Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Western bloting analysis of total PCLP1 expression in B cell lines (Raji, Ramos and Daudi), B cells from four healthy donors (B cells 1–4), and a T cell line (Jurkat). Actin is shown as a loading control. ( B ) PCLP1 expression on the surface of various B cell lines and Jurkat T cell line by flow cytometry. The grey line and the red line in the histograms represent isotype control (mouse IgG) and PCLP1 staining (anti-PCLP1 mAb), respectively. ( C ) Raji cells were stably transfected with pEGFP-PCLP1 (Raji-PCLP1) or pEGFP (Raji-Ctrl) and whole cell extracts from three different clones of each cell type were analyzed for the expression of PCLP1 by Western-blotting using an anti-hPCLP1 mAb. PCLP1: endogenous PCLP1. PCLP1-GFP: ectopic PCLP1. Actin is shown as a loading control. ( D ) Representative fluorescence microscope images of Raji cells stably expressing PCLP1-GFP fusion protein showing the localization of PCLP1-GFP (green) to cell surface and vesicles (I), and centrosome (II), using a 60× objective. Nuclei were stained with Hoechst 33342 (blue). ( E ) Representative fluorescence microscope images of Raji-PCLP1 cells showing the centrosomal localization of PCLP1-GFP fusion protein (green) at interphase (top) and mitosis (bottom). Cells were stained with an anti-γ-tubulin mAb to visualize the pericentriolar area of the centrosome (red). Nuclei were stained with DAPI (blue). Insets at lower right corner show amplified images of the centrosomal region. The size bar indicates 5 µm. ( F ) PCLP1 expression was determined on the surface of malignant and normal B cells from 11 patients with different lymphoma subtypes and 3 patients with ALL by flow cytometry. Median fluorescence intensity (MFI) of PCLP1 expression on malignant B cells was normalized to that detected on normal B cells from the same patient. N: normal B cells; M: malignant B cells; B-ALL: B-cell acute lymphoblastic leukemia; FL: follicular lymphoma; WM: Waldenström macroglobulinemia; HCL: hairy cell leukemia; CLL: chronic lymphocytic leukemia.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Staining, Stable Transfection, Transfection, Clone Assay, Fluorescence, Microscopy, Amplification

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Cell proliferation of Raji-PCLP1 and Raji-Ctrl cell clones was measured counting the number of viable cells at different time points of culture in complete medium using the trypan blue exclusion assay. ( B ) Colony formation of Raji-PCLP1 and Raji-Ctrl cells cultured in methylcellulose medium for 11 days. Colonies were stain with crystal violet and counted using an inverted microscope. The graph depicts the number of colonies per well from four independent experiments performed in triplicate. Representative microscope pictures and enlarged images of colony formation after crystal violet staining are shown. Statistical analysis of mean ± SD was calculated using two-tailed paired Student´s t -test. *** P < 0.001. ( C ) Cell-to-cell adhesion was calculated as the number of cell aggregates formed at 72 h of culture. Representative microscope images of cell aggregates using a 10× objective are shown. The size bar indicates 100 µm. ( D ) Cells were incubated in the presence of anti-CD62L mAb or isotype control (mIgG1) (left) or in the presence of anti-CD18 mAb or mIgG1 (right) and the number of aggregates were counted at 2 h. Cells were cultured in the presence of 1 mM EDTA, 1 mM EGTA or vehicle and the number of aggregates were counted at 24 h (middle). The graphs in C and D represents the mean ± SD of the number of aggregates per field containing more than 20 cells from 3 independent experiment and analyzing 10 fields in each experiment. Statistical analysis was calculated using no-paired Student´s t -test (C) and Mann-Whitney U test (D). ** P < 0.01 *** P < 0.001. ( E ) Chemotactic migration of Raji-PCLP1 or Raji-Ctrl toward 100 ng/ml CXCL12 using Transwell chambers. Data represents the mean chemotactic index ± SD normalized to control cells from six independent experiments. Statistical analysis was calculated using Mann-Whitney U test. ** P < 0.01.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Clone Assay, Trypan Blue Exclusion Assay, Cell Culture, Staining, Inverted Microscopy, Microscopy, Two Tailed Test, Incubation, Control, MANN-WHITNEY, Migration

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Raji-Ctrl and Raji-PCLP1 cells were treated with increasing concentrations of vehicle or dexamethasone for 72 hours and cell death was analyzed after Annexin V-PE/7´AAD staining by flow cytometry. The graph on the left shows the mean percentages ± SD of total specific cell death (including early apoptosis, late apoptosis and necrotic cells) from six independent experiments after subtracting the spontaneous cell death (vehicle). The percentage of specific cell death was calculated as follows: [(% lysis of target cell – % spontaneous cell death)/(100% – % spontaneous cell death)] × 100. Representative dot plots showing Annexin V/7AAD staining in cells treated with vehicle or 1000 µM dexamethasone are shown (right). As displayed in the dot plots, the spontaneous cell death (Annexin V PE positive/7ADD negative, Annexin V PE positive/7ADD positive and Annexin V PE negative/7ADD positive cells) in cells cultured with the vehicle was approximately 7.5% in Raji-Ctrl and 5.5% in Raji-PCLP1 cells. ( B ) Expression of PCLP1 on Annexin V and 7ADD double-negative population of cells treated with dexamethasone or vehicle by flow cytometry. ( C ) Raji-Ctrl and Raji-PCLP1 cells were treated with increasing concentrations of vehicle or H 2 O 2 for 6 h and cell death was analyzed as indicated in A. The graph on the left shows the mean percentages ± SD of total cell death from five independent experiments after subtracting the spontaneous cell death (vehicle). Representative dot plots showing Annexin V/7AAD staining in cells treated with vehicle or 200 µM H 2 O 2 are shown (left). As displayed in the dot plots, the spontaneous cell death in the presence of vehicle was approximately 17.0% in Raji-Ctrl and 5.9% in Raji-PCLP1 cells. ( D ) Cells were incubated overnight with increasing concentrations of obinutuzumab or vehicle and cell viability was measured based on Annexin V/7AAD double negative staining relative to vehicle-treated cells. Data show the mean percentages ± SD of viable cells from six independent experiments. ( E ) A representative image of the homotypic adhesion induced by obinutuzumab as visualized by light microscopy is shown. The size bar indicates 100 µm. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Staining, Flow Cytometry, Lysis, Cell Culture, Expressing, Incubation, Negative Staining, Light Microscopy, Two Tailed Test

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Measurement of pH of Raji-PCLP1 or Raji-Ctrl cell culture medium containing glutamine (complete medium) (left) or in the absence of glutamine (right) at the indicated time points. ( B ) Proliferation of Raji-PCLP1 and Raji-Ctrl cells in glucose-free culture medium (left) or glutamine-free culture medium (middle) at different time points. The number of viable cells was normalized to their counterparts grown in complete medium. Data show the mean ± SD of five independent experiments. Proliferation of Raji-PCLP1 and Raji-Ctrl cells in glucose-free conditions and decreasing concentrations of glutamine at 72 h of culture (right). The number of viable cells was normalized to their counterparts grown in complete medium. Data show the mean ± SD of six independent experiments. ( C ) Death of Raji-PCLP1 and Raji-Ctrl cells under glutamine-deprivation conditions. Cells were incubated in the presence of glucose and decreasing concentration of glutamine and the percentage of cell death was determined at 72 h of culture after Annexin V-PE/7´AAD staining by flow cytometry. Data show the mean ± SD of five independent experiments. Representative dot plots showing Annexin V/7AAD staining in cells treated with complete medium (2 mM glutamine/11 mM glucose) or glutamine free medium (0 mM glutamine/11 mM glucose) are depicted. ( D ) Effect of glucose depletion on PCLP1 expression in Raji cells. Cells were cultured in high-glucose medium (11 mM) or low-glucose medium (0.5 mM) for 72 h and the expression of PCLP1 on cell surface was determined by flow cytometry. Data show MFI of PCLP1 expression from five independent experiments. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Cell Culture, Incubation, Concentration Assay, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: Determination of lipid droplet content in Raji-PCLP1 and Raji-Ctrl cells ( A ) or Jurkat-PCLP1 and Jurkat-Ctrl cells ( B ) by fluorescence microscopy. Cells were cultured in complete medium for the indicated times, fixed and stained with Nile Red for the detection of neutral lipid droplets. Ten fields were randomly selected using a 60× oil objective and the total area of lipid droplets per cell was calculated employing ImageJ software. Bar graphs represent the quantitative analysis of lipid droplets content per cell. Mean ± SD of lipid droplet area per cell is shown. Statistical analysis was calculated using Mann-Whitney U test. * P < 0.05. ** P < 0.01. *** P < 0.001. Representative fluorescence microscopy images depicting lipid droplets stained with Nile Red (right panels, yellow). Nuclei were labeled with DAPI (left panels, blue). Digital zoom of a selected area is shown. The size bar indicates 10 µm.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Fluorescence, Microscopy, Cell Culture, Staining, Software, MANN-WHITNEY, Labeling

Journal: Oncotarget

Article Title: Podocalyxin promotes proliferation and survival in mature B-cell non-Hodgkin lymphoma cells

doi: 10.18632/oncotarget.21283

Figure Lengend Snippet: ( A ) Schematic representation of cancer cell metabolism. Once transported into cells, glucose is phosphorylated to glucose 6-phosphate by hexokinases. Glucose 6-phosphate can then be metabolized via aerobic glycolysis or PPP. Through the aerobic glycolysis, glucose 6-phosphate is sequentially converted to pyruvate. Afterwards, pyruvate is transformed to acetyl-CoA via TCA or to lactate. Acetyl-CoA can be used for the synthesis of fatty acids and lipids for biomembrane generation. Glucose 6-phosphate that enters PPP generates ribose, for nucleotides synthesis, and NADPH, for FA synthesis. Glutamine provides intermediates to maintain TCA function and contributes to lipid synthesis and the production of purines and pyrimidines for nucleotide synthesis. Glucose and glutamine also provide the carbon and nitrogen necessary for the synthesis of nonessential amino acids. Metabolic inhibitors are depicted in red color. 2DG: 2-deoxyglucose; 6AN: 6 aminonicotinamide. ( B ) Raji-PCLP1 and Raji-Ctrl cells were cultured for 72 h in complete medium in the absence or in the presence of increasing concentrations of the 2-DG (glycolysis inhibitor). ( C ) To determine the effect of compound 968 (glutaminase inhibitor) on cell proliferation, Raji-PCLP1 and Raji-Ctrl cells were cultured for 7 days in complete medium in the absence or in the presence of increasing concentrations of the inhibitor. ( D ) Raji-PCLP1 and Raji-Ctrl cell were cultured for 72 h in complete medium in the absence or in the presence of increasing concentrations of cerulenin (fatty acid synthase inhibitor). ( E ) Raji-PCLP1 and Raji-Ctrl cells were cultured for 72 h in complete medium in the absence or in the presence increasing concentrations of 6AN (PPP inhibitor). Cell proliferation was assessed by counting live cells on a hemocytometer using trypan blue exclusion dye. The number of viable cells was normalized to their counterparts grown in the absence of the inhibitor. Data show the mean ± SD percentage relative to non-treated cells of five independent experiments. Statistical analysis was calculated using two-tailed paired Student´s t -test. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Human Burkitt lymphoma cell lines Raji, Ramos and Daudi, human acute T cell leukemia cell line Jurkat, and human diffuse large B-cell lymphoma cell line Pfeiffer were obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA).

Techniques: Transformation Assay, Cell Culture, Two Tailed Test

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) p27 mRNA expression in CLL cells and in healthy B-cells determined by RT-qPCR. (B) Comparison of p27 mRNA expression (determined by RT-qPCR) and p27 protein expression (determined by immunoblot) in the same CLL samples. The black bars show the densitometric quantification of p27 protein levels normalized to actin expression. (C) p27 protein expression in CLL cells and in healthy B-cells (controls). (D) A representative immunoblot showing p27 and actin expression. The arrows mark examples of CLL patient samples with low p27 expression. T, tonsils. (E) Immnofluorescence analysis showing nuclear p27 in CLL cells from four patients. Nuclei are stained with DAPI.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot, Staining

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Myc mRNA expression in CLL cells and in healthy B-cells determined by RT-qPCR. (B) Comparison of Myc mRNA expression (determined by RT-qPCR) and p27 protein expression (determined by immunoblot) in the same CLL samples. The black bars show the densitometric quantification of Myc protein levels normalized to actin expression. (C) Myc protein expression in CLL cells and in healthy B-cells (controls). (D) Representative immunoblot showing Myc and actin expression. T, tonsils

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing p27, Myc and actin expression in cells from CLL patients and from tonsil cells (T, tonsils). (B) Expression levels of Myc and p27 in CLL patients. The protein levels of p27 and Myc as determined by film densitometry and normalized to actin, were classified in three categories with respect to the mean of expression in controls. (C) Classification of patients with low and high Myc protein expression according to their p27 levels.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Immunoblot showing p27 and Myc expression in MEC1 cell line transfected with a p27 expression vector (pCEFL-p27) and the corresponding empty vector. Proteins were analyzed 24 h after transfection. Actin levels are shown to asses protein loading. (B) MEC1 cells were transfected with pEYFPp27 vector and 24 h later the cell cycle of transfected cells was analysed by flow cytometry. The empty vector (pEYFP) was used as controls. The percentage of cells in the G0/G1 phase is indicated. (C) p27 expression rescued fludarabine-induced cell death. Cells were transfected with pEYFPp27 and pEYFP vector and 24 h after transfection the cells were treated with 10 μM fludarabine and cell death was determined 24 h later by trypan blue exclusion assay. (D) MEC1 cells were transfected and treated as in C, and the apoptosis was determined by annexin V binding, assessed by flow cytometry. (E) MEC1 cells were transfected and treated as in C and the levels of active caspase 3 were determined by immunoblot. (F) Correlation of high expression of p27 protein with higher Bcl2 levels in CLL cells.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Trypan Blue Exclusion Assay, Binding Assay

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing the levels of cyclins A and E in CLL cells with and without Myc/p27. Actin levels were also determined to assess protein loading. (B) Correlation between Myc and cyclin A protein levels in CLL cells. (C) Immunoblot showing Myc and p27 protein levels in four patient's cells selected for the experiments shown in D. (D) Molecular filtration chromatographic separation of CLL protein extracts followed by immunoblot for cyclins A, E and Cdk2. The elution of the 55 and 30 kDa proteins is shown at the top. (E) Immunoblot (IB) showing the levels of Myc and p27 in three CLL samples and MEC1 cells and kinase assay of Cdk2 in the same extracts. Proteins were immunoprecipitated with anti Cdk2 and the presence of both cyclin E, p27 and Cdk2 were determined by immunoblot. Lower panel: kinase assays were performed using HisCK − as kinase substrate. M, mock kinase reaction without extract. (F) Immunoprecipitation of Cdk2 and kinase assay of the immunoprecipitates of MEC1 cells and MEC1 cells incubated with lysates from a CLL sample (#01). (G) Immunoprecipitation of Cdk2 and kinase assay from MEC1 cells and from mixed lysates prepared with MEC1 cells and two CLL samples (#01 and #10).

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Filtration, Kinase Assay, Immunoprecipitation, Incubation

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing the protein levels of Myc, p27 and Skp2 in CLL cells. Actin levels are also shown as protein loading control. (B) p27 protein levels in CLL cells with high or low expression of Skp2. (C) Myc protein levels in CLL cells with high or low expression of Skp2.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Control, Expressing

Journal: Molecular Medicine Reports

Article Title: Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells

doi: 10.3892/mmr.2015.4715

Figure Lengend Snippet: ROS levels are increased in HepG2 cells following co-treatment with ABT-737 and curcumin. (A) HepG2 cells were separately treated with 1% dimethyl sulfoxide, 2 µ M curcumin, 10 µ M ABT-737 and 2 µ M curcumin+10 µ M ABT-737 for 24 h, following which a 2′,7′-dichlorofluorescin diacetate probe was used to measure the levels of cellular ROS. HepG2 cells were treated with 2 µ M curcumin and 10 µ M ABT-737 for 24 h in the presence or absence of the antioxidant, NAC (10 mM). Subsequently, the levels of HepG2 cell apoptosis were analyzed using (B) Hoechst 33258 staining (white arrows indicate cells undergoing apoptosis with nuclear fragmentation visualized by Hoechst staining), (C) Annexin V-fluorescein isothiocyanate/PI staining and (D) caspase-3 activity detection. Data are expressed as the mean ± standard deviation. * P<0.05. ROS, reactive oxygen species; PI, propidium iodide; NAC, N-acetyl-L-cysteine.

Article Snippet: An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China).

Techniques: Staining, Activity Assay, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells

doi: 10.3892/mmr.2015.4715

Figure Lengend Snippet: Increased levels of ROS promote the sustained activation of JNK, which leads to the apoptosis of HepG2 cells. (A) HepG2 cells were separately treated with 1% dimethyl sulfoxide, 2 µ M curcumin, 10 µ M ABT-737 and 2 µ M curcumin+10 µ M ABT-737 for 24 h, following which the total cellular proteins were extracted. Subsequently, the protein levels of p-JNK1/JNK2 and total JNK1/JNK2 were detected using western blotting. (B) HepG2 cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h in the presence or absence of 10 µ M SP600125 (a JNK inhibitor). Following treatment, apoptosis of the HepG2 cells was assessed using annexin V-fluorescein isothiocyanate/PI staining. (C) HepG2 cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h in the presence or absence of antioxidant, NAC (10 mM). Subsequently, the levels of p-JNK1/JNK2 and total JNK1/JNK2 were detected using western blotting. (D) HepG2 cells were separately transfected with control siRNA or ASK1 siRNA for 24 h, following which the cells were treated with 2 µ M curcumin+10 µ M ABT-737 for 24 h. A Trypan blue exclusion assay was then used to measure the total cell death ratio. * P<0.05 vs. control. ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; p-, phosphorylated; siRNA, small interfering RNA; PI, propidium iodide.

Article Snippet: An Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China).

Techniques: Activation Assay, Western Blot, Staining, Transfection, Control, Trypan Blue Exclusion Assay, Small Interfering RNA