Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: PLoS ONE

Article Title: Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function

doi: 10.1371/journal.pone.0173773

Figure Lengend Snippet: There was no difference in GFR of patients with predominantly methylated (>75% Foxp3 TSDR methylation) Treg and patients with a sizable proportion of demethylated (≤75% Foxp3 TSDR methylation) Treg (p>0.01; Mann-Whitney U test). Enriched IFNy+ and IFNy- Treg of male and female patients were tested separately using primer ADS783 (primer P1) and primer ADS3576 (primer P2).

Article Snippet: PCR was carried out in a 21 μl total volume containing: 2.1 μl PCR buffer, 0.27 μl MgCl 2 , 0.42 μl dNTPs, 1.05 μl SYBR Green dye (EpigenDx), 1.26 μl each of primers ADS783 and ADS3576 (Human Foxp3 Methylation Panel, EpigenDx), 0.12 μl Hot Start Taq polymerase (Qiagen), and 1.5 μl of bisulfite-treated genomic DNA (concentration 5 ng/μl).

Techniques: Methylation, MANN-WHITNEY

Journal: BioMed Research International

Article Title: RANKL Impairs the TLR4 Pathway by Increasing TRAF6 and RANK Interaction in Macrophages

doi: 10.1155/2022/7740079

Figure Lengend Snippet: RANKL promotes TRAF6 and RANK interaction in detriment to TLR4 in LPS-induced macrophages. (a) Representative images of RANK and TRAF6 colocalization by confocal microscopy in THP1 macrophages stimulated with LPS (10 ng/ml) and RANKL (10 ng/ml) and costimulated with both (LPS+RANKL) for 10 min or nonstimulated as the control group (CT), inset: colocalization scattergram graphs. The cells were labeled with RANK (green), TRAF6 (red), and DAPI (blue). (b) Quantification of protein interaction by RANK-TRAF6 immunofluorescence colocalization. Manders' correlation coefficient was used to calculate colocalization of the cell sections. A one-way ANOVA followed by Bonferroni's multiple comparisons test was performed to compare all groups, n = 6. All data are presented as mean ± SEM. ∗ p < 0.0001 vs. CT. # p < 0.0001 vs. LPS). (c, d, and e) Detection by western blot of RANK, TLR4, and TRAF6 co-immunoprecipitated with TRAF6 in protein extracts from THP1 macrophages stimulated with LPS (10 ng/ml) and RANKL (10 ng/ml) or costimulated with both (LPS+RANKL) for 10 min.

Article Snippet: Equivalent doses of proteins were then size-fractionated in 10% SDS-PAGE, transferred to a 0.45 μ m pore size PVDF Immobilon-P membrane (Millipore), subjected to 5% BSA blockage, and then incubated with the following antibodies: TLR4 (#ab183459, Abcam), RANK (#ab200369, Abcam), TRAF6 (#ab33915, Abcam), and β -actin (#sc-81178, Santa Cruz Biotechnologies.

Techniques: Confocal Microscopy, Control, Labeling, Immunofluorescence, Western Blot, Immunoprecipitation

Journal: Mediators of Inflammation

Article Title: The Long Noncoding RNA, LOC645166, in T Cells of Ankylosing Spondylitis (AS) Patients Regulates the FOXP3 Expression via the Axis of LOC645166/miR-188-5p/NFKBID

doi: 10.1155/mi/8574340

Figure Lengend Snippet: The effect of LOC645166/miR-188-5p/NFKBID axis on the protein levels of FOXP3 in Jurkat cells. (A) The effect of LOC645166 overexpressed in Jurkat cells on the protein levels FOXP3 ( N = 4). (B) The effect of miR-188-5p electro-transferred in Jurkat cells on the FOXP3 expression ( N = 4). (C) The effect of miR-188-5p co-transfected with its inhibitor in Jurkat cells on the protein levels of FOXP3 ( N = 4). (D) The effect of NFKBID overexpressed in Jurkat cells on protein levels of FOXP3 ( N = 4). (E) The effect of NFKBID knocked down by siRNA on the protein levels of FOXP3 ( N = 4).

Article Snippet: To examine the effect of NFKBID on the expression of FOXP3, the NFKBID mRNA of Jurkat cells was knocked down by using siRNA (sc-97928, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection

Journal: Mediators of Inflammation

Article Title: The Long Noncoding RNA, LOC645166, in T Cells of Ankylosing Spondylitis (AS) Patients Regulates the FOXP3 Expression via the Axis of LOC645166/miR-188-5p/NFKBID

doi: 10.1155/mi/8574340

Figure Lengend Snippet: The expression levels of FOXP3 and Treg cell frequencies in CD4 + T cells of AS patients and healthy control. (A) The levels of FOXP3 in CD4 + T cells of AS patients and healthy controls ( N = 5). (B) Flow cytometry analysis of the Treg cell frequencies in CD4 + T cells of AS patients ( N = 14) and healthy controls ( N = 13).

Article Snippet: To examine the effect of NFKBID on the expression of FOXP3, the NFKBID mRNA of Jurkat cells was knocked down by using siRNA (sc-97928, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Flow Cytometry

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Naive CD3 + CD4 + CD44 − CD62L + Foxp3 − naive T cells were isolated from the spleens of WT Foxp3 GFP and cultured for 72 h in the presence of CD3/CD28 beads, IL-2 and TGF-β. Culture plates were coated with 5 µg/mL PD-L2Fc or control IgG1Fc for 30 min prior culture. B Representative flow cytometry plots of Foxp3 induction in CD3 + CD4 + CD24 + Foxp3 GFP+ iTregs after 72 h of culture. C Absolute numbers of iTregs after 72 h of culture, presented as mean ± SEM. n = 3 biologically independent samples. D Foxp3 mean fluorescence intensity (MFI) of iTregs after 72 h of culture, presented as mean ± SEM. n = 3 biologically independent samples. E IL-10 secretion by iTregs after 72 h of culture, presented as the mean ± SEM. n = 8 biologically independent samples. F Following culture, cells were transferred to PDL-coated HS mini plates and a Mito Stress test was performed to measure oxygen levels in the culture medium. Oxygen consumption rate (OCR) in response to Oligomycin (oligo.), FCCP and Rotenone (Rot/AA) sequential injections. n = 3 biologically independent samples. G Basal respiration in PD-L2Fc or control IgG1Fc groups. H Spare respiratory capacity in PD-L2Fc or control IgG1Fc groups. I ATP production in PD-L2Fc or control IgG1Fc groups. J Schematic of the Foxp3 promoter region, showing the intron 1 TSDR region containing CpG#19, CpG#20, CpG#21, and CpG#22 within the CNS2. K Average methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. L Methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. n = 4 biologically independent samples. Data are representative of 2 independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Isolation, Cell Culture, Control, Flow Cytometry, Fluorescence, Methylation, Two Tailed Test

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Thymus of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were collected and analyzed by flow cytometry. Representative plots of CD25 + (CD25 + TregP cells) and Foxp3 lo Treg cell progenitors (Foxp3 lo TregP cells) analyzed based on CD25 and Foxp3 GFP expression. B Corresponding quantitation presented as mean ± SEM. n = 3 biologically independent mice. C Spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were collected and analyzed by flow cytometry. Representative plots of CD3 + CD4 + CD25 + Foxp3 GFP+ total Tregs. D Numbers of total Tregs in WT and PD-L2 KO mice presented as mean ± SEM. n = 5 biologically independent mice. E BALB/c (WT) BMDC were differentiated in vitro for 7–10 days, plated and further cultured with or without 50 ng/mL IL-4 for 24 h to measure PD-L2 expression by flow cytometry. F Representative plots of CD11c + BMDC, expression of PD-L2. G Numbers of PD-L2-expressing CD11c + BMDC presented as mean ± SEM. n = 4 biologically independent samples. H PD-L2 low and PD-L2 high BMDCs were intravenously injected or not to WT or PD-L2 KO Foxp3 GFP mice on day 0, and on day 3 the spleens of recipient mice were analyzed for CD3 + CD4 + CD25 + Foxp3 + total Tregs by flow cytometry. I Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ total Tregs per spleen at day 3 following BMDC transfer. n = 3 biologically independent mice. J – M PD-L2 high or PD-L2 null BMDCs were i.v. injected or not to WT or PD-L2 KO Foxp3 GFP mice on day 0, and on day 3 the spleens of recipient mice were analyzed for Tregs by flow cytometry. J Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ total Tregs. K Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − pTregs. L Numbers of CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 + tTregs. M Thymus of recipient mice were collected and analyzed by flow cytometry, and the frequencies of CD25 + TregP cells and Foxp3 lo TregP cells was analyzed based on CD25 and Foxp3 GFP expression, presented as mean ± SEM. n = 3 biologically independent mice. Data are representative of three independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse image provided with permission from Servier Medical Art.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Flow Cytometry, Expressing, Quantitation Assay, In Vitro, Cell Culture, Injection, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were collected and analyzed by flow cytometry. Representative plots of CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − pTregs and CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 + tTregs. B Quantitation of pTreg and tTreg numbers in the spleen presented as means ± SEM. n = 5 biologically independent mice. C – D pTregs and tTregs—gated as in ( A )—were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and cultured for 24 h with CD3/CD28 beads, IL-2 and TGF-β. IL-10 was measured by ELISA in the culture supernatants. C Levels of IL-10 in pTreg cultures. n = 3 biologically independent samples. D Levels of IL-10 in tTreg cultures. E CD3 + CD4 + CD44 − CD62L + naive T cells were isolated from the spleens of BALB/c mice and co-cultured with FACS-sorted CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs isolated from Foxp3 GFP or PD-L2 KO Foxp3 GFP mice for 72 h. Cell proliferation was assessed by measuring thymidine incorporation. F 3 H thymidine incorporation after 72 h of culture. n = 4 biologically independent samples. G CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β. Genomic DNA was extracted, followed by bisulfite conversion, purification, cloning, and the degree of methylation of CpG#19, CpG#20, CpG#21, and CpG#22 within the TSDR region was determined by bisulfite sequencing. H Foxp3 GFP expression within WT and PD-L2 KO pTregs. n = 3 biologically independent samples. I Average methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. n = 3 biologically independent samples. J Methylation of CpG#19, CpG#20, CpG#21, and CpG#22 in TSDR region. K 10 6 WT Foxp3 GFP iTregs were adoptively transferred to WT BALB/c or PD-L2 KO hosts on day 0 and on day 3, the frequencies of GFP + Tregs were analyzed in the spleen of recipients. L Representative plots of CD3 + CD4 + CD25 + Foxp3 GFP+ Tregs. M Numbers of transferred Tregs in the spleen presented as mean ± SEM. n = 3 biologically independent mice. Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse image provided with permission from Servier Medical Art.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Flow Cytometry, Quantitation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Incubation, Purification, Cloning, Methylation, Methylation Sequencing, Expressing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β. Cells were then lysed and mRNA was isolated to perform RNA-sequencing (RNA-seq). Volcano plot representation of differentially regulated genes in WT and PD-L2 KO pTregs. Dotted lines represent 2FC ( x axis) and p < 0.05 ( y axis) cutoffs. B – G Differentially regulated genes in selected pathways. B Treg function pathway. C Fatty acid (FA) degradation pathway. D Amino acid (AA) degradation pathway. E TCA cycle pathway. F Glycolysis pathway. G Pentose Phosphate Pathway (PPP) pathway. All red dots represent genes whose differences are p < 0.05. H – K CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTreg cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β. Following culture, cells were transferred to PDL-coated HS mini plates and a Mito Stress test was performed to measure oxygen levels in the culture medium. H Oxygen consumption rate (OCR) in response to Oligomycin (oligo.), FCCP, and Rotenone (Rot/AA) sequential injections. I Basal respiration in WT and PD-L2 KO pTregs. n = 3 biologically independent samples. J Spare respiratory capacity in WT and PD-L2 KO pTregs. K Respiration-coupled ATP production. L Representative flow cytometry plots of MitoTracker DR expression within WT and PD-L2 KO pTregs after 24 h of culture. M Quantitation of MitoTracker DR expression within WT and PD-L2 KO pTregs after 24 h of culture, presented as mean ± SEM. n = 3 biologically independent samples. N CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − splenic pTregs cells were FACS-sorted from the spleens of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and incubated for 24 h with CD3/CD28 beads, IL-2 and TGF-β with or without 2 mM methyl pyruvate. O Levels of IL-10 production in the culture supernatants. n = 3 biologically independent samples. H – O Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Incubation, Isolation, RNA Sequencing, Flow Cytometry, Expressing, Quantitation Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: PD-L2 controls peripherally induced regulatory T cells by maintaining metabolic activity and Foxp3 stability

doi: 10.1038/s41467-022-32899-5

Figure Lengend Snippet: A Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were i.n. challenged on days −10, −9 and −8 with 100 µg ovalbumin (OVA). On day 0, mice were intraperitoneally sensitized with 50 µg OVA emulsified in alum (1:1) and CD3 + CD4 + CD25 + Foxp3 GFP+ Nrp1 − pTreg numbers were analyzed by flow cytometry on day 7. B Representative plots of pTregs in the lungs of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice. C Numbers of pTregs in the lungs of Foxp3 GFP and PD-L2 KO Foxp3 GFP mice presented as mean ± SEM. n = 4 biologically independent mice. D Representative plots of PD-1 expression in lung pTregs from Foxp3 GFP and PD-L2 KO Foxp3 GFP . E Representative plots of Foxp3 expression in lung pTregs from Foxp3 GFP and PD-L2 KO Foxp3 GFP mice and corresponding quantitation presented as mean ± SEM. n = 3 biologically independent mice. F BALB/c (WT) and PD-L2 KO BMDC were differentiated in vitro for 7–10 days and cultured with FACS-sorted CD4 + CD44 − CD62L + KJ1-26 + naive T cells in the presence of IL-2, TGF-β, 100ng/mL OVA peptide and CD3/CD28 stimulation to induce Treg differentiation for 72 h at 1:20 ratio. G Representative plot of Foxp3 expression. H Foxp3 expression in T cells incubated with either WT or PD-L2 KO BMDC, presented as mean ± SEM. n = 3 biologically independent mice. I Levels of IL-10 in the supernatants, presented as mean ± SEM. n = 4 biologically independent samples. J Representative plots of BODIPY 493/503 expression in T cells incubated with either WT or PD-L2 KO BMDC and corresponding quantitation presented as mean ± SEM. n = 3 biologically independent samples. K Foxp3 GFP and PD-L2 KO Foxp3 GFP mice were challenged as in A . On days 7, 8, and 9 mice were further challenged or not with 100 mg/kg ethyl pyruvate intraperitoneally. L Lung resistance in response to 40 mg/mL of methacholine. M Numbers of CD11b + Ly6G + neutrophils (PMN), CD11c − SiglecF + eosinophils (eos.), CD11c + SiglecF + CD64 + macrophages (mac.) and CD3 + T cells in the BAL fluid. n = 3 biologically independent mice. Data are representative of two independent experiments, presented as means ± SEM. Source data are provided as a Source Data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse image provided with permission from Servier Medical Art.

Article Snippet: For the methylation analysis, Foxp3 DNA methylation was performed by bisulfite pyrosequencing by EpigenDX as described previously .

Techniques: Flow Cytometry, Expressing, Quantitation Assay, In Vitro, Cell Culture, Incubation, Two Tailed Test, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Serine protease inhibitor from the muscle larval Trichinella spiralis ameliorates non-alcoholic fatty liver disease in mice via anti-inflammatory properties and gut-liver crosstalk.

doi: 10.1016/j.biopha.2024.116223

Figure Lengend Snippet: Fig. 4. rTs-SPI attenuated macrophage infiltration and promoted CD4þCD25þFoxp3þ Tregs population in NAFLD mice. (A) Macrophage infiltration and (B) CD4+CD25+Foxp3+ Tregs in spleen using Flow cytometry analysis; Quantitative analysis of (C) F4/80+ macrophages and (D) CD4+CD25+Foxp3+ Tregs in spleen; (E) Microphotographs show total macrophages staining with F4/80 (red) and DAPI (blue) in liver (600x); (F) Quantitative analysis of F4/80+ macrophages in liver; The hepatic mRNA expression levels of (G) F4/80 and (H) Foxp3. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For Treg analysis, cells were stained with Percp/cy5.5 anti-mouse CD4 and APC anti-mouse CD25 antibodies (Elabscience, China), followed by fixing and permeabilizing using a Foxp3/transcription factor staining kit (Elabscience, China) and staining with PE anti-mouse Foxp3 antibody (Elabscience, China).

Techniques: Flow Cytometry, Staining, Expressing

Journal: The Indonesian Biomedical Journal

Article Title: 2-(3-(chloromethyl)benzoyloxy)benzoic Acid Increases CD4+ Regulatory T-Cell Population and FoxP3 Expression in Lipopolysaccharide-induced Mice

doi: 10.18585/inabj.v15i4.2429

Figure Lengend Snippet: Figure 3. Foxp3-expression in splenocytes of LPS- induced mice. Mice were induced with/without LPS and treated orally with/without ASA or 3-CH2Cl. Spleen of the mice were isolated, processed, and the splenic FoxP3 expression was measured with flow cytometry as described in Methods. A: Representative histogram with MFI value obtained from each group. B: MFI value of each group. **0.01≥p>0.001; ***0.001≥p>0.0001, tested with upaired t-test.

Article Snippet: Ten μg of APC Anti-Mouse Foxp3 Antibody (Elabscience) was added to the splenocytes.

Techniques: Expressing, Isolation, Flow Cytometry