Journal: Scientific Reports

Article Title: Osthol ameliorates obesity-associated lipid metabolic disorders by inhibiting ADRA1D-dependent Th17 cell differentiation

doi: 10.1038/s41598-025-20719-x

Figure Lengend Snippet: OST Attenuates Systemic Inflammation and Downregulates Th17-Related Factor Expression. ( A ) KEGG pathway enrichment analysis of downstream targets of OST. This figure is based on data from KEGG (Kyoto Encyclopedia of Genes and Genomes) at https://www.kegg.jp/ ; © Kanehisa Laboratories. ( B ) Flow cytometry was used to evaluate the proportion of CD4⁺IL-17A⁺ cells in the spleen. ( C ) Immunohistochemical staining of liver tissues was performed to assess IL-17A expression. ( D ) Immunofluorescence staining was conducted to detect CD4⁺RORγt⁺ double-positive cells in liver sections. ( E ) Serum levels of IL-6, IL-1β, TNF-α, and IL-17A were measured using ELISA. n = 6. **** P < 0.0001. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons.

Article Snippet: Cells were then fixed and permeabilized, followed by intracellular staining with a phycoerythrin (PE)-conjugated anti-IL-17A antibody (F21IL1702, Lianke Biotech, Hangzhou, China).

Techniques: Expressing, Flow Cytometry, Immunohistochemical staining, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Osthol ameliorates obesity-associated lipid metabolic disorders by inhibiting ADRA1D-dependent Th17 cell differentiation

doi: 10.1038/s41598-025-20719-x

Figure Lengend Snippet: OST Inhibits Th17 Cell Differentiation in Vitro. ( A ) Flow cytometry was used to assess the proportion of CD4⁺IL-17A⁺ cells in a Th17-polarized cell model treated with different doses of OST. ( B ) RT-qPCR was performed to evaluate mRNA expression levels of RORγt and IL-17A. ( C ) Western blot was performed to evaluate protein expression levels of RORγt, IL-17A, IL-17RA, TRAF6, and Act1. ( D ) ELISA was used to quantify the secretion of IL-6, IL-1β, TNF-α, IL-10, and TGF-β in the culture supernatants. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons.

Article Snippet: Cells were then fixed and permeabilized, followed by intracellular staining with a phycoerythrin (PE)-conjugated anti-IL-17A antibody (F21IL1702, Lianke Biotech, Hangzhou, China).

Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Osthol ameliorates obesity-associated lipid metabolic disorders by inhibiting ADRA1D-dependent Th17 cell differentiation

doi: 10.1038/s41598-025-20719-x

Figure Lengend Snippet: OST Suppresses Th17 Differentiation via an ADRA1D-Dependent Mechanism. ( A ) Volcano plot of differentially expressed genes (DEGs) in the GSE110729 obesity dataset. ( B ) Venn diagram showing the intersection between OST’s potential targets and DEGs from the GSE110729 dataset. ( C ) RT-qPCR was performed to assess the expression of ADRA1D, IL-17A, and RORγt in Th17-polarized CD4⁺ T cells with or without ADRA1D overexpression. ( D ) Western blot was conducted to evaluate the protein expression of ADRA1D, IL-17A, RORγt, IL-17RA, TRAF6, and Act1. ( E ) ELISA was used to measure the levels of IL-6, IL-1β, TNF-α, IL-10, and TGF-β in the culture supernatants. n = 3. *** P < 0.001, **** P < 0.0001. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons.

Article Snippet: Cells were then fixed and permeabilized, followed by intracellular staining with a phycoerythrin (PE)-conjugated anti-IL-17A antibody (F21IL1702, Lianke Biotech, Hangzhou, China).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Osthol ameliorates obesity-associated lipid metabolic disorders by inhibiting ADRA1D-dependent Th17 cell differentiation

doi: 10.1038/s41598-025-20719-x

Figure Lengend Snippet: ADRA1D Overexpression Attenuates the Metabolic and Immunomodulatory Effects of OST in Vivo. ( A ) Body weight changes were monitored in HFD-fed mice treated with OST. (B) Weights of adipose tissue and liver were measured to assess the effect on organ hypertrophy. ( C ) Serum levels of TG, TC, FFA, ALT, and AST were evaluated by biochemical assays. ( D ) Flow cytometry was performed to assess the proportion of CD4⁺IL-17A⁺ cells in the spleen. ( E ) Immunofluorescence staining of liver tissue was used to detect CD4⁺RORγt⁺ double-positive cell infiltration. ( F ) Immunohistochemistry was performed to evaluate hepatic IL-17A protein expression. ( G ) Western blotting was conducted to assess ADRA1D protein expression in liver tissue. ( H ) Western blot was used to detect the phosphorylation levels of ERK1/2 and PI3K in liver tissue. ( I ) H&E staining of adipose tissue was used to assess adipocyte morphology. (J) Oil red O staining of liver sections was performed to evaluate hepatic lipid deposition. n = 6. ** P < 0.01, **** P < 0.0001. For three or more groups, one-way or two-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant.

Article Snippet: Cells were then fixed and permeabilized, followed by intracellular staining with a phycoerythrin (PE)-conjugated anti-IL-17A antibody (F21IL1702, Lianke Biotech, Hangzhou, China).

Techniques: Over Expression, In Vivo, Flow Cytometry, Immunofluorescence, Staining, Immunohistochemistry, Expressing, Western Blot, Phospho-proteomics

Journal: Cell reports

Article Title: METTL14-dependent m 6 A modification controls iNKT cell development and function

doi: 10.1016/j.celrep.2022.111156

Figure Lengend Snippet: (A) Representative histogram of CD1d expression on DP thymocytes. CD1d was stained with α-CD1d or isotype control antibody (n = 8). (B) IL-2 detected by ELISA following 48-h co-culture of iNKT cell hybridoma DN32.D3 with irradiated thymocytes pulsed with α-GalCer ranging from 200 ng/mL to 12.5 ng/mL. Data representative of three independent experiments. (C) Flow cytometric analysis of iNKT cells in the Jα18 −/− recipient mice after 6 weeks of reconstitution with 1:1 mixture of bone marrow cells from WT (CD45.1) and T- Mettl14 −/− (CD45.2). (D) Quantification of iNKT cell reconstitution in thymus, spleen, and liver in bone marrow chimera recipients (n = 7). SEM is shown. ***p < 0.001.

Article Snippet: Purified rat anti-mouse IL-2 , BD Biosciences , RRID: AB_395383.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Irradiation

Journal: Cell reports

Article Title: METTL14-dependent m 6 A modification controls iNKT cell development and function

doi: 10.1016/j.celrep.2022.111156

Figure Lengend Snippet: (A) Relative expression of Cish in naive and activated (anti-CD3/anti-CD4) DP thymocytes (n = 5–9). (B) Relative expression of Cish in the m 6 A RNA immunoprecipitation of total thymocytes RNA in WT mice by qPCR quantification (n = 4). (C) Intracellular calcium flux in DP thymocytes in response to crosslinking of anti-CD3/anti-CD4 in T- Mettl14 −/− mice. (D and E) Quantification of maximum calcium flux on crosslinking and addition of Ca 2+ in T- Mettl14 −/− DP cells (n = 5–6). (F) Representative staining of thymic iNKT cell expansion at day 3 after stimulation with IL-2 or IL-15. (G and H) Percentage and cell number of thymic iNKT cells on D0 and D3 after stimulation with IL-2 or IL-15 (n = 5). (I) Representative figure of cell trace distribution in thymic iNKT cells on day 3 post-stimulation with IL-2 or IL-15 (n = 5–6). Unstimulated thymocytes were used as controls. (J) Bar graph of pSTAT5 in iNKT thymocytes after 20 min of IL-15 stimulation (n = 5). SEM is shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Purified rat anti-mouse IL-2 , BD Biosciences , RRID: AB_395383.

Techniques: Expressing, Immunoprecipitation, Staining

Journal: Cell reports

Article Title: METTL14-dependent m 6 A modification controls iNKT cell development and function

doi: 10.1016/j.celrep.2022.111156

Figure Lengend Snippet: (A) Dot plots of IFN-γ and IL-4 in residual iNKT cells from T- Mettl14 −/− mice after in vivo α -GalCer stimulation. (B) Quantification of the percentage of IFN-γ and IL-4 producing iNKT cells in α-GalCer-immunized WT and T- Mettl14 −/− mice (n = 4). (C) Expression of METTL14 in DN32.D3 cells transduced with lentivirus coding Mettl14 -specific shRNA (sh Mettl14 -1 or sh Mettl14 -2) or control shRNA (shNC). (D) m 6 A level in mRNA of DN32.D3 cells transduced with sh Mettl14 -2 and shNC. (E) Relative expression of Mettl14 and Cish in DN32.D3-sh Mettl14 -2 in medium or stimulation with α-GalCer for 24 h. (F) Production of IL-2 in DN32.D3-sh Mettl14 -2 after stimulation with α-GalCer for 24 h quantified by ELISA. (G) Intracellular calcium flux in DN32.D3-sh Mettl14 -2 cells in response to α-GalCer/Ca 2+ . (H) Quantification of maximum calcium flux upon α-GalCer stimulation. Data representative of three to six independent experiments. shNC or sh Mettl14 -2-treated DN32.D3 were spin-transduced with retrovirus carrying sh Cish or shNC. Zsgreen + cells were sorted and cultured. (I) Relative expression of Cish in the indicated groups. (J) IL-2 production in shNC/shNC-treated (‘‘WT’’ control) and shNC and shCish-treated Mettl14 KD DN32.D3 cells after stimulation with α-GalCer for 24 h. Vα14 Tg splenocytes were nucleofected with rCas9/gRNA complex and maintained in complete RPMI supplemented with IL-2 for 3 days. (K) Mettl14 and Cish expression by qPCR on day 3 after nucleofection. (L) Absolute cell numbers of iNKT and CD4 + T cells after nucleofection. (M) IFN-γ production in iNKT and CD4 + T cells after nucleofection (n = 3–4). SEM is shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Purified rat anti-mouse IL-2 , BD Biosciences , RRID: AB_395383.

Techniques: In Vivo, Expressing, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell reports

Article Title: METTL14-dependent m 6 A modification controls iNKT cell development and function

doi: 10.1016/j.celrep.2022.111156

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified rat anti-mouse IL-2 , BD Biosciences , RRID: AB_395383.

Techniques: Purification, shRNA, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, SYBR Green Assay, Software, Chromogenic In Situ Hybridization

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Genes differentially expressed in resident macrophages at 1 h postinfection with S. pyogenes

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Zinc-Fingers, Binding Assay

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Confirmation of microarray data by RT-PCR and protein expression. (A) RT-PCR analysis of selected gene transcription in resident macrophages uninfected or infected with S. pyogenes. Uninfected samples were loaded in lanes 1, and infected samples were loaded in lanes 2. β-Actin expression served as a control. (B) IL-6 protein expression by S. pyogenes-infected macrophages. Resident macrophages were isolated from the peritoneal cavity of mice after 1 h of infection with S. pyogenes and cultured in vitro for 2 h. Noninfected macrophages were used as a control. The levels of IL-6 in the supernatants were determined by ELISA. Each column represents the mean ± standard deviation of triplicate samples obtained from three independent experiments. P < 0.0001 (ANOVA).

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Isolation, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Response kinetics reveal novel features of ageing in murine T cells

doi: 10.1038/s41598-019-42120-1

Figure Lengend Snippet: Duration and strength of stimulation determine ageing-related cytokine profiles. Cytokine levels of IL-2 ( a ), IL-4 ( b ), IL-5 ( c ), IL-10 ( d ), IL-17 ( e ), IFN-γ ( f ), and TNF-α ( g ) were measured in supernatant of splenocytes exposed for two or four days to an intermediate (int) or high stimulatory strength. Data represent one experiment with young (n = 6, 2 months old) and aged (n = 6, 22–24 months old) mice. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 for difference between young and old mice at time points indicated using Two-way ANOVA.

Article Snippet: Cells were labelled intracellularly with the following antibodies according to the FoxP3 Transcription Factor staining buffer set protocol (eBioscience): anti-CD3zeta-FITC (clone H146-968) (Abcam, Cambridge, Cambridgeshire, UK); anti-CTLA-4-BV605 (clone UC10-4B9), and anti-TNF-α-BV785 (clone MP6-XT22) (BioLegend); anti-FoxP3-eFluor660 (clone 150D/E4), anti-GARP-PE (clone YGIC86), anti-IFN-γ-PE-Cy7 (clone XMG1.2), and IL-4-PE (clone 11B11) (eBioscience); anti-IL-5-PE (clone TRFK5) (BD Pharmingen).

Techniques: