Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Eomes promoted the expression of IFN-γ mRNA in EL4 cells. ( A , B ) Whole cell lysates were prepared from Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants and used for Western blotting using anti-FLAG, anti-RelA, and anti-NFATc2 antibodies. Blots were representative of five independent experiments ( A ). The amount of FLAG-Eomes, RelA, and NFATc2 was normalized to that of γ1-actin. The amount of the FLAG-Eomes protein in the Eomes #2 EL4 transfectant was set to 1-fold. The amounts of the RelA protein and NFATc2 protein in the Control #1 EL4 transfectant were set to 1-fold. The FLAG-Eomes protein (fold), RelA protein (fold), and NFATc2 protein (fold) are shown as the mean ± S.E. of five independent experiments. ( C , D ) Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants were treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 6 h. IFN-γ mRNA ( C ) and IL-2 mRNA ( D ) were measured by quantitative PCR. mRNA levels without PMA and IM in each EL4 transfectant were set to 1-fold. Data are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

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

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Binding of FLAG-Eomes, RelA, and NFATc2 to the IFN-γ promoter, CNS−22, and CNS+30 in EL4 transfectants. ( A – C ) The Control #2 EL4 transfectant (Control) and Eomes #2 EL4 transfectant (Eomes) were treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h. ChIP assays were performed for FLAG-Eomes ( A ), RelA ( B ), and NFATc2 ( C ). Quantitative PCR was used to measure the amounts of fifteen different DNA regions. The IFN-γ promoter (−0.1 kb), CNS−22, and CNS+30 are shown in this figure. Other regions for FLAG-Eomes, RelA, and NFATc2 are shown in , respectively. Immunoprecipitated (IP) DNA (% input) is shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Binding Assay, Control, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Effects of TPCA-1 on IFN-γ and IL-2 mRNA expression in EL4 transfectants. ( A – C ) Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants were pretreated with TPCA-1 for 1 h and then treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h in the presence or absence of TPCA-1 at the indicated final concentrations. Cell viability (%) is shown as the mean ± S.E. of three independent experiments ( A ). IFN-γ mRNA ( B ) and IL-2 mRNA ( C ) were measured by quantitative PCR. mRNA levels with PMA and IM in each EL4 transfectant were set to 100%. IFN-γ mRNA (%) and IL-2 mRNA (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Effects of IKK-16 on IFN-γ and IL-2 mRNA expression in EL4 transfectants. ( A – C ) Control #1, Control #2, Eomes #1, and Eomes #2 EL4 transfectants were pretreated with IKK-16 for 1 h and then treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h in the presence or absence of IKK-16 at the indicated final concentrations. Cell viability (%) is shown as the mean ± S.E. of three independent experiments ( A ). IFN-γ mRNA ( B ) and IL-2 mRNA ( C ) were measured by quantitative PCR. mRNA levels with PMA and IM in each EL4 transfectant were set to 100%. IFN-γ mRNA (%) and IL-2 mRNA (%) are shown as the mean ± S.E. of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores

doi: 10.3390/ijms222313098

Figure Lengend Snippet: Effects of TPCA-1 on the binding of RelA, FLAG-Eomes, and NFATc2 to the IFN-γ promoter, CNS−22, and CNS+30 in EL4 transfectants. ( A – C ) Eomes #2 EL4 transfectants were pretreated with (+) or without (−) TPCA-1 for 1 h and then treated with (+) or without (−) PMA (10 nM) and IM (1 µM) for 2 h in the presence or absence of TPCA-1 (20 µM). ChIP assays were performed for FLAG-Eomes ( A ), RelA ( B ), and NFATc2 ( C ) using control IgG (light color bars) or specific antibodies (dark color bars). Quantitative PCR was used to measure the amounts of DNA regions. Immunoprecipitated (IP) DNA (% input) is shown as the mean ± S.E. of four ( A ) and three ( B , C ) independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse lymphoma EL4 cells (RCB1641) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Binding Assay, Control, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: Current drug metabolism

Article Title: Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement

doi: 10.2174/1389200222666210831125041

Figure Lengend Snippet: Efficacy, tolerability, and pharmacokinetic assessment of GA-607 in EL4 tumor-bearing mice following GA-607 (3.2 mg/kg SC) treatment; dosed 5 consecutive days followed by 2 drug-free days. Tumors volumes and body weights were only measured on the day of dosing. (A) Complete tumor regression was observed following GA-607 administration. (B) No change in body weight was observed following GA-607 administration. (C) GA-607 and (D) GA-607-derived DON levels in plasma and tumors following GA-607 administration.

Article Snippet: EL4 mouse lymphoma cells were obtained as a gift from Dr. Jonathan Powell’s laboratory (Johns Hopkins University, Baltimore, MD).

Techniques: Derivative Assay, Clinical Proteomics

Journal: Current drug metabolism

Article Title: Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement

doi: 10.2174/1389200222666210831125041

Figure Lengend Snippet: Metabolomic analysis of GA-607-versus vehicle-treated EL4 tumors. GA-607 treatment caused an increase in FGAR, glucose, glutamine, and uridine 5′-diphospho-N-acetylglucosamine in the tumor and a decrease in succinate, aspartate, and nucleotide diphosphates.

Article Snippet: EL4 mouse lymphoma cells were obtained as a gift from Dr. Jonathan Powell’s laboratory (Johns Hopkins University, Baltimore, MD).

Techniques:

Journal: Current drug metabolism

Article Title: Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement

doi: 10.2174/1389200222666210831125041

Figure Lengend Snippet: Representative high-resolution mass spectrum and chromatographic spectra of FGAR and IS. (A) High-resolution full scan mass spectrum of FGAR in negative mode with < 2 ppm error; (B) high-resolution full scan mass spectrum of internal standard (NAA-d3) in negative mode with < 2 ppm error; (C) extracted chromatogram of FGAR spiked in plasma at LLOQ (0.03 nmol/mL); (D) extracted chromatogram for internal standard (NAA-d3) in plasma; (E) extracted chromatogram of FGAR spiked in tumor at LLOQ (1 nmol/g); (F) extracted chromatogram for internal standard (NAA-d3) in EL4 tumor.

Article Snippet: EL4 mouse lymphoma cells were obtained as a gift from Dr. Jonathan Powell’s laboratory (Johns Hopkins University, Baltimore, MD).

Techniques: Clinical Proteomics

Journal: Current drug metabolism

Article Title: Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement

doi: 10.2174/1389200222666210831125041

Figure Lengend Snippet: FGAR quantification following GA-607 treatment (3.2 mg/kg SC daily for 4 days) in EL4 tumor-bearing mice. (A) (i) FGAR levels in plasma at 1 and 4 hours after GA-607 or vehicle administration; (ii) FGAR levels in tumor at 1 and 4 hours after GA-607 or vehicle administration. Mean ± S.D. ***p > .001 (two-way ANOVA with Tukey’s post hoc test) (B) Schematic representation of the biochemical pathway regulating FGAR metabolism in tumor tissues and the effect of GA therapy. FGAR-AT, an enzyme in de novo purine synthesis catalyzes the conversion of FGAR, ATP, and glutamine to FGAM, ADP, Pi, ammonia, and glutamate, respectively. Upon cleavage in tumors, GA-607 releases DON. The inhibition of FGAR-AT results in elevated FGAR levels.

Article Snippet: EL4 mouse lymphoma cells were obtained as a gift from Dr. Jonathan Powell’s laboratory (Johns Hopkins University, Baltimore, MD).

Techniques: Clinical Proteomics, Inhibition

Journal: Scientific Reports

Article Title: In vivo imaging of T cell lymphoma infiltration process at the colon

doi: 10.1038/s41598-018-22399-2

Figure Lengend Snippet: Localization of EL4-EGFP cells in the blood vessels adjacent to crypts in the colon of C57BL6/J mice. ( A ) EL4 cells stably expressing EGFP in vitro under fluorescence microscopy (LSM710, Carl Zeiss). Bar indicates 50 μm. ( B ) EGFP fluorescence intensity measured using a cell analyzer (SH800, Sony). ( C ) At 1 to 3 weeks after EL4-EGFP cell injection, the colon was removed from the body and observed on living tissues. ( D ) EL4-EGFP cell imaging of in the colon. Green color indicates EGFP of EL4-EGFP cells, as shown with white arrows. Blood vessels were stained with rhodamine B–conjugated dextran (M.W. 70,000) (red). Bar indicates 50 μm. Right upper panel shows an enlarged image of an elongated EL4 cell. Bar indicates 10 μm. Right lower panel shows an EL4 cell lodged in the T-junction of blood vessels. Bar indicates 10 μm. These images were representative from 3 mice examined. ( E ). Imaging of EL4-EGFP cells localized in large blood vessels under the crypts in the mucosal layer. EL4-EGFP cells are indicated by white arrows. Bar indicates 50 μm. ( F ). Crypts visualized using green mice (C57BL/6-Tg[CAG-EGFP]). Crypts and blood vessels are shown in green and red, respectively. Bar indicates 50 μm.

Article Snippet: EL4 mouse lymphoma cells were purchased from Japanese Collection of Research Bioresources.

Techniques: Stable Transfection, Expressing, In Vitro, Fluorescence, Microscopy, Injection, Imaging, Staining

Journal: Scientific Reports

Article Title: In vivo imaging of T cell lymphoma infiltration process at the colon

doi: 10.1038/s41598-018-22399-2

Figure Lengend Snippet: Localization of EL4-DsRed2 cells in blood vessels adjacent to crypts in the colon of green mice. ( A ) EL4 cells stably expressing DsRed2 in vitro observed under a fluorescence microscope. Bar indicates 50 μm. (pCAG-DsRed2 sequence is in supplementary data2). ( B ) DsRed2 fluorescence intensity measured using a cell analyzer (SH800, Sony). ( C ) Imaging of EL4-DsRed2 cells in the colon. Red color indicates DsRed2 in EL4-DsRed2 cells, as shown by white arrowheads. Green color mainly shows endothelial cells of blood vessels. Bar indicates 50 μm. This image was representative from 4 mice examined. ( D ) Analyses of the diameter of EL4-EGFP cells in vitro and in colonic blood vessels. Error values and bars indicate standard error (S.E.). ( E ) Hematoxylin and eosin staining and fluorescence analysis of the infiltration of EL4 cells into colon tissues. EL4 cells (5 × 10 5 ) were injected into 9-week-old C57BL6/J mice via the tail vein. The colon was removed and fixed with 4% paraformaldehyde at 14 days after EL4 cell injection. The colon tissue was stained with hematoxylin and eosin. Bar indicates 50 μm. ( F ) Infiltration of EL4 cells was also examined by fluorescence microscopy with the same sample used in ( E ).

Article Snippet: EL4 mouse lymphoma cells were purchased from Japanese Collection of Research Bioresources.

Techniques: Stable Transfection, Expressing, In Vitro, Fluorescence, Microscopy, Sequencing, Imaging, Staining, Injection

Journal: Scientific Reports

Article Title: In vivo imaging of T cell lymphoma infiltration process at the colon

doi: 10.1038/s41598-018-22399-2

Figure Lengend Snippet: Time-lapse imaging of EL4-EGFP cells in colonic blood vessels. ( A ) Time-lapse (20-min intervals) imaging of EL4-EGFP cells (green) in small blood vessels (red). EL4-EGFP cells (5 × 10 5 ) were injected into the tail vein of C57BL6/J mice and observed 10 days after injection. White arrows show lodged EL4-EGFP cells. Blue arrows indicate moving EL4 cells. Bar indicates 100 μm. ( B ) Tracking analysis of EL4-EGFP cell movement for 1 h. Traces of moving cells are shown in red. Bar indicates 100 μm. ( C ). Ratio of moving and lodged cells determined at 20-min intervals. A total of 23.3 ± 0.32% of the EL4 cells exhibited motility. Analyses were performed from 3 mice studied in the early stage of metastasis.

Article Snippet: EL4 mouse lymphoma cells were purchased from Japanese Collection of Research Bioresources.

Techniques: Imaging, Injection

Journal: Scientific Reports

Article Title: In vivo imaging of T cell lymphoma infiltration process at the colon

doi: 10.1038/s41598-018-22399-2

Figure Lengend Snippet: Time-lapse imaging of highly motile EL4-EGFP cells in colonic blood vessels. ( A ) Dynamics of EL4 cells examined at subcellular resolution. Migration of two EL4 cells is shown with blue and orange lines. Bar indicates 50 μm. ( B ) Analysis of distance traversed by EL4 cells each min. ( C ). Observation of two individual EL4 cells in small vessel in the colon. Note that we observed a pseudopod (white arrow) extending from one EL4 cell (blue arrow) and touching the other cell (orange arrow). Bar indicates 50 μm.

Article Snippet: EL4 mouse lymphoma cells were purchased from Japanese Collection of Research Bioresources.

Techniques: Imaging, Migration

Journal: Scientific Reports

Article Title: In vivo imaging of T cell lymphoma infiltration process at the colon

doi: 10.1038/s41598-018-22399-2

Figure Lengend Snippet: Formation of small nodules composed of several EL4-EGFP cells in crypts and blood vessels. ( A ) 3D image of a small nodule composed of EGFP-EL4 cells that infiltrated into crypts, as shown by a white arrow. Bar indicates 20 μm. ( B ) Image of a small EGFP-EL4 cell nodule in a blood vessel. Bar indicates 20 μm. This image is representative from 3 mice examined. ( C ). Percentage of EL4-EGFP nodules in crypts and blood vessels. ( D ). Schematic representation of the formation of nodules composed of EL4-EGFP cells. Green and red color indicate EL4-EGFP cells and blood vessels, respectively.

Article Snippet: EL4 mouse lymphoma cells were purchased from Japanese Collection of Research Bioresources.

Techniques: