Journal: Immunology letters

Article Title: "In vitro" studies on galectin-3 in human natural killer cells.

doi: 10.1016/j.imlet.2017.12.004

Figure Lengend Snippet: Fig. 2. Gal-3 protein is expressed in human NK cells and its expression increases upon cell activation. Gal- 3 protein expression was evaluated by western blot analysis in NK cells, unstimulated/stimulated (24 h) with IL-2 (100 U/ml) and IL-15 (20 ng/ml), THP-1 (positive control) and Jurkat (negative control) cells. Total cellular proteins (35 μg) were resolved by 12% polyacrylamide gel and immunoblotted with specific anti-human galectin-3 antibody. β-Actin was used as internal control. Left panels are representative of typical immunoblottings from NK cells expressing high (A) or low (B) basal levels of Gal-3. The western blot signals were densitometrically analyzed by Image Lab software and results expressed as fold increase of Gal-3 signal in stimulated cells compared to unstimulated cells set at 1 (C). The results re- present the mean ± SEM of at least six independent experiments. **P ≤0.01 vs. unstimulated cells.

Article Snippet: Mingari, Department of Experimental Medicine, IRCCS AOU San Martino-IST, Genova, Italy), human monocytic leukemia THP1 cells (ATCC TIB-202; American Type culture Collection, Manassas, VA, USA) and lymphoblastic leukemia Jurkat cells (ATCC TIB-152) were cultured (1× 106 cells/ml) in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Carlo Erba, Cornaredo, Italy), 2 mM L-glutamine, 100 μg/ml kanamycin, 1 mM sodium pyruvate e 1% MEM amino acid solution (Sigma-Aldrich, Milan, Italy) in a humidified incubator at 5% CO2 and 37 °C.

Techniques: Expressing, Activation Assay, Western Blot, Positive Control, Negative Control, Control, Software

Journal: Immunology letters

Article Title: "In vitro" studies on galectin-3 in human natural killer cells.

doi: 10.1016/j.imlet.2017.12.004

Figure Lengend Snippet: Fig. 3. Gal-3 is intracellularly expressed in human NK cells and its expression increases upon cell activation. NK cells, unstimulated/stimulated for 24 h with IL-2 (100 U/ml) and IL-15 (20 ng/ml), THP-1 (positive control) and Jurkat (negative control) cells were harvested, stained with anti-human Gal-3 and analyzed by FACS. (A) Representative histograms of extracellular (left panels) and intracellular (right panels) Gal-3 expression in human NK with high (a) and low basal (b) Gal-3 expression, THP-1 and Jurkat cells. Resting cells are shown as blue histograms, stimulated NK cells as red, while the isotype controls as black histograms. (B) Mean Fluorescence Intensity (MFI) levels of Gal-3 in human NK cells expressed as fold increase in stimulated cells compared to unstimulated cells set at 1. The results represent the mean ± SEM of at least six independent experiments. **P ≤0.01 vs. unstimulated cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mingari, Department of Experimental Medicine, IRCCS AOU San Martino-IST, Genova, Italy), human monocytic leukemia THP1 cells (ATCC TIB-202; American Type culture Collection, Manassas, VA, USA) and lymphoblastic leukemia Jurkat cells (ATCC TIB-152) were cultured (1× 106 cells/ml) in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Carlo Erba, Cornaredo, Italy), 2 mM L-glutamine, 100 μg/ml kanamycin, 1 mM sodium pyruvate e 1% MEM amino acid solution (Sigma-Aldrich, Milan, Italy) in a humidified incubator at 5% CO2 and 37 °C.

Techniques: Expressing, Activation Assay, Positive Control, Negative Control, Staining, Fluorescence

Journal: Immunology letters

Article Title: "In vitro" studies on galectin-3 in human natural killer cells.

doi: 10.1016/j.imlet.2017.12.004

Figure Lengend Snippet: Fig. 4. Confocal microscopy analysis of cytoplasmic and nuclear Gal-3 subcellular distribution in human NK cells. (A) NK cells, unstimulated/stimulated for 24 h with IL-2 (100 U/ml) and IL-15 (20 ng/ml), THP-1 (positive control) and Jurkat (negative control) cells were harvested, stained with anti-human Gal-3, and analyzed by confocal microscopy. Representative images of Gal-3 expression in: THP-1 cells (positive control; a and d), Jurkat cells (negative control; g), resting NK cells (b, e and f), stimulated NK cells (c, h and i). Gal-3 is shown as red dots, perforin as green dots. Nuclei are shown in blue. Scale bar, 10 μm. (B). Representative images of Gal-3 localization in the nucleus and/or in the cytoplasm (a-b) or co-localization of Gal-3 with perforin (c). Gal-3 is shown as red, perforin as green dots. Yellow dots represent co-localization of Gal-3 with perforin. Nuclei are shown in blue. Scale bar, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mingari, Department of Experimental Medicine, IRCCS AOU San Martino-IST, Genova, Italy), human monocytic leukemia THP1 cells (ATCC TIB-202; American Type culture Collection, Manassas, VA, USA) and lymphoblastic leukemia Jurkat cells (ATCC TIB-152) were cultured (1× 106 cells/ml) in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Carlo Erba, Cornaredo, Italy), 2 mM L-glutamine, 100 μg/ml kanamycin, 1 mM sodium pyruvate e 1% MEM amino acid solution (Sigma-Aldrich, Milan, Italy) in a humidified incubator at 5% CO2 and 37 °C.

Techniques: Confocal Microscopy, Positive Control, Negative Control, Staining, Expressing

Journal: Immunology letters

Article Title: "In vitro" studies on galectin-3 in human natural killer cells.

doi: 10.1016/j.imlet.2017.12.004

Figure Lengend Snippet: Fig. 5. Gal-3 inhibition increases the degree of degranulation in human activated NK cells. A classical degranulation assay was used to measure NK cell activity (see Methods, 2.6). (A) The first three panels are representative contour plots of resting and cytokine-stimulated NK cells in the absence/presence of the 30 μM Gal-3 inhibitor (dashed gates represent the percentage of total CD56/CD107a positive cells, while solid gates those highly positive) incubated (4 h) with K562 cells. The fourth panel on the right represents overlapped histograms of CD107a expression in resting (black histogram, 5Aa), cytokine-stimulated (blue histogram, 5Ab), cytokine-stimulated plus Gal-3 inhibitor (red histogram, 5Ac) NK cells incubated (4 h) with K562 cells. (B) Percentage of highly positive CD107a cells in activated NK cells untreated/treated with increasing concentrations (1–30 μM) of the Gal-3 inhibitor. The percentage increases in CD107a highly positive cells are calculated vs the value of inhibitor-untreated cells set at 1. The results represent the mean ± SEM of at least four independent experiments. *P ≤0.05 vs. inhibitor-untreated cells. (C) Representative Gal-1 protein expression evaluated by western blot analysis in NK cells, unstimulated/stimulated (24 h) with IL-2 (100 U/ml) and IL-15 (20 ng/ml), K562 (positive control) and Jurkat (negative control) cells. Total cellular proteins (35 μg) were resolved by 12% polyacrylamide gel and immunoblotted with specific anti-human galectin-1 antibody. β-Actin was used as internal control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mingari, Department of Experimental Medicine, IRCCS AOU San Martino-IST, Genova, Italy), human monocytic leukemia THP1 cells (ATCC TIB-202; American Type culture Collection, Manassas, VA, USA) and lymphoblastic leukemia Jurkat cells (ATCC TIB-152) were cultured (1× 106 cells/ml) in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Carlo Erba, Cornaredo, Italy), 2 mM L-glutamine, 100 μg/ml kanamycin, 1 mM sodium pyruvate e 1% MEM amino acid solution (Sigma-Aldrich, Milan, Italy) in a humidified incubator at 5% CO2 and 37 °C.

Techniques: Inhibition, Degranulation Assay, Activity Assay, Incubation, Expressing, Western Blot, Positive Control, Negative Control, Control

Journal: International Journal of Molecular Sciences

Article Title: A Dual Role for FADD in Human Precursor T-Cell Neoplasms

doi: 10.3390/ijms232315157

Figure Lengend Snippet: Pharmacological arrest at the G2/M stage of the cell cycle. The cell cycle of the FADD-expressing (FADD) and FADD-deficient (NEG) JURKAT cell lines was evaluated without any treatment (untreated) or after treatment with 100 nM etoposide, 50 ng/mL nocodazole, and 10 nM paclitaxel for 18 h. ( A ) Representative histograms of cells stained with propidium iodine showing DNA content distribution. Cell cycle phase distribution was analyzed by flow cytometry. ( B ) Bar chart of cell cycle distribution from six independent experiments. One-way ANOVA was used to test for statistical significance: * p ≤ 0.05; ** p ≤ 0.01. Error bars represent the standard error of the mean (SEM).

Article Snippet: Additionally, we corroborated that FADD expression of stable cell lines was equivalent to the FADD endogenous level of the parental clone JURKAT A3 (A3; ATCC, CRL-2570) ( ).

Techniques: Expressing, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: A Dual Role for FADD in Human Precursor T-Cell Neoplasms

doi: 10.3390/ijms232315157

Figure Lengend Snippet: Interactome analysis using DIA-MS confirms FADD participation in energy metabolism processes. ( A ) Workflow of co-Immunoprecipitation-Mass Spectrometry. Endogenous FADD protein was co-immunoprecipitated from FADD-expressing (FADD) and FADD-deficient (NEG) JURKAT cell lines using magnetic beads. Samples were trypsin-digested and injected into the mass spectrometer for data-independence acquisition. Finally, the raw data were analyzed with Spectronaut software and R. ( B ) STRING interaction map of significant proteins that interact with FADD in JURKAT cells compared to JURKAT-deficient FADD (FADD NEG). Proteins were clustered based on K-means into five clusters. The functional enrichment analysis and adjusted p -value (adj. p -value) are shown for the three main clusters. ( C ) Validation of DIABLO–FADD interaction. The input and immunoprecipitated (IP) fractions were separated by SDS-PAGE and blotted with antibodies against anti-FADD and -DIABLO. The densitometry values are shown below. ( D ) Schematic representation to show interactions of FADD with proteins involved in different biological processes. Under non-apoptotic conditions, FADD interacts with DIABLO/Smac, which hampers FADD function inducing apoptosis. FADD also interacts with several proteins involved in the energy metabolism.

Article Snippet: Additionally, we corroborated that FADD expression of stable cell lines was equivalent to the FADD endogenous level of the parental clone JURKAT A3 (A3; ATCC, CRL-2570) ( ).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Magnetic Beads, Injection, Software, Functional Assay, Biomarker Discovery, SDS Page

Journal: European journal of biochemistry

Article Title: Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.

doi: 10.1046/j.1432-1033.2002.02748.x

Figure Lengend Snippet: Fig. 1. All-trans-RA represses the induction of FasL expression. (A) The effects of all-trans-RA on FasL transcription were examined using RT-PCR. Jurkat cells were incubated with the indicated concentra- tions of all-trans-RA for 24 h and then treated with PMA (10 ngÆmL)1) and ionomycin (0.5 lM) for 6 h. The expression of b-actin was monitored as a control. (B) The FasL (nucleotides )2306 to )2)-Luc reporter, together with the b-gal expression vector, was transiently transfected into Jurkat cells as described in the Experi- mental procedures. Transfected cells were treated with PMA (10 ngÆmL)1) and ionomycin (0.5 lM) in the presence or absence of 1.0 lM RA for 6 h. The luciferase activity was measured and nor- malized by b-gal activity. Data are shown as the mean ±SE of three independent measurements.

Article Snippet: The Jurkat human T-cell leukemia (ATCC, CRL1990), and HeLa human cervical carcinoma (ATCC, CCL-2) cell lines were obtained from the American Type Culture Collection.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Control, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: European journal of biochemistry

Article Title: Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.

doi: 10.1046/j.1432-1033.2002.02748.x

Figure Lengend Snippet: Fig. 2. Delineation of all-trans-RA-responsive cis-acting elements in the FasL promoter. (A) Schematic representation of the deletions in the 5¢ terminus of the FasL promoter that were cloned upstream of a luciferase reporter gene. The 3¢ end of the FasL promoter contains nucleotide )2, counted from the translation initiation site, and tran- scription starts from nucleotide )181 [22]. (B) Each reporter construct was transiently transfected into Jurkat cells. Transfected cells were stimulated with PMA (25 ngÆmL)1) and ionomycin (0.5 lM) in the absence (empty bar) or presence (filled bar) of all-trans-RA (2.0 lM) for 6 h. Luciferase activity was measured and normalized by b-gal activity. To establish the reporter construct basal expression, pTK-luc, which contains a minimal promoter of thymidine kinase, was also used in the transfection assay.

Article Snippet: The Jurkat human T-cell leukemia (ATCC, CRL1990), and HeLa human cervical carcinoma (ATCC, CCL-2) cell lines were obtained from the American Type Culture Collection.

Techniques: Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Expressing

Journal: European journal of biochemistry

Article Title: Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.

doi: 10.1046/j.1432-1033.2002.02748.x

Figure Lengend Snippet: Fig. 3. The effect of all-trans-RA is mediated by an NFAT binding motif present in the FasL promoter region. (A) Schematic representation of the FasL promoter (nucleotides )318 to )2) reporter construct, along with NFAT and SP-1 binding sites. The nucleotide sequences of the NFAT- and SP-1- binding sites and of mutations in these sites are shown. (B) The indicated reporter constructs together with a b-gal expression vector were transiently transfected into Jurkat cells as described in the Experimental procedures. Transfected cells were treated with PMA (10 ngÆmL)1) and ionomycin (0.5 lM) in the pres- ence or absence of RA (1.0 lM) for 6 h. Luciferase activity was mea- sured and normalized by b-gal activity. (C) The NFAT(FasL)-Luc construct was transfected into Jurkat cells and incubated for 6 h with PMA (10 ngÆmL)1) and ionomycin (0.5 lM) in the absence or presence of all-trans-RA (1.0 lM). Luciferase activity was measured and nor- malized by b-gal activity. (D) Jurkat-NFAT cells were treated with PMA (25Æng mL)1)/ionomycin (0.5 lM), CsA (1 lgÆmL)1), and RA (2.0 lM) for 6 h, as indicated. b-Gal activity was measured and nor- malized with the protein concentrations of cell extracts. All data from the reporter gene assays are shown as the mean ±SE of more than three independent measurements.

Article Snippet: The Jurkat human T-cell leukemia (ATCC, CRL1990), and HeLa human cervical carcinoma (ATCC, CCL-2) cell lines were obtained from the American Type Culture Collection.

Techniques: Binding Assay, Construct, Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Incubation

Journal: European journal of biochemistry

Article Title: Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.

doi: 10.1046/j.1432-1033.2002.02748.x

Figure Lengend Snippet: Fig. 5. All-trans-RA represses the DNA-binding activity of NFAT. A, PBMCs (7 · 106 cells) obtained from a healthy donor were stimulated in a 100-cm2 plate that was precoated with anti-CD3 Ig for 4 h with or without 1.0 lM all-trans-RA. B, Jurkat cells (3 · 106 cells) were treated with PMA (10 ngÆmL)1) and ionomycin (0.5 lM) for 4 h, in the presence or absence of a 24-h pretreatment with all-trans-RA, as indicated. The reaction mixture containing 5 lg nuclear extract was incubated with 32P-labeled oligonucleotide and analyzed by gel shift assay, as described in the Experimental procedures. The designations for cNFAT(FasL), cNFAT(IL-2), and cSP-1 indicate a 100-fold excess of the competing unlabeled oligonucleotides.

Article Snippet: The Jurkat human T-cell leukemia (ATCC, CRL1990), and HeLa human cervical carcinoma (ATCC, CCL-2) cell lines were obtained from the American Type Culture Collection.

Techniques: Binding Assay, Activity Assay, Incubation, Labeling, Gel Shift