1 cells . a. PANC-1 cells were grown in 6-well plates and infected with GLV-1h153 or GLV-1h68 at an MOI of 0.01 and 1.0. Three wells of each virus were harvested at 1, 24, 48, and 72 hours postinfection. GLV-1h153 replicated in a similar manner to GLV-1h68, with a 4-log increase in viral load at an MOI of 0.01 by 72 hours, reaching similar levels as that in cells infected with an MOI of 1.0. This demonstrates that GLV-1h153 is able to replicate efficiently within PANC-1 cells in vitro as well as parental virus GLV-1h68. b. GFP expression was quantified via flow cytometry in PANC-1 cells infected with GLV-1h153 at MOIs of 1.0 and 0.01 and was shown to be MOI dependent. GFP expression mimicked the viral replication growth curve, with GFP expression in the MOI 0.01 infected cells reaching similar levels as the MOI of 1.0 by 72 hours after infection. c. GFP expression was quantified via flow cytometry in PANC-1 cells infected with an MOI of 0.01, 0.1, 0.5, 1.0 2.0, and 5.0 at 24 hours after infection, and was shown to be MOI-dependent.

    Article Snippet: Virus and cell culture African green monkey kidney fibroblast CV-1 cells and human pancreatic ductal carcinoma PANC-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% antibiotic-antimycotic solution (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS) (Mediatech, Inc.) at 37°C under 5% CO2 .

    Techniques: Proliferation Assay, Infection, In Vitro, Expressing, Flow Cytometry, Cytometry

    Assessment of hNIS expression in GLV-1h153-infected PANC-1 cells . a. Microarray analysis of cells infected with an MOI of 5.0 of GLV-1h153 yielded an almost 2000-fold increase by 6 hours and an almost 5000-fold increase by 24 hours in hNIS mRNA production as compared to noninfected control. b. PANC-1 cells were either mock infected or infected with GLV-1h68 at an MOI of 1.0 or infected with GLV-1h153 at an MOI of 1.0 or 5.0 for 24 hours. The hNIS protein was detected by Western blot analysis using monoclonal anti-hNIS antibody. Only GLV-1h153-infected cells expressed the hNIS protein, but cells either mock infected or infected with GLV-1h68 did not. The molecular weight marker bands (in kiloDaltons) are shown on the left. c. PANC-1 cells were mock infected or infected with GLV-1h68 or GLV-1h153 at a MOI of 1.0 for 24 hours. The hNIS protein was detected by immunofluorescence microscopy using monoclonal anti-hNIS antibody, which recognizes the intracellular domain of the protein. Mock- or GLV-1h68-infected cells (as demonstrated by GFP expression) did not express the hNIS protein, whereas the hNIS protein on the cell membrane of PANC-1 cells infected with GLV-1h153 was readily detectable.

    Journal: Journal of Translational Medicine

    Article Title: Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    doi: 10.1186/1479-5876-9-36

    Figure Lengend Snippet: Assessment of hNIS expression in GLV-1h153-infected PANC-1 cells . a. Microarray analysis of cells infected with an MOI of 5.0 of GLV-1h153 yielded an almost 2000-fold increase by 6 hours and an almost 5000-fold increase by 24 hours in hNIS mRNA production as compared to noninfected control. b. PANC-1 cells were either mock infected or infected with GLV-1h68 at an MOI of 1.0 or infected with GLV-1h153 at an MOI of 1.0 or 5.0 for 24 hours. The hNIS protein was detected by Western blot analysis using monoclonal anti-hNIS antibody. Only GLV-1h153-infected cells expressed the hNIS protein, but cells either mock infected or infected with GLV-1h68 did not. The molecular weight marker bands (in kiloDaltons) are shown on the left. c. PANC-1 cells were mock infected or infected with GLV-1h68 or GLV-1h153 at a MOI of 1.0 for 24 hours. The hNIS protein was detected by immunofluorescence microscopy using monoclonal anti-hNIS antibody, which recognizes the intracellular domain of the protein. Mock- or GLV-1h68-infected cells (as demonstrated by GFP expression) did not express the hNIS protein, whereas the hNIS protein on the cell membrane of PANC-1 cells infected with GLV-1h153 was readily detectable.

    Article Snippet: Virus and cell culture African green monkey kidney fibroblast CV-1 cells and human pancreatic ductal carcinoma PANC-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% antibiotic-antimycotic solution (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS) (Mediatech, Inc.) at 37°C under 5% CO2 .

    Techniques: Expressing, Infection, Microarray, Western Blot, Molecular Weight, Marker, Immunofluorescence, Microscopy

    Assessment of in vitro 131 I radiouptake of GLV-1h153-infected PANC-1 cells . a. PANC-1 cells were infected with an MOI of 0, 0.01, 0.1, and 1.0 of GLV-1h153 and MOI of 1.0 of GLV-1h68. PCCL3 was used as a positive control. Twenty-four hours after infection, there is a > 70-fold enhanced radiouptake at an MOI of 1.0 as compared to an MOI of 0 in GLV-1h153, and radiouptake is shown to be MOI dependent and hNIS specific (as shown with blocking with competitive inhibitor of hNIS, NaClO4). b. Maximum radiouptake with an MOI of 1.0 24 hrs after infection corresponded to maximum GFP expression.

    Journal: Journal of Translational Medicine

    Article Title: Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    doi: 10.1186/1479-5876-9-36

    Figure Lengend Snippet: Assessment of in vitro 131 I radiouptake of GLV-1h153-infected PANC-1 cells . a. PANC-1 cells were infected with an MOI of 0, 0.01, 0.1, and 1.0 of GLV-1h153 and MOI of 1.0 of GLV-1h68. PCCL3 was used as a positive control. Twenty-four hours after infection, there is a > 70-fold enhanced radiouptake at an MOI of 1.0 as compared to an MOI of 0 in GLV-1h153, and radiouptake is shown to be MOI dependent and hNIS specific (as shown with blocking with competitive inhibitor of hNIS, NaClO4). b. Maximum radiouptake with an MOI of 1.0 24 hrs after infection corresponded to maximum GFP expression.

    Article Snippet: Virus and cell culture African green monkey kidney fibroblast CV-1 cells and human pancreatic ductal carcinoma PANC-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% antibiotic-antimycotic solution (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS) (Mediatech, Inc.) at 37°C under 5% CO2 .

    Techniques: In Vitro, Infection, Positive Control, Blocking Assay, Expressing

    PET imaging of enhanced radiouptake in GLV-1h153-infected PANC-1 xenografts . Two × 10 7 PFU of GLV-1h153, GLV-1h68, or PBS was injected intratumorally into PANC-1 hindleg tumor-bearing mice. 124 I-PET scanning was obtained 48 hours after infection and 1 hour after radiotracer administration. GLV-1h153-infected PANC-1 tumors were easily visualized, while no enhanced signal was seen in the PBS- or GLV-1h68 injected tumors. The stomach and thyroid were also imaged due to native NIS expression, and the bladder due to tracer excretion.

    Journal: Journal of Translational Medicine

    Article Title: Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    doi: 10.1186/1479-5876-9-36

    Figure Lengend Snippet: PET imaging of enhanced radiouptake in GLV-1h153-infected PANC-1 xenografts . Two × 10 7 PFU of GLV-1h153, GLV-1h68, or PBS was injected intratumorally into PANC-1 hindleg tumor-bearing mice. 124 I-PET scanning was obtained 48 hours after infection and 1 hour after radiotracer administration. GLV-1h153-infected PANC-1 tumors were easily visualized, while no enhanced signal was seen in the PBS- or GLV-1h68 injected tumors. The stomach and thyroid were also imaged due to native NIS expression, and the bladder due to tracer excretion.

    Article Snippet: Virus and cell culture African green monkey kidney fibroblast CV-1 cells and human pancreatic ductal carcinoma PANC-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% antibiotic-antimycotic solution (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS) (Mediatech, Inc.) at 37°C under 5% CO2 .

    Techniques: Positron Emission Tomography, Imaging, Infection, Injection, Mouse Assay, Expressing

    The effect of DHA on monosialotetrahexosylganglioside (GM1) and EGFR levels in the lipid rafts of PANC-1 cells. Cells were treated for 4 h with 150 µM DHA ( A ) or the indicated concentrations of DHA ( B ). ( A ) Confocal microscope images. Fixed cells were stained with Alexa-fluor 555-conjugated cholera toxin subunit B (CTB-555) to identify the GM1 of lipid rafts, and with 4′,6-diamidino-2-phenylindole (DAPI) to locate the nucleus. The row of images labeled “None” corresponds to the extract from untreated cells and that labeled “DHA” to the extracts of cells treated with 150 µM DHA. ( B ) Western blot detection of EGFR associated with the soluble vs. insoluble fractions of PANC-1 cellular membranes generated using the detergent Triton X-100. Caveolin-1 was used as a marker for the plasma membrane. The column labeled “None” corresponds to the extract from untreated cells, and the columns labeled “50”, “100”, and “150” correspond to the extracts of cells treated with 50, 100, and 150 µM DHA, respectively.

    Journal: Nutrients

    Article Title: Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB

    doi: 10.3390/nu10111621

    Figure Lengend Snippet: The effect of DHA on monosialotetrahexosylganglioside (GM1) and EGFR levels in the lipid rafts of PANC-1 cells. Cells were treated for 4 h with 150 µM DHA ( A ) or the indicated concentrations of DHA ( B ). ( A ) Confocal microscope images. Fixed cells were stained with Alexa-fluor 555-conjugated cholera toxin subunit B (CTB-555) to identify the GM1 of lipid rafts, and with 4′,6-diamidino-2-phenylindole (DAPI) to locate the nucleus. The row of images labeled “None” corresponds to the extract from untreated cells and that labeled “DHA” to the extracts of cells treated with 150 µM DHA. ( B ) Western blot detection of EGFR associated with the soluble vs. insoluble fractions of PANC-1 cellular membranes generated using the detergent Triton X-100. Caveolin-1 was used as a marker for the plasma membrane. The column labeled “None” corresponds to the extract from untreated cells, and the columns labeled “50”, “100”, and “150” correspond to the extracts of cells treated with 50, 100, and 150 µM DHA, respectively.

    Article Snippet: Experimental Protocol PANC-1 cells were incubated with DHA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in ethanol (final concentration of 50, 100 or 150 µM) for 4 h prior to the determination of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the assays of NF-κB DNA-binding activity and STAT3 DNA-binding activity, and the measurement of lipid raft EGFR levels.

    Techniques: Microscopy, Staining, CtB Assay, Labeling, Western Blot, Generated, Marker

    The effect of DHA on cyclin D1 and survivin gene expression in PANC-1 cells. Cells were treated with DHA for 24 h. ( A , B ) A plot of the relative levels of cyclin D and survivin messenger RNA (mRNA), determined by using real-time PCR analysis. * p

    Journal: Nutrients

    Article Title: Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB

    doi: 10.3390/nu10111621

    Figure Lengend Snippet: The effect of DHA on cyclin D1 and survivin gene expression in PANC-1 cells. Cells were treated with DHA for 24 h. ( A , B ) A plot of the relative levels of cyclin D and survivin messenger RNA (mRNA), determined by using real-time PCR analysis. * p

    Article Snippet: Experimental Protocol PANC-1 cells were incubated with DHA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in ethanol (final concentration of 50, 100 or 150 µM) for 4 h prior to the determination of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the assays of NF-κB DNA-binding activity and STAT3 DNA-binding activity, and the measurement of lipid raft EGFR levels.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Effect of DHA on nuclear transcription factor-κB (NF-κB) activation and IκBα levels in PANC-1 cells. Cells were pre-treated with the indicated concentrations of DHA for 4 h. ( A ) DNA-binding activity of NF-κB was examined by the luciferase assay. * p

    Journal: Nutrients

    Article Title: Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB

    doi: 10.3390/nu10111621

    Figure Lengend Snippet: Effect of DHA on nuclear transcription factor-κB (NF-κB) activation and IκBα levels in PANC-1 cells. Cells were pre-treated with the indicated concentrations of DHA for 4 h. ( A ) DNA-binding activity of NF-κB was examined by the luciferase assay. * p

    Article Snippet: Experimental Protocol PANC-1 cells were incubated with DHA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in ethanol (final concentration of 50, 100 or 150 µM) for 4 h prior to the determination of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the assays of NF-κB DNA-binding activity and STAT3 DNA-binding activity, and the measurement of lipid raft EGFR levels.

    Techniques: Activation Assay, Binding Assay, Activity Assay, Luciferase

    Effect of DHA on the levels of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the interaction of EGFR and STAT3, STAT3 DNA-binding activity, and the effect of AG1478 on p-STAT3 and STAT3 levels in PANC-1 cells. Cells were treated with 150 µM DHA for various time periods ( A ) and with 50, 100, and 150 µM DHA for 4 h ( B – D ). The cells were treated with 10 μM tyrophostin AG1478 for 4 h ( E ). ( A ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated with 150 µM DHA for 2, 4, and 6 h. “-” means without DHA treatment and “+” represents with DHA treatment. ( B ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated for 4 h with the concentrations of DHA indicated. Column “None” corresponds to the extract from untreated cells, column “50”, “100”, and “150” to the extracts from cells treated with 50, 100, and 150 µM DHA, respectively. ( C ) Western blot (WB) of PANC-1 cells treated with 150 µM DHA for 4 h. The total cell lysates were immunoprecipitated (IP) with EGFR antibody and then immunoblotted (by Western blotting, WB) using STAT3 antibody. ( D ) DNA-binding activity of STAT3 in PANC-1 cells treated for 4 h with the indicated concentrations of DHA. The description of the columns is the same as in ( B ). ( E ) Plot of the protein levels of p-STAT3 and STAT3 (and the protein standard actin) in the cells treated with 10 µM AG1478 for 4 h. The column labeled “None” corresponds to the extracts from untreated cells and the column labeled “AG1478” corresponds to the extracts from cells treated with 10 µM AG1478. EGFR = epidermal growth factor receptor (EGFR); STAT3 = signal transducer and activator of transcription factor 3.

    Journal: Nutrients

    Article Title: Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB

    doi: 10.3390/nu10111621

    Figure Lengend Snippet: Effect of DHA on the levels of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the interaction of EGFR and STAT3, STAT3 DNA-binding activity, and the effect of AG1478 on p-STAT3 and STAT3 levels in PANC-1 cells. Cells were treated with 150 µM DHA for various time periods ( A ) and with 50, 100, and 150 µM DHA for 4 h ( B – D ). The cells were treated with 10 μM tyrophostin AG1478 for 4 h ( E ). ( A ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated with 150 µM DHA for 2, 4, and 6 h. “-” means without DHA treatment and “+” represents with DHA treatment. ( B ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated for 4 h with the concentrations of DHA indicated. Column “None” corresponds to the extract from untreated cells, column “50”, “100”, and “150” to the extracts from cells treated with 50, 100, and 150 µM DHA, respectively. ( C ) Western blot (WB) of PANC-1 cells treated with 150 µM DHA for 4 h. The total cell lysates were immunoprecipitated (IP) with EGFR antibody and then immunoblotted (by Western blotting, WB) using STAT3 antibody. ( D ) DNA-binding activity of STAT3 in PANC-1 cells treated for 4 h with the indicated concentrations of DHA. The description of the columns is the same as in ( B ). ( E ) Plot of the protein levels of p-STAT3 and STAT3 (and the protein standard actin) in the cells treated with 10 µM AG1478 for 4 h. The column labeled “None” corresponds to the extracts from untreated cells and the column labeled “AG1478” corresponds to the extracts from cells treated with 10 µM AG1478. EGFR = epidermal growth factor receptor (EGFR); STAT3 = signal transducer and activator of transcription factor 3.

    Article Snippet: Experimental Protocol PANC-1 cells were incubated with DHA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in ethanol (final concentration of 50, 100 or 150 µM) for 4 h prior to the determination of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the assays of NF-κB DNA-binding activity and STAT3 DNA-binding activity, and the measurement of lipid raft EGFR levels.

    Techniques: Binding Assay, Activity Assay, Western Blot, Immunoprecipitation, Labeling

    Effect of MFX and CFX on apoptotic and survival pathway proteins. Western blot analysis of apoptotic and survival pathway protein in MIA PaCa-2 ( a ), and panc-1 cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: Effect of MFX and CFX on apoptotic and survival pathway proteins. Western blot analysis of apoptotic and survival pathway protein in MIA PaCa-2 ( a ), and panc-1 cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Western Blot

    MFX and CFX effects S-phase associated regulatory proteins. Western blot analysis of S-phase regulatory Cyclins and CDKs in MIA PaCa-2 ( a ), and Panc-1 cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: MFX and CFX effects S-phase associated regulatory proteins. Western blot analysis of S-phase regulatory Cyclins and CDKs in MIA PaCa-2 ( a ), and Panc-1 cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Western Blot

    Effects of MFX and CFX on MAPK signalling pathway proteins Western blot analysis of MAPK pathway protein in MIA PaCa-2 ( a ), and panc-1 cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: Effects of MFX and CFX on MAPK signalling pathway proteins Western blot analysis of MAPK pathway protein in MIA PaCa-2 ( a ), and panc-1 cells ( b ), treated with MFX and CFX in a dose dependent manner. GAPDH was used as loading control. The protein bands were quantified and normalized to GAPDH intensities. Data are representative of typical experiment repeated three times with similar results. Bar Graph represents the mean ± SEM of the fold change from three independent experiments. *p

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Western Blot

    Antiproliferative effects of MFX and CFX on cultured pancreatic cancer cells. Dose and time dependent response of MFX and CFX on MIA PaCa-2 (i) , and Panc-1 (ii) cells, as assessed by MTT assay. Cells were seeded in 96 well plates (1 × 10 4 cells/well) which were allowed to adhere overnight and were subsequently treated with increasing concentration of MFX and CFX for 24 h ( a ) and 48 h ( b ). Vertical axis represents % proliferation rate whereas Horizontal axis represents increasing concentration of MFX and CFX in μg/ml. Data are mean ± SEM three independent experiments performed in triplicate. *p

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: Antiproliferative effects of MFX and CFX on cultured pancreatic cancer cells. Dose and time dependent response of MFX and CFX on MIA PaCa-2 (i) , and Panc-1 (ii) cells, as assessed by MTT assay. Cells were seeded in 96 well plates (1 × 10 4 cells/well) which were allowed to adhere overnight and were subsequently treated with increasing concentration of MFX and CFX for 24 h ( a ) and 48 h ( b ). Vertical axis represents % proliferation rate whereas Horizontal axis represents increasing concentration of MFX and CFX in μg/ml. Data are mean ± SEM three independent experiments performed in triplicate. *p

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Cell Culture, MTT Assay, Concentration Assay

    Effects of MFX and CFX on biochemical events associated with apoptosis. a As described in material and method, caspase-8, 9, 3 activities were measured in MIA PaCa-2 (i) , and Panc-1 cells (ii) , in presence and absence of MFX/CFX for 48 h. The enzyme activity was measured by extent of cleavage of the caspase substrates Ac-IETD-pNA, Ac-LEHD-pNA and Ac-DEVD-pNA respectively. Bar graph represents the mean ± SEM of the fold increase in enzyme activity versus untreated control of three independent experiments performed in duplicates. Here vertical axis represents fold change in caspase activity. *p

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: Effects of MFX and CFX on biochemical events associated with apoptosis. a As described in material and method, caspase-8, 9, 3 activities were measured in MIA PaCa-2 (i) , and Panc-1 cells (ii) , in presence and absence of MFX/CFX for 48 h. The enzyme activity was measured by extent of cleavage of the caspase substrates Ac-IETD-pNA, Ac-LEHD-pNA and Ac-DEVD-pNA respectively. Bar graph represents the mean ± SEM of the fold increase in enzyme activity versus untreated control of three independent experiments performed in duplicates. Here vertical axis represents fold change in caspase activity. *p

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Activity Assay

    MFX and CFX perturb mitochondrial membrane potential. Mitochondrial membrane potential disruption was estimated using DiOC 6 . 20 min prior to harvesting, cells were incubated with 40 nM DiOC 6 and after incubation MIA PaCa-2 and Panc-1 cells were harvested, and the change in fluorescence was measured by flowcytometry. The X-axis represents green fluorescence, and the Y-axis represents the count scale. The illustrated histograms are representative of the three independent experiments with similar results. Results were also validated using mCCCp as a positive control in both the cell lines

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: MFX and CFX perturb mitochondrial membrane potential. Mitochondrial membrane potential disruption was estimated using DiOC 6 . 20 min prior to harvesting, cells were incubated with 40 nM DiOC 6 and after incubation MIA PaCa-2 and Panc-1 cells were harvested, and the change in fluorescence was measured by flowcytometry. The X-axis represents green fluorescence, and the Y-axis represents the count scale. The illustrated histograms are representative of the three independent experiments with similar results. Results were also validated using mCCCp as a positive control in both the cell lines

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Incubation, Fluorescence, Positive Control

    MFX and CFX induced apoptosis is caspase-8 dependent in both the cell lines. a MFX and CFX induced Caspase-8 activity in a time dependent manner in MIA Paca-2 (i) , and Panc-1 cells (ii) . Here vertical axis represents fold change in caspase activity and horizontal axis represents time in hours. *p

    Journal: BMC Cancer

    Article Title: Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    doi: 10.1186/s12885-015-1560-y

    Figure Lengend Snippet: MFX and CFX induced apoptosis is caspase-8 dependent in both the cell lines. a MFX and CFX induced Caspase-8 activity in a time dependent manner in MIA Paca-2 (i) , and Panc-1 cells (ii) . Here vertical axis represents fold change in caspase activity and horizontal axis represents time in hours. *p

    Article Snippet: Cell culture MIA PaCa-2 and Panc-1 cells were obtained from National Centre for Cell Science, Pune, India and maintained in DMEM medium containing 10 % (v/v) FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin-B in a humidified 5 % CO2 atmosphere.

    Techniques: Activity Assay

    Chmp1A knockdown abolished ATRA mediated growth inhibition and the increase of protein levels of Chmp1A, CRBP, P53 and phospho-P53 . (A) Chmp1A shRNA was cloned into shRNA RNAintro™ pSM2 retroviral vector to generate stable PanC-1 clones expressing Chmp1A shRNA. Western blot analysis demonstrated the knockdown efficiency of two colonies (KD1 and KD2). Non-silencing shRNA was used as control. (B) Chmp1A expression was reduced in Chmp1A silenced PanC-1 cells more than 50% on Day 1 and 40% on Day 2 regardless of ATRA treatment, compared with non-silencing shRNA expressing cells that were treated with vehicle DMSO. ATRA treatment increased Chmp1A expression slightly. (C) Silencing of Chmp1A induced growth promotion in the presence or absence of ATRA, compared with control cells that were treated with either DMSO or ATRA. ATRA did not have an effect on growth upon stable expression of Chmp1A shRNA compared to control shRNA. (D) Control shRNA expressing cells showed an increase in P53 and phospho-P53 at serine 15 and 37 without exhibiting an increase in CRBP-1. However, Chmp1A shRNA expressing cells exhibited a decline in CRBP-1 and phospho-P53 expression at serine 15 and 37 when compared with control shRNA expressing cells that were treated with DMSO. ATRA increased P53 expression when Chmp1A was silenced, however, Chmp1A depletion did not decrease P53 expression. D: DMSO, R: ATRA

    Journal: Molecular Cancer

    Article Title: Chmp 1A is a mediator of the anti-proliferative effects of All-trans Retinoic Acid in human pancreatic cancer cells

    doi: 10.1186/1476-4598-8-7

    Figure Lengend Snippet: Chmp1A knockdown abolished ATRA mediated growth inhibition and the increase of protein levels of Chmp1A, CRBP, P53 and phospho-P53 . (A) Chmp1A shRNA was cloned into shRNA RNAintro™ pSM2 retroviral vector to generate stable PanC-1 clones expressing Chmp1A shRNA. Western blot analysis demonstrated the knockdown efficiency of two colonies (KD1 and KD2). Non-silencing shRNA was used as control. (B) Chmp1A expression was reduced in Chmp1A silenced PanC-1 cells more than 50% on Day 1 and 40% on Day 2 regardless of ATRA treatment, compared with non-silencing shRNA expressing cells that were treated with vehicle DMSO. ATRA treatment increased Chmp1A expression slightly. (C) Silencing of Chmp1A induced growth promotion in the presence or absence of ATRA, compared with control cells that were treated with either DMSO or ATRA. ATRA did not have an effect on growth upon stable expression of Chmp1A shRNA compared to control shRNA. (D) Control shRNA expressing cells showed an increase in P53 and phospho-P53 at serine 15 and 37 without exhibiting an increase in CRBP-1. However, Chmp1A shRNA expressing cells exhibited a decline in CRBP-1 and phospho-P53 expression at serine 15 and 37 when compared with control shRNA expressing cells that were treated with DMSO. ATRA increased P53 expression when Chmp1A was silenced, however, Chmp1A depletion did not decrease P53 expression. D: DMSO, R: ATRA

    Article Snippet: Stable over-expression of Chmp1A in PanC-1 cells Tet-On advanced inducible gene expression system was used to generate conditional stable clones of PanC-1 cells (Clontech).

    Techniques: Inhibition, shRNA, Clone Assay, Plasmid Preparation, Expressing, Western Blot

    The growth inhibition of PanC-1 cells by ATRA was accompanied by an increase of Chmp1A, CRBP-1, P53 and phospho-P53 protein expression . (A) PanC-1 cells were counted and equal numbers of cells were seeded in 10 cm tissue culture dishes. Next day, cells were treated with DMSO or ATRA. The following 4 days, cells were counted and plotted for the graph. Dashed line with square represents DMSO treated and straight line with circle represents ATRA treated. P value indicates a significant growth inhibition by ATRA treatment. (B, C) The cells were processed for Western blot analysis after lysis. Notice the increase of Chmp1A and CRBP-1, P53 and phospho-P53 upon ATRA treatment.

    Journal: Molecular Cancer

    Article Title: Chmp 1A is a mediator of the anti-proliferative effects of All-trans Retinoic Acid in human pancreatic cancer cells

    doi: 10.1186/1476-4598-8-7

    Figure Lengend Snippet: The growth inhibition of PanC-1 cells by ATRA was accompanied by an increase of Chmp1A, CRBP-1, P53 and phospho-P53 protein expression . (A) PanC-1 cells were counted and equal numbers of cells were seeded in 10 cm tissue culture dishes. Next day, cells were treated with DMSO or ATRA. The following 4 days, cells were counted and plotted for the graph. Dashed line with square represents DMSO treated and straight line with circle represents ATRA treated. P value indicates a significant growth inhibition by ATRA treatment. (B, C) The cells were processed for Western blot analysis after lysis. Notice the increase of Chmp1A and CRBP-1, P53 and phospho-P53 upon ATRA treatment.

    Article Snippet: Stable over-expression of Chmp1A in PanC-1 cells Tet-On advanced inducible gene expression system was used to generate conditional stable clones of PanC-1 cells (Clontech).

    Techniques: Inhibition, Expressing, Western Blot, Lysis

    Chmp1A was translocated to the nucleus in ATRA responsive cells but to the membrane in ATRA resistant cells upon ATRA treatment (A and B), and Model (C) . (A) In the presence of DMSO, Chmp1A expression was modestly detected both in the nucleus and cytoplasm in PanC-1 cells (a, c). However, Chmp1A protein expression became robust in the presence of ATRA, especially in the nucleus (n in c, d). (a, b) and (c, d) is one and two days after vehicle or ATRA treatment, respectively. (B) Chmp1A was initially distributed ubiquitously in CRL-2151 cells in the presence of DMSO or ATRA (a, b). From Day 3 on, however, Chmp1A protein was mainly detected and remained at the membrane (arrows in c, d) in both DMSO and ATRA treated cells. (a, b) is for Day 2 and (c, d) is for Day 3 after DMSO and ATRA treatment, respectively. (C) Model: Chmp1A mediates growth inhibition of ATRA signaling. Chmp1A positively regulates the expression of CRBP-1. In turn, CRBP-1 controls the activity of ATRA via regulating the storage and metabolism of retinol A. ATRA treatment produces an increase in the expression level of Chmp1A in the nucleus, which leads to the accumulation of total and 'active' P53 resulting in a decrease in cell proliferation.

    Journal: Molecular Cancer

    Article Title: Chmp 1A is a mediator of the anti-proliferative effects of All-trans Retinoic Acid in human pancreatic cancer cells

    doi: 10.1186/1476-4598-8-7

    Figure Lengend Snippet: Chmp1A was translocated to the nucleus in ATRA responsive cells but to the membrane in ATRA resistant cells upon ATRA treatment (A and B), and Model (C) . (A) In the presence of DMSO, Chmp1A expression was modestly detected both in the nucleus and cytoplasm in PanC-1 cells (a, c). However, Chmp1A protein expression became robust in the presence of ATRA, especially in the nucleus (n in c, d). (a, b) and (c, d) is one and two days after vehicle or ATRA treatment, respectively. (B) Chmp1A was initially distributed ubiquitously in CRL-2151 cells in the presence of DMSO or ATRA (a, b). From Day 3 on, however, Chmp1A protein was mainly detected and remained at the membrane (arrows in c, d) in both DMSO and ATRA treated cells. (a, b) is for Day 2 and (c, d) is for Day 3 after DMSO and ATRA treatment, respectively. (C) Model: Chmp1A mediates growth inhibition of ATRA signaling. Chmp1A positively regulates the expression of CRBP-1. In turn, CRBP-1 controls the activity of ATRA via regulating the storage and metabolism of retinol A. ATRA treatment produces an increase in the expression level of Chmp1A in the nucleus, which leads to the accumulation of total and 'active' P53 resulting in a decrease in cell proliferation.

    Article Snippet: Stable over-expression of Chmp1A in PanC-1 cells Tet-On advanced inducible gene expression system was used to generate conditional stable clones of PanC-1 cells (Clontech).

    Techniques: Expressing, Inhibition, Activity Assay

    Chmp1A over-expression positively regulates CRBP-1 . (A) HEK 293T cells were transfected with either empty CS2+ or Chmp1A-CS2+ plasmid. 18 hours after transfection cells were processed for total RNA isolation. Reverse transcriptase PCR (RT-PCR) indicates that Chmp1A and CRBP-1 transcripts were increased 1.5 and 3.3 folds respectively at 31 cycles compared to control. Gapdh was used as RT-PCR control. (B) Chmp1A expression was induced by the addition of doxycycline to the media in two independent stable clones of PanC-1 cells. Cells were lysed two days after the doxycycline supplement and processed for Western blot analysis. Chmp1A and CRBP-1 protein was increased upon Chmp1A over-expression. Con: Control

    Journal: Molecular Cancer

    Article Title: Chmp 1A is a mediator of the anti-proliferative effects of All-trans Retinoic Acid in human pancreatic cancer cells

    doi: 10.1186/1476-4598-8-7

    Figure Lengend Snippet: Chmp1A over-expression positively regulates CRBP-1 . (A) HEK 293T cells were transfected with either empty CS2+ or Chmp1A-CS2+ plasmid. 18 hours after transfection cells were processed for total RNA isolation. Reverse transcriptase PCR (RT-PCR) indicates that Chmp1A and CRBP-1 transcripts were increased 1.5 and 3.3 folds respectively at 31 cycles compared to control. Gapdh was used as RT-PCR control. (B) Chmp1A expression was induced by the addition of doxycycline to the media in two independent stable clones of PanC-1 cells. Cells were lysed two days after the doxycycline supplement and processed for Western blot analysis. Chmp1A and CRBP-1 protein was increased upon Chmp1A over-expression. Con: Control

    Article Snippet: Stable over-expression of Chmp1A in PanC-1 cells Tet-On advanced inducible gene expression system was used to generate conditional stable clones of PanC-1 cells (Clontech).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, Western Blot

    Selinexor treatment or miR-145 mimic transfection suppressed the migration activity of PDAC cells Selinexor treated or miR-145 mimic transfected MiaPaCa-2 (A) , AsPC-1 (B) and PANC-1 (C) PDAC cells were seeded in 6 well plate and subjected to wound healing assay for 3 days as described in the section of “Materials and Methods”. The cells were photographed in each day.

    Journal: Oncotarget

    Article Title: Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration

    doi: 10.18632/oncotarget.19285

    Figure Lengend Snippet: Selinexor treatment or miR-145 mimic transfection suppressed the migration activity of PDAC cells Selinexor treated or miR-145 mimic transfected MiaPaCa-2 (A) , AsPC-1 (B) and PANC-1 (C) PDAC cells were seeded in 6 well plate and subjected to wound healing assay for 3 days as described in the section of “Materials and Methods”. The cells were photographed in each day.

    Article Snippet: miRNA array and data analysis Five micrograms of each total RNA sample from HPDE, Colo357, and PANC-1 cells were sent to a service provider for completion of the miRNA arrays (LCSciences, Houston, TX).

    Techniques: Transfection, Migration, Activity Assay, Wound Healing Assay

    Treatment of PDAC cells with selinexor increased the expression of miR-145 MiaPaCa-2 (A) , AsPC-1 (B) , L3.6pl (C) , PANC-1 (D) and HPAC cells (E) were treated with 500 nM selinexor for 48 hours. MiaPaCa-2 cells were also treated with gemcitabine or paclitaxel for 48 hours. (F) (Gem: gemcitabine; Pac: paclitaxel). The total RNAs from each sample were extracted and subjected to real-time RT-PCR for detection of miR-145 expression (*: p

    Journal: Oncotarget

    Article Title: Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration

    doi: 10.18632/oncotarget.19285

    Figure Lengend Snippet: Treatment of PDAC cells with selinexor increased the expression of miR-145 MiaPaCa-2 (A) , AsPC-1 (B) , L3.6pl (C) , PANC-1 (D) and HPAC cells (E) were treated with 500 nM selinexor for 48 hours. MiaPaCa-2 cells were also treated with gemcitabine or paclitaxel for 48 hours. (F) (Gem: gemcitabine; Pac: paclitaxel). The total RNAs from each sample were extracted and subjected to real-time RT-PCR for detection of miR-145 expression (*: p

    Article Snippet: miRNA array and data analysis Five micrograms of each total RNA sample from HPDE, Colo357, and PANC-1 cells were sent to a service provider for completion of the miRNA arrays (LCSciences, Houston, TX).

    Techniques: Expressing, Quantitative RT-PCR

    Selinexor treatment or miR-145 mimic transfection inhibited the proliferation of PDAC cells MiaPaCa-2 (A) , AsPC-1 (B) , PANC-1 (C) and HPAC (D) cells were treated with 500 nM selinexor or transfected with miR-145 mimic or control mimic. The cell proliferation index was measured by MTT assay as described in the section of “Materials and Methods”.

    Journal: Oncotarget

    Article Title: Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration

    doi: 10.18632/oncotarget.19285

    Figure Lengend Snippet: Selinexor treatment or miR-145 mimic transfection inhibited the proliferation of PDAC cells MiaPaCa-2 (A) , AsPC-1 (B) , PANC-1 (C) and HPAC (D) cells were treated with 500 nM selinexor or transfected with miR-145 mimic or control mimic. The cell proliferation index was measured by MTT assay as described in the section of “Materials and Methods”.

    Article Snippet: miRNA array and data analysis Five micrograms of each total RNA sample from HPDE, Colo357, and PANC-1 cells were sent to a service provider for completion of the miRNA arrays (LCSciences, Houston, TX).

    Techniques: Transfection, MTT Assay

    Selinexor treatment or miR-145 mimic transfection inhibited the expression of miR-145 target or downstream genes at protein or RNA level (A-D) MiaPaCa-2, AsPC-1, PANC-1, Colo357 and HPAC cells were treated with 500nM selinexor or transfected with miR-145 mimic or control mimic for 72 hours. Total protein was extracted from each sample and subjected to Western Blot analysis for detection of EGFR, MMP1, MT-MMP, c-Myc, Pak4 and p21 WAF1 expression at protein level (A, C and D). Total RNA was extracted and subjected to real-time PCR for detection of Sox-2 and Pak4 (B).

    Journal: Oncotarget

    Article Title: Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration

    doi: 10.18632/oncotarget.19285

    Figure Lengend Snippet: Selinexor treatment or miR-145 mimic transfection inhibited the expression of miR-145 target or downstream genes at protein or RNA level (A-D) MiaPaCa-2, AsPC-1, PANC-1, Colo357 and HPAC cells were treated with 500nM selinexor or transfected with miR-145 mimic or control mimic for 72 hours. Total protein was extracted from each sample and subjected to Western Blot analysis for detection of EGFR, MMP1, MT-MMP, c-Myc, Pak4 and p21 WAF1 expression at protein level (A, C and D). Total RNA was extracted and subjected to real-time PCR for detection of Sox-2 and Pak4 (B).

    Article Snippet: miRNA array and data analysis Five micrograms of each total RNA sample from HPDE, Colo357, and PANC-1 cells were sent to a service provider for completion of the miRNA arrays (LCSciences, Houston, TX).

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

    miR-145 was significantly down-regulated in PDAC cells (A) and transfection of XPO1 siRNA into PDAC cells induced the expression of miR-145. MiaPaCa-2 (B) , AsPC-1 (C) , L3.6pl (D) , PANC-1 (E) and HPAC (F) PDAC cells were transfected with XPO1 siRNA or control siRNA for 48 hours. The total RNAs from each sample were extracted and subjected to real-time RT-PCR for detection of miR-145 expression (*: p

    Journal: Oncotarget

    Article Title: Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration

    doi: 10.18632/oncotarget.19285

    Figure Lengend Snippet: miR-145 was significantly down-regulated in PDAC cells (A) and transfection of XPO1 siRNA into PDAC cells induced the expression of miR-145. MiaPaCa-2 (B) , AsPC-1 (C) , L3.6pl (D) , PANC-1 (E) and HPAC (F) PDAC cells were transfected with XPO1 siRNA or control siRNA for 48 hours. The total RNAs from each sample were extracted and subjected to real-time RT-PCR for detection of miR-145 expression (*: p

    Article Snippet: miRNA array and data analysis Five micrograms of each total RNA sample from HPDE, Colo357, and PANC-1 cells were sent to a service provider for completion of the miRNA arrays (LCSciences, Houston, TX).

    Techniques: Transfection, Expressing, Quantitative RT-PCR

    Panc-1 cells treated with Ni NWs which potentiates apoptosis. Cells were treated with 200 μL Ni NWs for 8, 16, and 24 hours and stained with ethidium bromide (EB) and acridine orange (AO) and were visualized and photographed immediately with fluorescence microscope. ( A ) Control; ( B ) 8 hours after treatment – blue arrow indicates initial membrane blebbing of the apoptosed cells; ( C ) 16 hours after treatment – the white arrow indicates late membrane blebbing; ( D ) 24 hours after treatment. Cells were stained with AO and EB where viable cells were green nuclear fluorescence and apoptotic cells exhibit a red nucleus.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Panc-1 cells treated with Ni NWs which potentiates apoptosis. Cells were treated with 200 μL Ni NWs for 8, 16, and 24 hours and stained with ethidium bromide (EB) and acridine orange (AO) and were visualized and photographed immediately with fluorescence microscope. ( A ) Control; ( B ) 8 hours after treatment – blue arrow indicates initial membrane blebbing of the apoptosed cells; ( C ) 16 hours after treatment – the white arrow indicates late membrane blebbing; ( D ) 24 hours after treatment. Cells were stained with AO and EB where viable cells were green nuclear fluorescence and apoptotic cells exhibit a red nucleus.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: Staining, Fluorescence, Microscopy

    Quantification of reactive oxygen species (ROS) generation on Ni NW-induced Panc-1 cells stained with DCF-DA dye and measured by flow cytometry. This bar graph represents the concentration dependent ROS generation where the x-axis represents the concentration of Ni NWs and the y-axis denotes the percentage of ROS (DCF)-positive Panc-1 cells.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Quantification of reactive oxygen species (ROS) generation on Ni NW-induced Panc-1 cells stained with DCF-DA dye and measured by flow cytometry. This bar graph represents the concentration dependent ROS generation where the x-axis represents the concentration of Ni NWs and the y-axis denotes the percentage of ROS (DCF)-positive Panc-1 cells.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: Staining, Flow Cytometry, Cytometry, Concentration Assay

    Qualitative morphological study of apoptosis by MTT assay. Phase contrast pictures of formazon in Ni NWs treated Panc-1 cells where ( A ) control, ie, untreated cells and ( B ) treated cells. Viable cells exhibited a deep purple cell nucleus with rod shaped formazon whereas apoptotic cells lacked formazon growth because of the lack of mitochondrial reductase and thus did not change in color. Dead cells are indicated by black arrows and live cells are identified by white arrows.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Qualitative morphological study of apoptosis by MTT assay. Phase contrast pictures of formazon in Ni NWs treated Panc-1 cells where ( A ) control, ie, untreated cells and ( B ) treated cells. Viable cells exhibited a deep purple cell nucleus with rod shaped formazon whereas apoptotic cells lacked formazon growth because of the lack of mitochondrial reductase and thus did not change in color. Dead cells are indicated by black arrows and live cells are identified by white arrows.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: MTT Assay

    Effects of concentration of Ni NWs on apoptosis of Panc-1 cells after 24-hour exposure.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Effects of concentration of Ni NWs on apoptosis of Panc-1 cells after 24-hour exposure.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: Concentration Assay

    Quantitative 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of cell viability and proliferation. Cell viability assay shows the effect of Ni NWs as concentration dependent reduction of viability in Panc-1 cells.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Quantitative 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of cell viability and proliferation. Cell viability assay shows the effect of Ni NWs as concentration dependent reduction of viability in Panc-1 cells.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: MTT Assay, Viability Assay, Concentration Assay

    Morphological characteristics of Panc-1 cells which were treated with 200 μl of Ni NWs suspension for 0 (control), 8, 16, and 24 hours and visualized with phase contrast microscope (magnification 40×). Increasing detachment of cells from the well surface is seen with increasing exposure time. ( A ) Adherent cells without Ni NWs (control); ( B ) adherent (black arrows) and suspended (red arrows) cells with internalized Ni NWs after 8 hours; ( C ) adherent and a greater number of suspended cells with internalized Ni NWs after 16 hours; ( D ) Adherent and suspended cells with internalized Ni NWs after 24 hours. ( E – G ) represent more specific cellular morphological changes. ( E ) Uptake of Ni NWs by endocytosis to the cell cytoplasm; ( F ) internalization of Ni NWs to the cell nucleus followed by cell shrinkage, round shaped, and initiation of membrane blebbing; ( G ) apoptotic body formation through final membrane blebbing.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Morphological characteristics of Panc-1 cells which were treated with 200 μl of Ni NWs suspension for 0 (control), 8, 16, and 24 hours and visualized with phase contrast microscope (magnification 40×). Increasing detachment of cells from the well surface is seen with increasing exposure time. ( A ) Adherent cells without Ni NWs (control); ( B ) adherent (black arrows) and suspended (red arrows) cells with internalized Ni NWs after 8 hours; ( C ) adherent and a greater number of suspended cells with internalized Ni NWs after 16 hours; ( D ) Adherent and suspended cells with internalized Ni NWs after 24 hours. ( E – G ) represent more specific cellular morphological changes. ( E ) Uptake of Ni NWs by endocytosis to the cell cytoplasm; ( F ) internalization of Ni NWs to the cell nucleus followed by cell shrinkage, round shaped, and initiation of membrane blebbing; ( G ) apoptotic body formation through final membrane blebbing.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: Microscopy

    Cell cycle analysis after induction of apoptosis in Panc-1 cells by Ni NWs. The cells were incubated with Ni NWs for 24 hours, stained with PI and analyzed by Cell Quest Pro ® on FC (BD-FACS Calibur ® ). A portrays untreated cells with all phases of the cell cycle, whereas B shows an exclusively increased sub-G 1 peak with Ni NWs treatment.

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Cell cycle analysis after induction of apoptosis in Panc-1 cells by Ni NWs. The cells were incubated with Ni NWs for 24 hours, stained with PI and analyzed by Cell Quest Pro ® on FC (BD-FACS Calibur ® ). A portrays untreated cells with all phases of the cell cycle, whereas B shows an exclusively increased sub-G 1 peak with Ni NWs treatment.

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: Cell Cycle Assay, Incubation, Staining, FACS

    Ni NWs-induced reactive oxygen species generation in Panc-1 cells were measured by flow cytometry. The fluorescence intensities of 10,000 cells were analyzed by BD-FACS Calibur ® . Representative histograms show cell number on the y-axis and increases of fluorescence (oxidant production) as right shifts on the x-axis (mean fluorescence intensity). ( A ) Cells without treatment; ( B ) cells treated with 0.3% H 2 O 2 ; ( C ) Ni NWs treated cells; ( D ) overlay histogram of A – C .

    Journal: International Journal of Nanomedicine

    Article Title: Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    doi: 10.2147/IJN.S21697

    Figure Lengend Snippet: Ni NWs-induced reactive oxygen species generation in Panc-1 cells were measured by flow cytometry. The fluorescence intensities of 10,000 cells were analyzed by BD-FACS Calibur ® . Representative histograms show cell number on the y-axis and increases of fluorescence (oxidant production) as right shifts on the x-axis (mean fluorescence intensity). ( A ) Cells without treatment; ( B ) cells treated with 0.3% H 2 O 2 ; ( C ) Ni NWs treated cells; ( D ) overlay histogram of A – C .

    Article Snippet: Detection and quantification of intracellular ROS production To determine the intracellular ROS generation, the Panc-1 cells were labeled with the fluorescent DCF-DA probe and measured with a BD FACSCalibur® flow cytometer.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, FACS

    Ectopic expression of RhoB, not RhoA, inhibits Ras/PI3K/Akt-mediated transformation and migration. (a) Parental NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-PI3K, or CA-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The cells were split, and an aliquot of the cells was evaluated for expression of transfected RhoA and RhoB by Western blotting. The rest of the cells were then cultured for another 4 weeks. The plates were examined for focus formation by staining. H-Ras/NIH 3T3 (b) and PANC-1 (c) cells were transiently transfected with pcDNA3 vector control, CA-Akt, or WT-Akt in the presence or absence of RhoA or RhoB or pcDNA3 for 36 h, internal ribosome entry site-EGFP was cotransfected as a transfection indicator. The cells were then split and analyzed for their migration capabilities through collagen type I-coated transfilters as described in Materials and Methods. (d and e) NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-Akt, or WT-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The monolayers were then scratched with a yellow tip and microphotographed at the time points indicated to analyze their capability to migrate into and fill the wounded area.

    Journal: Molecular and Cellular Biology

    Article Title: Akt Mediates Ras Downregulation of RhoB, a Suppressor of Transformation, Invasion, and Metastasis

    doi: 10.1128/MCB.24.12.5565-5576.2004

    Figure Lengend Snippet: Ectopic expression of RhoB, not RhoA, inhibits Ras/PI3K/Akt-mediated transformation and migration. (a) Parental NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-PI3K, or CA-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The cells were split, and an aliquot of the cells was evaluated for expression of transfected RhoA and RhoB by Western blotting. The rest of the cells were then cultured for another 4 weeks. The plates were examined for focus formation by staining. H-Ras/NIH 3T3 (b) and PANC-1 (c) cells were transiently transfected with pcDNA3 vector control, CA-Akt, or WT-Akt in the presence or absence of RhoA or RhoB or pcDNA3 for 36 h, internal ribosome entry site-EGFP was cotransfected as a transfection indicator. The cells were then split and analyzed for their migration capabilities through collagen type I-coated transfilters as described in Materials and Methods. (d and e) NIH 3T3 cells were transiently transfected with pcDNA3 vector control, H-Ras61L, CA-Akt, or WT-Akt in the presence or absence of RhoA, RhoB, or pcDNA3 for 36 h. The monolayers were then scratched with a yellow tip and microphotographed at the time points indicated to analyze their capability to migrate into and fill the wounded area.

    Article Snippet: In contrast, -treated H-Ras/NIH 3T3 and PANC-1 cells did not invade.

    Techniques: Expressing, Transformation Assay, Migration, Transfection, Plasmid Preparation, Western Blot, Cell Culture, Staining

    Tracking individual cells in heterogeneous populations. ( a ) Comparison of ROC curves based on data from Harmony AUC = 0.93, CI 95% = [0.87,0.98] (blue), CellProfiler, AUC = 0.92, CI 95% = [0.84, 0.99] (yellow) and Imaris, AUC = 0.89 95%CI = [0.81–0.97] (green). ( b ) Cell not rejected due to TrAM outlined in green (τ = 2.79). Below outlined in red, cell excluded from evaluation due to TrAM value > 4.81 threshold (τ = 36.33). Nuclear area and roundness are plotted over time. Smoothed curves represent typical fluctuation between adjacent time points. Cell segmentation images correspond to highlighted data points. ( c ) Adjustment of PC3 cell motility to high density imaging areas (R2 = 0.66). Plot correlates number of cells per imaging field with current speed. Each dot represents an imaging well of a 96-well plate. ( d ) Speed distribution of PC3, HeLa and Panc-1 cells before (blue) and after (yellow) TrAM filtering. PC3 data points indicate average speed of examples of high and low τ shown in b . ( e ) Speed distribution of HeLa (n = 26 fields, SEM = 0.005) vs. Panc-1 (6 fields, SEM = 0.006) cells post-filtering, p

    Journal: Scientific Reports

    Article Title: Single cell dynamic phenotyping

    doi: 10.1038/srep34785

    Figure Lengend Snippet: Tracking individual cells in heterogeneous populations. ( a ) Comparison of ROC curves based on data from Harmony AUC = 0.93, CI 95% = [0.87,0.98] (blue), CellProfiler, AUC = 0.92, CI 95% = [0.84, 0.99] (yellow) and Imaris, AUC = 0.89 95%CI = [0.81–0.97] (green). ( b ) Cell not rejected due to TrAM outlined in green (τ = 2.79). Below outlined in red, cell excluded from evaluation due to TrAM value > 4.81 threshold (τ = 36.33). Nuclear area and roundness are plotted over time. Smoothed curves represent typical fluctuation between adjacent time points. Cell segmentation images correspond to highlighted data points. ( c ) Adjustment of PC3 cell motility to high density imaging areas (R2 = 0.66). Plot correlates number of cells per imaging field with current speed. Each dot represents an imaging well of a 96-well plate. ( d ) Speed distribution of PC3, HeLa and Panc-1 cells before (blue) and after (yellow) TrAM filtering. PC3 data points indicate average speed of examples of high and low τ shown in b . ( e ) Speed distribution of HeLa (n = 26 fields, SEM = 0.005) vs. Panc-1 (6 fields, SEM = 0.006) cells post-filtering, p

    Article Snippet: PC3 and HeLa cells were maintained in RPMI1640 (Corning, cat. no. 10-040), Panc-1 cells in DMEM (Corning, cat. no. 10-013), all supplemented with 10% heat-inactivated GemCell bovine serum (Gemini Bio-Products, cat. no. 100-500), Penicillin-Streptomycin (Gemini Bio-Products, cat. no. 400-109).

    Techniques: Imaging

    α -Mangostin modulates the expression of EMT-related genes in pancreatic cancer cells cells. (a) Panc-1 and BxPC-3 cells were treated with α -mangostin 16 μ M at the indicated concentrations for 24 h. Protein (a) and mRNA (b) levels of MMP-2, MMP-9, E-cadherin, vimentin, and N-cadherin were measured by Western blotting and qRT-PCR, respectively. * P

    Journal: BioMed Research International

    Article Title: α-Mangostin Suppresses the Viability and Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells by Downregulating the PI3K/Akt Pathway

    doi: 10.1155/2014/546353

    Figure Lengend Snippet: α -Mangostin modulates the expression of EMT-related genes in pancreatic cancer cells cells. (a) Panc-1 and BxPC-3 cells were treated with α -mangostin 16 μ M at the indicated concentrations for 24 h. Protein (a) and mRNA (b) levels of MMP-2, MMP-9, E-cadherin, vimentin, and N-cadherin were measured by Western blotting and qRT-PCR, respectively. * P

    Article Snippet: Cell Culture BxPc-3 and Panc-1 cells were purchased from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS); hTERT-HPNE cell was purchased from ATCC (Manassas, VA) and cultured at 37°C and 5% CO2 in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM/High Glucose) (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, USA), 100 μ g/mL ampicillin, and 100 μ g/mL streptomycin.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    α -Mangostin induces apoptosis of pancreatic cancer cells. (a) Panc-1 and BxPC-3 cells were treated with α -mangostin (0 μ M, 8 μ M, or 16 μ M) for 24 h. Apoptotic cells were evaluated by Annexin V-FITC/PI staining and flow cytometry. Representative FACS plots are shown. (b) Apoptosis rate of the early and late apoptosis was quantified by flow cytometry. * P

    Journal: BioMed Research International

    Article Title: α-Mangostin Suppresses the Viability and Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells by Downregulating the PI3K/Akt Pathway

    doi: 10.1155/2014/546353

    Figure Lengend Snippet: α -Mangostin induces apoptosis of pancreatic cancer cells. (a) Panc-1 and BxPC-3 cells were treated with α -mangostin (0 μ M, 8 μ M, or 16 μ M) for 24 h. Apoptotic cells were evaluated by Annexin V-FITC/PI staining and flow cytometry. Representative FACS plots are shown. (b) Apoptosis rate of the early and late apoptosis was quantified by flow cytometry. * P

    Article Snippet: Cell Culture BxPc-3 and Panc-1 cells were purchased from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS); hTERT-HPNE cell was purchased from ATCC (Manassas, VA) and cultured at 37°C and 5% CO2 in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM/High Glucose) (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, USA), 100 μ g/mL ampicillin, and 100 μ g/mL streptomycin.

    Techniques: Staining, Flow Cytometry, Cytometry, FACS

    Treatment of pancreatic cancer cells with α -mangostin results in loss of cell viability. Pancreatic cancer BxPc-3 (a), Panc-1 (b), and hTERT-HPNE (c) cells were treated with α -mangostin at the indicated concentrations for 6, 12, 24, and 48 h. Cell viability relative to control was assessed using the MTT assay.

    Journal: BioMed Research International

    Article Title: α-Mangostin Suppresses the Viability and Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells by Downregulating the PI3K/Akt Pathway

    doi: 10.1155/2014/546353

    Figure Lengend Snippet: Treatment of pancreatic cancer cells with α -mangostin results in loss of cell viability. Pancreatic cancer BxPc-3 (a), Panc-1 (b), and hTERT-HPNE (c) cells were treated with α -mangostin at the indicated concentrations for 6, 12, 24, and 48 h. Cell viability relative to control was assessed using the MTT assay.

    Article Snippet: Cell Culture BxPc-3 and Panc-1 cells were purchased from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS); hTERT-HPNE cell was purchased from ATCC (Manassas, VA) and cultured at 37°C and 5% CO2 in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM/High Glucose) (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, USA), 100 μ g/mL ampicillin, and 100 μ g/mL streptomycin.

    Techniques: MTT Assay

    α -Mangostin suppresses activation of the PI3K/Akt pathway in pancreatic cancer cells. Protein levels of total Akt and pAkt-S473 were detected by Western blotting. (a) Panc-1 and BxPC-3 cells were treated with α -mangostin (16 μ M) for 0 h, 3 h, 6 h, 12 h, and 24 h. (b) Panc-1 and BxPC-3 cells were treated with α -mangostin at the indicated concentrations for 24 h. (c) Panc-1 and BxPC-3 cells were treated with 5 ng/mL TGF- β either alone or in combination with 16 μ M α -mangostin for 24 h. Protein levels of total Akt, pAkt-S473, E-cadherin, vimentin, and N-cadherin were measured by Western blotting with β -actin as loading control.

    Journal: BioMed Research International

    Article Title: α-Mangostin Suppresses the Viability and Epithelial-Mesenchymal Transition of Pancreatic Cancer Cells by Downregulating the PI3K/Akt Pathway

    doi: 10.1155/2014/546353

    Figure Lengend Snippet: α -Mangostin suppresses activation of the PI3K/Akt pathway in pancreatic cancer cells. Protein levels of total Akt and pAkt-S473 were detected by Western blotting. (a) Panc-1 and BxPC-3 cells were treated with α -mangostin (16 μ M) for 0 h, 3 h, 6 h, 12 h, and 24 h. (b) Panc-1 and BxPC-3 cells were treated with α -mangostin at the indicated concentrations for 24 h. (c) Panc-1 and BxPC-3 cells were treated with 5 ng/mL TGF- β either alone or in combination with 16 μ M α -mangostin for 24 h. Protein levels of total Akt, pAkt-S473, E-cadherin, vimentin, and N-cadherin were measured by Western blotting with β -actin as loading control.

    Article Snippet: Cell Culture BxPc-3 and Panc-1 cells were purchased from Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS); hTERT-HPNE cell was purchased from ATCC (Manassas, VA) and cultured at 37°C and 5% CO2 in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM/High Glucose) (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, USA), 100 μ g/mL ampicillin, and 100 μ g/mL streptomycin.

    Techniques: Activation Assay, Western Blot

    Activation of RhoG GTPase in hTERT-HPNE, BxPC3 and PANC-1 pancreatic cell lines. Cells were starved 6 hours and confronted with rhEGF for the period of 0, 2 and 10 minutes (Marked as 0, 2 and 10). Fluorescence microscopic staining of RhoG (green) was carried out in hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoG activity was performed using RhoG pulldown assay. Immunoblots for RhoG, Rac-1 and GAPDH proteins for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) are shown. To quantify the amount of RhoG-GTP and bound-Rac-1 through the temporal course, densitometric analysis was performed using Image Lab software, hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I). For comparison of RhoG activity, the total amount of RhoG in cell lysates was normalized to total RhoG. GAPDH was used as a protein loading control. ELMO1-GST beads coomassie are shown as beads loading control. Arrowheads denote the localization of RhoG into the peripheral membrane; boxes with number represent the number of cells with this phenotype. Scale bar 100 μm.

    Journal: PLoS ONE

    Article Title: Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

    doi: 10.1371/journal.pone.0166370

    Figure Lengend Snippet: Activation of RhoG GTPase in hTERT-HPNE, BxPC3 and PANC-1 pancreatic cell lines. Cells were starved 6 hours and confronted with rhEGF for the period of 0, 2 and 10 minutes (Marked as 0, 2 and 10). Fluorescence microscopic staining of RhoG (green) was carried out in hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoG activity was performed using RhoG pulldown assay. Immunoblots for RhoG, Rac-1 and GAPDH proteins for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) are shown. To quantify the amount of RhoG-GTP and bound-Rac-1 through the temporal course, densitometric analysis was performed using Image Lab software, hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I). For comparison of RhoG activity, the total amount of RhoG in cell lysates was normalized to total RhoG. GAPDH was used as a protein loading control. ELMO1-GST beads coomassie are shown as beads loading control. Arrowheads denote the localization of RhoG into the peripheral membrane; boxes with number represent the number of cells with this phenotype. Scale bar 100 μm.

    Article Snippet: Immunofluorescence (IF) and Confocal Microscopy Imaging of Pancreatic Cell Lines hTERT-HPNE, BxPC3 and PANC-1 cells were grown on coverslips (Tedpela 26020, Redding, CA) coated with poly-D-Lysine, washed twice with PBS, and then fixed with 4% PFA for 20 min. After a second wash, the cells were permeabilized with 0.1% Triton X-100 for 3 minutes at 37°C.

    Techniques: Activation Assay, Fluorescence, Staining, Activity Assay, Western Blot, Software

    The expression of RhoG and RhoB proteins is altered in cancerous pancreatic cell lines. Immunofluorescence microscopy analysis of RhoGDI3 protein (green); RhoG (A) and RhoB (B) (Red) and Nuclei (DAPI, blue) of hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. Representative Immunoblot using antibodies anti-RhoG (C), anti RhoB (D), GAPDH was used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. Total amount of RhoG (E) and RhoB (F) proteins was normalized to GAPDH (n = 3). Immunoblot densitometric analysis was performed with Image Lab software. Values are means ± SEM, **P

    Journal: PLoS ONE

    Article Title: Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

    doi: 10.1371/journal.pone.0166370

    Figure Lengend Snippet: The expression of RhoG and RhoB proteins is altered in cancerous pancreatic cell lines. Immunofluorescence microscopy analysis of RhoGDI3 protein (green); RhoG (A) and RhoB (B) (Red) and Nuclei (DAPI, blue) of hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. Representative Immunoblot using antibodies anti-RhoG (C), anti RhoB (D), GAPDH was used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. Total amount of RhoG (E) and RhoB (F) proteins was normalized to GAPDH (n = 3). Immunoblot densitometric analysis was performed with Image Lab software. Values are means ± SEM, **P

    Article Snippet: Immunofluorescence (IF) and Confocal Microscopy Imaging of Pancreatic Cell Lines hTERT-HPNE, BxPC3 and PANC-1 cells were grown on coverslips (Tedpela 26020, Redding, CA) coated with poly-D-Lysine, washed twice with PBS, and then fixed with 4% PFA for 20 min. After a second wash, the cells were permeabilized with 0.1% Triton X-100 for 3 minutes at 37°C.

    Techniques: Expressing, Immunofluorescence, Microscopy, Software

    The dynamic role of RhoGDI3 in the pancreatic cell lines. Cells were starved for 6 hours and activated with rhEGF for the period of 0, 2 and 10 minutes (marked above the images as 0, 2 and 10 min). An immunofluorescence microscopy analysis of RhoGDI3 (green), RhoG (A) and RhoB (B) (red, not shown) was carried out on hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines, at the time point of 10 min it is shown the detail of RhoGDI3 staining to highlight the signal at the lamellipodial protrusions evident only in the cell lines hTERT-HPNE and PANC-1 (white arrowheads). The colocalization index was determined using the confocal Olympus software FluoView 300 for RhoGDI3 with RhoG and RhoB. Overlap index is shown in Fig 5C and 5D, respectively, index 1 corresponds to maximum overlap and cero corresponds to negative overlap. Scale bar 10 μm.

    Journal: PLoS ONE

    Article Title: Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

    doi: 10.1371/journal.pone.0166370

    Figure Lengend Snippet: The dynamic role of RhoGDI3 in the pancreatic cell lines. Cells were starved for 6 hours and activated with rhEGF for the period of 0, 2 and 10 minutes (marked above the images as 0, 2 and 10 min). An immunofluorescence microscopy analysis of RhoGDI3 (green), RhoG (A) and RhoB (B) (red, not shown) was carried out on hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines, at the time point of 10 min it is shown the detail of RhoGDI3 staining to highlight the signal at the lamellipodial protrusions evident only in the cell lines hTERT-HPNE and PANC-1 (white arrowheads). The colocalization index was determined using the confocal Olympus software FluoView 300 for RhoGDI3 with RhoG and RhoB. Overlap index is shown in Fig 5C and 5D, respectively, index 1 corresponds to maximum overlap and cero corresponds to negative overlap. Scale bar 10 μm.

    Article Snippet: Immunofluorescence (IF) and Confocal Microscopy Imaging of Pancreatic Cell Lines hTERT-HPNE, BxPC3 and PANC-1 cells were grown on coverslips (Tedpela 26020, Redding, CA) coated with poly-D-Lysine, washed twice with PBS, and then fixed with 4% PFA for 20 min. After a second wash, the cells were permeabilized with 0.1% Triton X-100 for 3 minutes at 37°C.

    Techniques: Immunofluorescence, Microscopy, Staining, Software

    GTPase RhoB shows differential activation in PDAC cell lines. Cells were starved for 6 hours and treated with rhEGF for a period of 0, 2 and 10 minutes (Marked as 0, 2 and 10 min). An immunofluorescence microscopy analysis of RhoB (green) was carried out on hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoB activity was performed using RhoB pulldown assay. Immunoblots for RhoB and GAPDH, as loading control for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) cell lines are shown. To quantify the amount of RhoB-GTP, densitometric analysis (n = 3) was performed using Image Lab software for samples of hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I) cell lines. For comparison of RhoB activity, GTP-RhoB was normalized to total RhoB. GAPDH was used as a protein loading control. Coomassie of RBD-GST beads are shown as beads loading control. Arrowheads denote the localization of RhoB into the peripheral membrane; boxes with number represent the quantity of cells per field with this phenotype. Scale bar = 100 μm.

    Journal: PLoS ONE

    Article Title: Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

    doi: 10.1371/journal.pone.0166370

    Figure Lengend Snippet: GTPase RhoB shows differential activation in PDAC cell lines. Cells were starved for 6 hours and treated with rhEGF for a period of 0, 2 and 10 minutes (Marked as 0, 2 and 10 min). An immunofluorescence microscopy analysis of RhoB (green) was carried out on hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cells lines. To show the cytoskeleton reorganization, F-Actin was stained with rhodamine phalloidin. Measurement of RhoB activity was performed using RhoB pulldown assay. Immunoblots for RhoB and GAPDH, as loading control for hTERT-HPNE (D), BxPC3 (E) and PANC-1 (F) cell lines are shown. To quantify the amount of RhoB-GTP, densitometric analysis (n = 3) was performed using Image Lab software for samples of hTERT-HPNE (G), BxPC3 (H) and PANC-1 (I) cell lines. For comparison of RhoB activity, GTP-RhoB was normalized to total RhoB. GAPDH was used as a protein loading control. Coomassie of RBD-GST beads are shown as beads loading control. Arrowheads denote the localization of RhoB into the peripheral membrane; boxes with number represent the quantity of cells per field with this phenotype. Scale bar = 100 μm.

    Article Snippet: Immunofluorescence (IF) and Confocal Microscopy Imaging of Pancreatic Cell Lines hTERT-HPNE, BxPC3 and PANC-1 cells were grown on coverslips (Tedpela 26020, Redding, CA) coated with poly-D-Lysine, washed twice with PBS, and then fixed with 4% PFA for 20 min. After a second wash, the cells were permeabilized with 0.1% Triton X-100 for 3 minutes at 37°C.

    Techniques: Activation Assay, Immunofluorescence, Microscopy, Staining, Activity Assay, Western Blot, Software

    Cancerous and non-cancerous pancreatic cell lines show different expression patterns of RhoGDI3 protein. (A) Immunofluorescence microscopy analysis of RhoGDI3 protein (green); 58 kDa protein, Golgi apparatus marker (Red) and Nuclei (DAPI, blue) was performed on hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. (B) Representative Immunoblot using antibodies anti-RhoGDI3, anti-RhoGDI2 and anti-GAPDH were used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. (C) Densitometric analysis of the bands detected in the Western blots of RhoGDI3 (n = 3) of protein extracts from all three cell lines, the data was normalized to GAPDH. Densitometric analysis was determined with Image Lab software. Values are means ± SEM, **P

    Journal: PLoS ONE

    Article Title: Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

    doi: 10.1371/journal.pone.0166370

    Figure Lengend Snippet: Cancerous and non-cancerous pancreatic cell lines show different expression patterns of RhoGDI3 protein. (A) Immunofluorescence microscopy analysis of RhoGDI3 protein (green); 58 kDa protein, Golgi apparatus marker (Red) and Nuclei (DAPI, blue) was performed on hTERT-HPNE (upper panel), BxPC3 (middle panel) and PANC-1 (bottom panel) cells lines. (B) Representative Immunoblot using antibodies anti-RhoGDI3, anti-RhoGDI2 and anti-GAPDH were used as loading control. Total lysates from hTERT-HPNE, BxPC3 and PANC-1 cell lines were analyzed. (C) Densitometric analysis of the bands detected in the Western blots of RhoGDI3 (n = 3) of protein extracts from all three cell lines, the data was normalized to GAPDH. Densitometric analysis was determined with Image Lab software. Values are means ± SEM, **P

    Article Snippet: Immunofluorescence (IF) and Confocal Microscopy Imaging of Pancreatic Cell Lines hTERT-HPNE, BxPC3 and PANC-1 cells were grown on coverslips (Tedpela 26020, Redding, CA) coated with poly-D-Lysine, washed twice with PBS, and then fixed with 4% PFA for 20 min. After a second wash, the cells were permeabilized with 0.1% Triton X-100 for 3 minutes at 37°C.

    Techniques: Expressing, Immunofluorescence, Microscopy, Marker, Western Blot, Software

    Nuclear localization of RhoGDI3 in rhEGF treated hTERT-HPNE cells. Subcellular fractionation was performed after cells were treated with rhEGF (marked above the images as 0, 2 and 10 rhEGF Min). Nuclear (N) and cytosolic (C) fractions from hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cell lines were obtained and analyzed by immunoblotting, using anti-RhoGDI3, anti-RhoG, anti-RhoB antibodies. Anti-histone H3 antibody was used as a nuclear control and anti-Aldolase B antibody was used as a cytosol control. 20 μg of cell lysates were loaded.

    Journal: PLoS ONE

    Article Title: Immunological and Functional Characterization of RhoGDI3 and Its Molecular Targets RhoG and RhoB in Human Pancreatic Cancerous and Normal Cells

    doi: 10.1371/journal.pone.0166370

    Figure Lengend Snippet: Nuclear localization of RhoGDI3 in rhEGF treated hTERT-HPNE cells. Subcellular fractionation was performed after cells were treated with rhEGF (marked above the images as 0, 2 and 10 rhEGF Min). Nuclear (N) and cytosolic (C) fractions from hTERT-HPNE (A), BxPC3 (B) and PANC-1 (C) cell lines were obtained and analyzed by immunoblotting, using anti-RhoGDI3, anti-RhoG, anti-RhoB antibodies. Anti-histone H3 antibody was used as a nuclear control and anti-Aldolase B antibody was used as a cytosol control. 20 μg of cell lysates were loaded.

    Article Snippet: Immunofluorescence (IF) and Confocal Microscopy Imaging of Pancreatic Cell Lines hTERT-HPNE, BxPC3 and PANC-1 cells were grown on coverslips (Tedpela 26020, Redding, CA) coated with poly-D-Lysine, washed twice with PBS, and then fixed with 4% PFA for 20 min. After a second wash, the cells were permeabilized with 0.1% Triton X-100 for 3 minutes at 37°C.

    Techniques: Fractionation

    AXL knockdown in the Panc-1 pancreatic cancer cell line affects cell viability, colony formation, migration and impairs tumor growth. Analysis of cell viability after five days in culture by MTS (A), colony formation after 14 days by Giemsa staining (B),

    Journal: Oncogene

    Article Title: Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy

    doi: 10.1038/onc.2013.487

    Figure Lengend Snippet: AXL knockdown in the Panc-1 pancreatic cancer cell line affects cell viability, colony formation, migration and impairs tumor growth. Analysis of cell viability after five days in culture by MTS (A), colony formation after 14 days by Giemsa staining (B),

    Article Snippet: Panc-1 cells were provided by Prof L. Buscail (INSERM U858, Toulouse, France) and were authenticated using short tandem repeat analysis.

    Techniques: Migration, Staining

    The E8 and D9 anti-AXL mAbs are internalized, down-regulate AXL and inhibit the AKT signaling pathway and the MAPK pathway in a KRAS -wt cell line. (A) Internalization of the anti-AXL D9 and E8 mAbs in Panc-1 cells was detected by immunofluorescence. Cells

    Journal: Oncogene

    Article Title: Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy

    doi: 10.1038/onc.2013.487

    Figure Lengend Snippet: The E8 and D9 anti-AXL mAbs are internalized, down-regulate AXL and inhibit the AKT signaling pathway and the MAPK pathway in a KRAS -wt cell line. (A) Internalization of the anti-AXL D9 and E8 mAbs in Panc-1 cells was detected by immunofluorescence. Cells

    Article Snippet: Panc-1 cells were provided by Prof L. Buscail (INSERM U858, Toulouse, France) and were authenticated using short tandem repeat analysis.

    Techniques: Immunofluorescence

    Cell size specific delivery. A) Diameters of the three different cell types (T-cells, BxPc3, PANC-1 cells). B) Percentage delivery and viability of T-cell (left), BxPc3 (center), and PANC-1 (right) when passed through chips with different constriction widths at 100 psi. The average and standard deviation of 3 to 5 independent experiments are shown. C) Delivery efficacy of PANC-1, where delivery efficacy is the product of percent viability and percent delivery at each condition (constriction width and pressure). D) Heat map of delivery efficacy when a suspension of one cell type (x-axis) is passed through chips with different constriction widths (y-axis) at 50 and 100 psi. Darker colors indicate higher delivery efficacy and lighter colors indicates lower delivery efficacy. E) Flow cytometry plot of a mixture of T-cells (low FITC region) and PANC-1 (high FITC region) cells incubated with the dextran blue dye (Pacific Blue), but not processed through the microfluidic delivery platform (left panel) demonstrating negative controls to establish the basal levels of surface receptor-mediated binding and nonspecific uptake of dye. Flow cytometry plot of a mixture of T-cell and PANC-1 cells passed through a chip with constrictions that were 6 µm wide (and 10 µm long) at 100 psi (right panel). F) Percentage delivery of a mixture of T-cell and GFP-labeled PANC-1 cells when passed through chips with different constriction widths at 100 psi. Each condition represents the average and standard deviation of three independent experiments.

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: A Size-Selective Intracellular Delivery Platform

    doi: 10.1002/smll.201601155

    Figure Lengend Snippet: Cell size specific delivery. A) Diameters of the three different cell types (T-cells, BxPc3, PANC-1 cells). B) Percentage delivery and viability of T-cell (left), BxPc3 (center), and PANC-1 (right) when passed through chips with different constriction widths at 100 psi. The average and standard deviation of 3 to 5 independent experiments are shown. C) Delivery efficacy of PANC-1, where delivery efficacy is the product of percent viability and percent delivery at each condition (constriction width and pressure). D) Heat map of delivery efficacy when a suspension of one cell type (x-axis) is passed through chips with different constriction widths (y-axis) at 50 and 100 psi. Darker colors indicate higher delivery efficacy and lighter colors indicates lower delivery efficacy. E) Flow cytometry plot of a mixture of T-cells (low FITC region) and PANC-1 (high FITC region) cells incubated with the dextran blue dye (Pacific Blue), but not processed through the microfluidic delivery platform (left panel) demonstrating negative controls to establish the basal levels of surface receptor-mediated binding and nonspecific uptake of dye. Flow cytometry plot of a mixture of T-cell and PANC-1 cells passed through a chip with constrictions that were 6 µm wide (and 10 µm long) at 100 psi (right panel). F) Percentage delivery of a mixture of T-cell and GFP-labeled PANC-1 cells when passed through chips with different constriction widths at 100 psi. Each condition represents the average and standard deviation of three independent experiments.

    Article Snippet: Two negative controls were employed for these experiments: (1) RBC-depleted whole blood that was not spiked with PANC-1 cells was incubated with the dextran dye and processed through the microfluidic delivery platform to establish delivery of dye into leukocytes and (2) GFP-expressing PANC-1 cells incubated with the dextran dye, but not processed through the microfluidic delivery platform, to establish the basal levels of surface receptor-mediated binding and non-specific uptake of dye.

    Techniques: Standard Deviation, Flow Cytometry, Cytometry, Incubation, Binding Assay, Chromatin Immunoprecipitation, Labeling

    Selective labeling of blood spiked with tumor cells. A) Schematic diagram for CTC isolation from whole blood. 1. GFP-expressing PANC-1 cells were spiked into whole blood and then depleted of red blood cells. 2. Red blood cell-depleted sample was delivered through device in the presence of tetramethylrhodamine dextran-labeled dye. 3. Cells were counterstained with an anti-CD45 antibody (APC) and GFP-positive CD45-negative cells were isolated by FACS. B) FACS plot demonstrating high specificity in tagging PANC-1 cells when PANC-1 cells are spiked into whole blood at high concentration (2000 cells per mL). GFP-expressing PANC-1 cells tagged with the rhodamine fluorophore were independently verified based on GFP fluorescence. The P4 gate [high rhodamine, low CD45 region] was used as a basis for sorting high candidate CTCs, and the P5 gate [high GFP, low CD45 region] was used to sort for GFP-expressing PANC-1 cells. The light blue dots within P5 are accurate hits (i.e., cells that are present within both P4 P5 gates), such that those are GFP-expressing PANC-1 cells with intracellular rhodamine; 92% of the cells within P4 are accurate hits. False positive hits are red, and false negative hits are black. Within a mixture of about 1.2 million viable cells, we were able to isolate 227 GFP-expressing PANC-1 cells using the size-selective delivery platform.

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    Article Title: A Size-Selective Intracellular Delivery Platform

    doi: 10.1002/smll.201601155

    Figure Lengend Snippet: Selective labeling of blood spiked with tumor cells. A) Schematic diagram for CTC isolation from whole blood. 1. GFP-expressing PANC-1 cells were spiked into whole blood and then depleted of red blood cells. 2. Red blood cell-depleted sample was delivered through device in the presence of tetramethylrhodamine dextran-labeled dye. 3. Cells were counterstained with an anti-CD45 antibody (APC) and GFP-positive CD45-negative cells were isolated by FACS. B) FACS plot demonstrating high specificity in tagging PANC-1 cells when PANC-1 cells are spiked into whole blood at high concentration (2000 cells per mL). GFP-expressing PANC-1 cells tagged with the rhodamine fluorophore were independently verified based on GFP fluorescence. The P4 gate [high rhodamine, low CD45 region] was used as a basis for sorting high candidate CTCs, and the P5 gate [high GFP, low CD45 region] was used to sort for GFP-expressing PANC-1 cells. The light blue dots within P5 are accurate hits (i.e., cells that are present within both P4 P5 gates), such that those are GFP-expressing PANC-1 cells with intracellular rhodamine; 92% of the cells within P4 are accurate hits. False positive hits are red, and false negative hits are black. Within a mixture of about 1.2 million viable cells, we were able to isolate 227 GFP-expressing PANC-1 cells using the size-selective delivery platform.

    Article Snippet: Two negative controls were employed for these experiments: (1) RBC-depleted whole blood that was not spiked with PANC-1 cells was incubated with the dextran dye and processed through the microfluidic delivery platform to establish delivery of dye into leukocytes and (2) GFP-expressing PANC-1 cells incubated with the dextran dye, but not processed through the microfluidic delivery platform, to establish the basal levels of surface receptor-mediated binding and non-specific uptake of dye.

    Techniques: Labeling, Isolation, Expressing, FACS, Concentration Assay, Fluorescence