Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: Generation of iPSCs and NPCs from human skin fibroblasts (NB1RGB). ( A ) To identify the PSC status of the established iPSC clone 2 (C2) from human skin fibroblasts (NB1RGB), cells were immunostained using PSC markers Nanog, SSEA4, OCT4 and KLF4. The cell nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ( B ) iPSC-like colonies were stained with alkaline phosphatase staining. ( C ) To confirm pluripotency, iPSCs were differentiated into three germ layers and stained with germ-layer markers Otx2, Brachury and Sox17 for the ectoderm, mesoderm and endoderm, respectively. ( D ) iPSCs were differentiated to neural progenitor cells (NPCs) and stained with neural stem cell markers GFAP, Nestin, Pax6, Sox1 and Sox2 and the differentiated neuron marker β-III Tubulin (Tuj1). NPCs were positive for all the NPC markers except Tuj1. Scale bar represents 25 μm or 50 μm, as indicated. iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, PSC, pluripotent stem cell = DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Staining, Marker

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: DDR after IR in iPSCs and NPCs. ( A ) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 2 Gy and fixed with 4% PFA at designated times. Cells were immunostained with 53BP1 (green) and γ-H2AX (red) antibodies. The cell nucleus was counterstained with DAPI. ( B ) Immunostained cells containing >10 53BP1- and γ-H2AX-positive foci (dot foci) were counted and graphed. ( C ) γ-H2AX apoptotic foci (pan staining) were also counted. At least 200 cells were counted and all experiments were performed three times. Scale bar represents 25 μm. Error bars represent the standard error of the mean (SEM). Welch’s (one-tailed) t -test was performed and statistical significance is indicated by asterisks ( * P < 0.05; ** P < 0.01; *** P < 0.001). DDR = DNA damage response, IR = ionizing radiation, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, PFA = paraformaldehyde, 53BP1 = p53 binding protein 1, γ-H2AX = γ-H2A histone family member X, DAPI = 4′,6-diamidino-2-phenylindole.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Irradiation, Staining, One-tailed Test, Binding Assay

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: Apoptosis detection by TUNEL assay in iPSCs. ( A ) Fibroblasts (NB1RGB) and iPSCs (NB1RGB C2) were irradiated at 2 Gy and fixed at designated times after IR. Apoptosis was detected via the TUNEL assay. ( B ) At least 200 cells were counted and all experiments were performed three times and graphed. Scale bar represents 25 μm. Error bars represent the SEM. Welch’s (one-tailed) t -test was performed and statistical significance is indicated by asterisks ( * P < 0.05). iPSCs = induced pluripotent stem cells, TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling, SEM = standard error of the mean.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: TUNEL Assay, Irradiation, One-tailed Test

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: RNA-Seq analysis revealed transcriptional alteration in fibroblasts, iPSCs and NPCs. ( A ) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 5 Gy. One hour after IR, RNA samples were extracted and analyzed using the Illumina HiSeq 2500 next-generation sequencer for RNA-Seq. Raw data were quality controlled and aligned to the reference hg19. Heat maps classed as HR repair, NHEJ repair and apoptosis were obtained using R software. Red, upregulated; green, downregulated. ( B ) Differential expression patterns of each gene were represented by FPKM. Expression differences were classified according to molecular machinery components: DNA repair, cell cycle checkpoints and apoptosis. ( C ) Differential expression patterns by RNA-Seq were confirmed by immunoblotting. ATM, NBS1, CHK1, p53 and p21 antibodies were used for immunoblotting. GAPDH antibody was used as loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in and . RNA-Seq = RNA sequencing, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, HR = homologous recombination, NHEJ = nonhomologous end joining, FPKM = fragments per kilobase of exon per million reads mapped.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Irradiation, Software, Quantitative Proteomics, Expressing, Western Blot, Control, Homologous Recombination

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: IR sensitivity and persistent activation of DDR after IR in iPSCs . ( A ) To determine cell sensitivity to IR exposure, colony formation assay was performed. Fibroblasts (NB1RGB) and iPSCs (NB1RGBC2 and 201B7) were used. Compared with fibroblasts, iPSCs showed hypersensitivity to IR exposure. Error bars represent the SEM. ( B ) After IR exposure, cell extracts were immunoblotted with ATM, p53, serine 15 phospho-p53, KAP1 and serine 824 phospho-KAP1 antibodies. GAPDH antibody was used as a loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in . DDR hyperactivation increased in a time-dependent manner in iPSCs but not in fibroblasts and NPCs. IR = ionizing radiation, DDR = DNA damage response, iPSCs = induced pluripotent stem cells, SEM = standard error of the mean, ATM = ataxia-telangiectasia-mutated, KAP1 = Kruppel-associated box domain (KRAB)-associated protein-1, GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Colony Assay, Control, Western Blot

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 1. Overexpression of Mov10 decreases HIV-1 infectivity. (A) 293T cells were transfected with different amounts of Mov10 plasmid, and the expression of Mov10 was determined by Western blot. (B) 293T cells were transfected with 0.5 mg of either pCMV6-XL5 plasmid (control), Mov10 or APOBEC3G in the presence or absence of 0.5 mg vif as well as 1 mg HIV-1-GFP (Denv, Dvif, Dvpr, Dnef) and 0.5 mg p-L-VSV-G. Virus was collected 24 h later, and then added to Jurkat T cells. Virus transfer was standardized across treatment conditions by p24 levels as described (Materials and Methods). Percent infected cells was then determined using FACS analysis for GFP-expression after virus was allowed to incubate with target cells for 72 hours. Error bars represent one standard deviation. doi:10.1371/journal.pone.0009081.g001

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Over Expression, Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Virus, Standard Deviation

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 2. Mov10 decreases specific infectivity of HIV-1. Supernatants of 293T cells that had been transfected with varying amounts of Mov10- expressing plasmid were assayed for (A) HIV-1.Luc p24 levels and (B) after standardization by p24 content, infectivity of target cells was determined by luciferase activity from VSVG.HIV.Luc, which encodes all the HIV accessory genes. For simplicity, the amount of Mov10 plasmid that was transfected is expressed as a ratio of HIV-1 plasmid to Mov10 plasmid and plotted logarithmically. In the experiment (see Materials and Methods for further explanation) HIV-1 plasmid levels remained constant, while Mov10 plasmid was used at levels of 1/6 to 1/1500 that of the HIV-1 plasmid. Error bars represent one standard deviation. (C) Representative plots of Jurkat T cells infected with GFP-expressing virus produced in the presence of either pcDNA3 or Mov10 (ratio of Mov10 to HIV-1 plasmid was 1/25) three days after infection. Quantification of experiments performed using GFP- expressing virus is shown in supplemental figure 1. doi:10.1371/journal.pone.0009081.g002

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Virus, Produced

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 3. Mov10 impairs HIV-1 replication in primary CD4+ T cells. Supernatants of activated CD4+ cells that had been nucleofected with a replication competent, CCR5-tropic HIV-1 viral plasmid and either Mov10 or control (pcDNA3) plasmid ([A] schematic) were assayed for p24 production (B) and then standardized by p24 concentration and used to infect CCR5+ Hut cells. (C) Infection success was determined by flow cytometry analysis of GFP expression. doi:10.1371/journal.pone.0009081.g003

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Plasmid Preparation, Control, Concentration Assay, Infection, Flow Cytometry, Expressing

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 5. An optimal concentration of Mov10 is required for HIV-1 infectivity. 293T cells were transfected with a non-targeting siRNA or Mov10-specific siRNA. At 48 h post-siRNA transfection, the cells were transfected with 1.5 mg of pHIV-RFP, 0.7 mg of p-L-VSV-G and increasing amounts of the Mov10 expression plasmid. At 96 h post-siRNA transfection: (A) the cell lysates were examined by Western blot for Mov10 levels using an anti-Mov10 antibody. Equal protein loading was confirmed by probing with anti-tubulin antibody. (B) After normalizing for p24 values, virus obtained from the transfections was used to infect HeLa cells and infectivity was measured by FACS. The % infected cells represents the percentage of RFP-positive cells in the cell population. Error bars represent one standard deviation. doi:10.1371/journal.pone.0009081.g005

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Concentration Assay, Infection, Transfection, Expressing, Plasmid Preparation, Western Blot, Virus, Standard Deviation

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 4. Broad inhibition of infectious retroviruses by Mov10. Virions derived from 293T cells transfected with various viral plasmids (as described in Materials and Methods) and either pcDNA3 or pcDNA3- Mov10 were used to infect HeLa cells. The % infected cells represents the percentage of GFP-positive cells in the cell population. doi:10.1371/journal.pone.0009081.g004

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Inhibition, Derivative Assay, Transfection, Infection

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 6. Viral glycoprotein incorporation is not affected by Mov10 overexpression. 293T cells were transfected with plasmids necessary for the production of VSV-G pseudotyped Virion-Like Particles (VLPs) containing a GFP tag (as a Gag-GFP fusion) as well as either control or Mov10. Supernatant from these cells was then collected and added to Jurkat T cells and assayed for binding to target cells. Cells bound by one or more VLPs are GFP+ by FACS. doi:10.1371/journal.pone.0009081.g006

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Over Expression, Transfection, Control, Binding Assay

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 7. Early and late reverse transcription is suppressed in virus produced from cells overexpressing Mov10. Synthesis of reverse transcripts by real-time PCR after infection by HIV-1 produced in the presence of either empty vector (pcDNA3) or Mov10 expressing plasmid was measured. No significant difference between production of (A) Early (R-U5) or (B) Late (R-Gag) DNA products of reverse transcription was observed. (C) Infectivity of virions produced in the presence of Mov10 was significantly inhibited. Input viruses were normalized for p24. Results are representative of one out of three similar experiments. doi:10.1371/journal.pone.0009081.g007

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Reverse Transcription, Virus, Produced, Real-time Polymerase Chain Reaction, Infection, Plasmid Preparation, Expressing

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 8. The helicase domain of Mov10 is not required for HIV-1 restriction. Virions were produced by cotransfection of 293T cells with HIV-1 vector and either empty vector or Mov10, Mov10 N-terminus, Mov10 C-terminus or the putative helicase motif mutant expression plasmids. (A) Schematic of wild-type and mutated human Mov10 constructs. (B) Cell lysates were probed with anti-HA, anti-Mov10, and anti-tubulin. Lanes: 1) pcDNA3, 2) Mov10, 3) Mov10 Nterm, 4) Mov10 Cterm, and 5) Mov10-EQ. Arrows indicate molecular weight of native Mov10. (C) Infectivity of virions produced was examined by FACS after infection of HeLa cells. The % infectivity represents the percentage of RFP-positive cells in the cell population. Error bars represent one standard deviation. doi:10.1371/journal.pone.0009081.g008

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Produced, Cotransfection, Plasmid Preparation, Mutagenesis, Expressing, Construct, Molecular Weight, Infection, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cloning, expression, and functional characterization of TL1A-Ig.

doi: 10.4049/jimmunol.1201908

Figure Lengend Snippet: FIGURE 2. Characterization of purified TL1A-Ig by SDS-PAGE, Western blot, and gel filtration. (A) Purified TL1A-Ig was quantified using an mIgG-targeted ELISA. The nonreduced (22-ME) and reduced (+2-ME) TL1A-Ig samples were separated on SDS gel and stained with Coomassie blue or analyzed by immunoblotting using Abs to mTL1A or mIgG (B) SDS-PAGE separation of reduced TL1A-Ig after enzymatic deglycosyla- tion. Protein samples were treated with glycanases and separated on SDS- PAGE followed by staining with Coomassie blue stain. Denatured TL1A- Ig was loaded in each lane. Lane 1, Untreated control; lane 2, treated with N-Glycanase only; lane 3, treated with N-Glycanase and Sialidase A; and lane 4, treated with N-Glycanase, Sialidase A, and O-Glycanase. (C and D) Gel filtration analysis and model of expected hexameric oligomerization of TL1A-Ig. Purified TL1A-Ig (100 mg) was applied on a Superdex 200 column and subjected to size exclusion chromatography. The apparent molecular mass was calculated based on the elution volumes of the standards thyroglobulin (670 kDa), aldolase (158 kDa), and OVA (44 kDa).

Article Snippet: The mTL1A cDNAwas purchased from OriGene and the extracellular domain (ECD), containing amino acids 68–252, was PCR amplified using primers that were located upstream and downstream of the cloning sites.

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, SDS-Gel, Staining, Control, Size-exclusion Chromatography

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cloning, expression, and functional characterization of TL1A-Ig.

doi: 10.4049/jimmunol.1201908

Figure Lengend Snippet: FIGURE 3. In vitro functional activity of TL1A-Ig. (A) P815 cells were stably transfected with expression vectors encoding mTNFRSF25. TNFRSF25- P815 cells were incubated with isotype mIgG1, TL1A-Ig (in culture supernatant or purified), or 4C12 and stained with fluorochrome-conjugated anti-IgG. TNFRSF25+ cells were then visualized by flow cytometry. (B) TNFRSF25-P815 cells were exposed to titrating concentrations of IgG1, TL1A-Ig, or 4C12. Cells were directly incubated with caspase substrate solution and free rhodamine 110 was determined fluorometrically. These data are representative of more than five experiments. (C) CD4+ T cells were purified from FIR mice and activated using plate-bound anti-CD3 for 4 d. Cells were harvested and incubated with isotype IgG or TL1A-Ig followed by staining with FITC anti-mouse IgG. mTL1A-Ig–bound TNFRSF25 was analyzed using flow cytometry by gating on CD4+FIR2 Tconvs and CD4+FIR+ Tregs. (D) CD4+CD252 Tconvs or (E) CD4+FIR+ Tregs were cultured in proliferation assays with indicated stimuli: anti- CD3 (2 mg/ml; 2C11), mIL-2 (10 U/ml), 4C12 (10 mg/ml), or purified TL1A-Ig (0.1 mg/ml) in triplicates for each condition. Data are representative of three independent experiments. (F) CD4+FIR+ Tregs were cultured in titrating concentration of cyclosporin A. Cultures in (D)–(F) were pulsed with [3H]thymidine for the last 6 h of 72-h incubation and incorporated isotope was measured by liquid scintillation counting. (G) For iTreg induction, FIR Tconvs were cultured with plate-bound anti-CD3, TGF-b, retinoic acid, mIL-2, and 4C12 or TL1A-Ig, and OT-II Tconvs were cultured as above plus 1:2 APCs and OVA. Cultures were analyzed for Foxp3+ cells in the CD4 gate. One representative analysis of three independent experiments is shown. ***p , 0.001 versus appropriate control. Significance was determined by one-way ANOVA with Tukey posttest (D, E). Error bars indicate mean 6 SEM. NV, Not visible.

Article Snippet: The mTL1A cDNAwas purchased from OriGene and the extracellular domain (ECD), containing amino acids 68–252, was PCR amplified using primers that were located upstream and downstream of the cloning sites.

Techniques: In Vitro, Functional Assay, Activity Assay, Stable Transfection, Transfection, Expressing, Incubation, Staining, Cytometry, Cell Culture, Concentration Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cloning, expression, and functional characterization of TL1A-Ig.

doi: 10.4049/jimmunol.1201908

Figure Lengend Snippet: FIGURE 4. mTL1A-Ig stimulates rapid proliferation of CD4+Foxp3+ Tregs in vivo. (A) The time-related serum concentrations of 4C12 and TL1A-Ig in C57BL/6 mice (n = 3–5) was determined after a single i.p injection of 100 mg 4C12 or TL1A-Ig. Protein concentration was measured in serum samples collected at indicated time points by a sandwich ELISA specific for Armenian hamster IgG or TL1A. Concentration at each time point was calculated as a percentage of the average initial serum concentration (stable agonist concentration after equilibration between blood and tissues). Unique symbol represents each 4C12 treated (solid) mouse or TL1A-Ig (hollow) mouse. (B) The kinetics and dose-dependent expansion of Tregs in peripheral blood were determined after i.p injection of 100 mg IgG (n = 3) or TL1A-Ig (n = 4) in FIR mice for 3 consecutive days as indicated by the arrows. Mice were bled daily and the percentage of peripheral Tregs relative to total CD4+ cells was determined by flow cytometry. (C) Treg expansion was monitored in the peripheral blood while administering daily i.p injections of 100 mg IgG (n = 2) or TL1A-Ig (n = 4) starting on day 0 and ending on day 20. (D) Ten million CD4+ cells were highly purified by FACS sorting from FIR mice and adoptively transferred into CD742/2 or CD42/2 mice. After 3 d (day 0), recipient mice were treated with 100 mg TL1A-Ig or IgG followed by two consecutive doses on days 1 and 2. The percentage of Foxp3+ cells was analyzed in the spleen and pooled lymph nodes on day 6. Data are representative of two independent experiments, with two or more mice per group. Statistical analysis was performed by an unpaired two-tailed Student t test (B, C). All data are means 6 SEM (B–D). *p , 0.05, **p , 0.01, ***p , 0.001 versus control.

Article Snippet: The mTL1A cDNAwas purchased from OriGene and the extracellular domain (ECD), containing amino acids 68–252, was PCR amplified using primers that were located upstream and downstream of the cloning sites.

Techniques: In Vivo, Injection, Protein Concentration, Sandwich ELISA, Concentration Assay, Cytometry, Two Tailed Test, Control