Journal: Medical mycology

Article Title: A fucose specific lectin from Aspergillus flavus induced interleukin-8 expression is mediated by mitogen activated protein kinase p38.

doi: 10.1093/mmy/myw066

Figure Lengend Snippet: Figure 3. Interleukin-8 expression profile upon FFL treatment: Cells were treated with indicated concentrations of FFL for different time period. Total RNA was isolated and cDNA was synthesized as mentioned, mRNA expression level was determined by Q-PCR method. (A) Cell viability assay: Cells were treated with varying concentrations of FFL for 24 hours. Cell viability was determined as explained in methods. The untreated cells were considered as 100% viable. (B) Cell number assay: Cells were stimulated with varying concentrations of FFL for 24 hours. The cell number in untreated sample was taken as 100%. Trypan blue exclusion assay was used to count the cells. (C) Time dependent expression of IL-8 mRNA in L-132 cells. Cells without treatment were used as control. (D) Dose dependent expression of IL-8 mRNA in L-132 cells. (E) Inhibition of IL-8 mRNA expression by L-fucose: The 40 ng/ml of FFL was preincubated with different concentrations of L-fucose (0.1 mM, 0.2 mM, and 0.3 mM) for 1 hour prior treatment to the cells for indicated time. RNA was isolated and mRNA expression level was determined as explained earlier. (F, G) Expression of IL-8 mRNA in L-132, U-937, and PBMCs: Cells were treated with FFL for 6 and 12 hour. The mRNA expression was determined as explained earlier. (H) Measurements of IL-8 production by sandwich ELISA in L-132, U-937, and PBMCs: Cells were treated with 40 ng/ml of FFL for indicated time. After each time course supernatant was collected and IL-8 concentration was determined as mentioned in kit. The cells treated with 100 nM TPA for 4 hours was used as positive control and untreated cells considered as negative control. The data are presented as mean ± SD of three individual experiments that gave similar results. ∗∗P < .01, ∗∗∗P < .001 versus control. #P < .05, ##P < .01, ###P < .001 versus FFL (40 ng/ml).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) Interleukin-8 concentration in cell culture supernatant was determined by sandwich ELISA using human IL-8 ELISA kit from Elabscience, Beijing (E-EL-H0048), following manufacturers protocol.

Techniques: Expressing, Isolation, Synthesized, Viability Assay, Trypan Blue Exclusion Assay, Control, Inhibition, Sandwich ELISA, Concentration Assay, Positive Control, Negative Control

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: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: Knockdown of UCA1 inhibited cell growth and migration in MHCC97 cells. MHCC97 cells were transfected with shNC, sh-UCA1#1, and sh-UCA1#2. (A) The efficiency of transfection was quantified by RT-qPCR analysis. (B) Cell viability was measured by trypan blue exclusion assay. (C) Cell migration and (D) invasion were detected by Transwell migration assay. (E) Apoptotic cells and (F) the levels of apoptosis-related proteins were measured by flow cytometry and Western blot, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Knockdown, Migration, Transfection, Quantitative RT-PCR, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry, Western Blot

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: miR-301a overexpression blocked the effect of UCA1 on cell growth and migration in MHCC97 cells. MHCC97 cells were transfected with sh-UCA1 or shNC, or cotransfected with sh-UCA1 and miR-301a mimic or mimic control. (A) Cell viability, (B) cell migration, and (C) invasion were assessed by trypan blue exclusion assay and Transwell migration assay, respectively. (D) Apoptotic cells were quantified by flow cytometry assay. (E) Western blot was conducted to measure the expression of apoptosis-related core factors. * p < 0.05, ** p < 0.01.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Over Expression, Migration, Transfection, Control, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry, Western Blot, Expressing

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: CXCR4 was involved in the regulatory effect of miR-301a on cell growth and migration. MHCC97 cells were transfected with sh-CXCR4 or sh-NC, or cotransfected with sh-CXCR4 and miR-301a mimic, or mimic control. The (A) mRNA and (B) protein expression levels of CXCR4 were tested by RT-qPCR analysis and Western blot analysis, respectively. (C) Cell viability, (D) cell migration, (E) invasion, and (F) apoptotic cells were assessed by trypan blue exclusion assay, Transwell migration assay, and flow cytometry assay, respectively. (G) The protein levels of apoptosis-related factors were measured by Western blot. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Migration, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry