Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of hScFv-IL-17A proteins expressed in various E. coli strains. (a) SDS-PAGE of insoluble fractions; (b) SDS-PAGE of soluble fractions; (c) Western blot of insoluble fractions; (d) Western blot of soluble fractions. Lane 1: M (molecular weight marker); Lanes 2–4: hScFv-IL-17A-WT expressed in E. coli Origami B (DE3), SHuffle, and BL21 (DE3) with DisCoTune, respectively; Lanes 5–7: hScFv-IL-17A-C97S expressed in the same strains; Lane 8: purified hScFv M6-1B9 (positive control). Western blot detection was performed using an anti-His tag antibody. The arrow indicates the expected size of hScFv-IL-17A (∼28 kDa). Estimated amounts of hScFv-IL-17A are labeled. < LOD indicates signals below the limit of detection.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Purification, Positive Control, Labeling

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: The expression efficiency of soluble hScFv-IL-17A-WT (orange) and hScFv-IL-17A-C97S (green) in different expression systems: (WT/Ori and C97S/Ori) from Origami B (DE3); (WT/SHuffle and C97S/SHuffle) from SHuffle; and (WT/BL21Dc and C97S/BL21Dc) from BL21 (DE3) with DisCoTune. Data represent mean ± SD from three independent biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Expressing

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Indirect ELISA of hScFv-IL-17A binding to hIL-17A using an avidin–biotin system. Purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S were tested at 1 and 5 µg/mL, expressed from SHuffle (WT/SHuffle-1, WT/SHuffle-5; C97S/SHuffle-1, C97S/SHuffle-5; light/dark green) and BL21 (DE3) with DisCoTune (WT/BL21Dc-1, WT/BL21Dc-5; C97S/BL21Dc-1, C97S/BL21Dc-5; light/dark orange). An IL-17A mAb (dark grey) was used as a positive control. Data represent mean ± SD from three independent biological replicates. Statistical analysis was determined by unpaired two-tailed Student’s t -test. Significance levels are denoted as: ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Indirect ELISA, Binding Assay, Avidin-Biotin Assay, Purification, Positive Control, Two Tailed Test

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Binding assessment of hScFv-IL-17A-WT (a) and hScFv-IL-17A-C97S (b) from BL21 (DE3) with DisCoTune by competitive ELISA. Various concentrations (0.1, 1, and 2 µg/mL) of IL-17A mAb (neutralizing antibody) were tested in competition with hScFv-IL-17A. WT was used at 1 µg/mL, whereas C97S was used at 5 µg/mL to compensate for its reduced binding activity observed in indirect ELISA. An anti-IFN-γ mAb (clone B27) was used as an irrelevant antibody control. Data represent mean ± SD from three independent biological replicates.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Binding Assay, Competitive ELISA, Activity Assay, Indirect ELISA, Control

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Structural alignment of hScFv-IL-17A-WT and hScFvIL-17A-C97S. The predicted structures were generated using ABodyBuilder2. The conformational structures of hScFv-IL-17A-WT and hScFv-IL-17A-C97S were compared using UCSF ChimeraX software. The red ribbon indicates hScFv-IL-17A-WT structure, and the gold ribbon indicates hScFv-IL-17A-C97S structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Generated, Software

Journal: Gut Microbes

Article Title: Antibiotic cocktail-induced changes in gut microbiota drive alteration of bile acid metabolism to restrain Th17 differentiation through the FXR–NLRP3 axis

doi: 10.1080/19490976.2025.2582944

Figure Lengend Snippet: Antibiotic therapy modifies gut microbes and mitigates EAP (A) Timeline for EAP induction and ABX feeding. (B) Gut microbiota α diversity calculated via Chao1. (C) PCoA ( β -diversity) of bacterial 16S rRNA genes in feces from EAP mice and EAP + ABX mice. (D) Relative abundance of gut microbes at the genus level. (E) LEfSe analysis of the differentially abundant genera in the two groups. (F) Predicted outcomes of the KEGG pathway analysis (G) Assessment of pelvic pain in mice using von Frey filaments. (H) Body weights of the mice. (I) H&E staining revealing alterations in prostate tissue following ABX therapy. The red arrow indicates the invasion of inflammatory cells. Inflammation score assessment in the EAP and EAP + ABX groups. (J) IHC of IL-17A in prostate tissue. (K) The percentages of CD4+ IL-17A + cells in the spleens of EAP and EAP + ABX mice. (L) Relative level of IL-17A mRNA in prostate tissue from EAP and EAP + ABX mice. (M) The percentages of CD4+ RORγt + cells in the spleens of EAP and EAP + ABX mice. ( N ) Relative level of RORγt mRNA in prostate tissue from EAP and EAP + ABX mice. (O) Serum concentrations of IL-17A, GM-CSF, and IFN- γ in the EAP and EAP + ABX groups measured via ELISA ( n = 5–10; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The sections were incubated with mouse anti-IL-17A antibodies (Proteintech, 66148−1-lg), rabbit anti-FXR antibodies (Santa Cruz, sc−25309), rabbit anti-caspase-1β antibodies (Affinity, AF5418), rabbit anti-ASC antibodies (Affinity, DF6304), rabbit anti-NLRP3 antibodies (Santa Cruz, sc−134306), rabbit anti-cleaved caspase−1 antibodies (Affinity, AF4005), mouse anti-IL-1β antibodies (Affinity, BF8021) and cleaved-IL-1β antibodies (Affinity, AF4006) overnight at 4 °C, and appropriate biotinylated goat anti-rabbit/mouse IgG (Servicebio, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Gut Microbes

Article Title: Antibiotic cocktail-induced changes in gut microbiota drive alteration of bile acid metabolism to restrain Th17 differentiation through the FXR–NLRP3 axis

doi: 10.1080/19490976.2025.2582944

Figure Lengend Snippet: FMT from EAP + ABX mice changes the gut microbiome and alleviates prostatitis in recipient EAP mice (A) Schedule for EAP mice receiving FMT from EAP or EAP + ABX mice. (B) Gut microbiota α diversity calculated via Chao1. (C) PCoA ( β -diversity) of bacterial 16S rRNA genes between FMT from the EAP group and FMT from the EAP + ABX group. (D) Relative abundance of gut microbes at the genus level. (E) LEfSe analysis of the two groups. (F) Predicted outcomes of the KEGG pathway analysis. (G) Assessment of pelvic pain in mice using von Frey filaments. (H) H&E staining of prostate tissue: Representative H&E-stained sections of prostate tissue after FMT from the EAP group vs. FMT from the EAP + ABX group. Red arrows indicate the infiltration of inflammatory cells in the prostate tissue. (I, J) The percentages of CD4+ IL-17A + and CD4+ RORγt + cells among the splenic lymphocytes of FMT from the EAP group and FMT from the EAP + ABX group. (K, L) Relative mRNA expression levels of IL-17A and RORγt mRNA in prostate tissue from mice after FMT from the EAP group and FMT from the EAP + ABX group, as measured by qRT‒PCR. (M) IHC for IL-17A in prostate tissue from the two groups. ( N , O) Serum and prostate concentrations of IL-17A, GM-CSF, and IFN- γ determined by ELISA in the two groups ( n = 5–8; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The sections were incubated with mouse anti-IL-17A antibodies (Proteintech, 66148−1-lg), rabbit anti-FXR antibodies (Santa Cruz, sc−25309), rabbit anti-caspase-1β antibodies (Affinity, AF5418), rabbit anti-ASC antibodies (Affinity, DF6304), rabbit anti-NLRP3 antibodies (Santa Cruz, sc−134306), rabbit anti-cleaved caspase−1 antibodies (Affinity, AF4005), mouse anti-IL-1β antibodies (Affinity, BF8021) and cleaved-IL-1β antibodies (Affinity, AF4006) overnight at 4 °C, and appropriate biotinylated goat anti-rabbit/mouse IgG (Servicebio, China).

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Gut Microbes

Article Title: Antibiotic cocktail-induced changes in gut microbiota drive alteration of bile acid metabolism to restrain Th17 differentiation through the FXR–NLRP3 axis

doi: 10.1080/19490976.2025.2582944

Figure Lengend Snippet: DCA inhibits FXR induced Th17 cell differentiation (A) Feeding schedule for EAP mice treated with EAP, ABX or ABX + DCA. (B) Assessment of pelvic pain in mice using von Frey filaments. (C) H&E staining reveals alterations in prostate tissue. The red arrow indicates the invasion of inflammatory cells. Inflammation score assessment in the EAP, EAP + ABX and EAP + ABX + DCA groups. (D) Relative level of RORγt mRNA in prostate tissue from EAP, EAP + ABX and EAP + ABX + DCA mice. (E, F) The percentages of CD4+ IL-17A + and CD4+ RORγt + cells among the splenic lymphocytes from EAP, EAP + ABX and EAP + ABX + DCA mice. (G) The relative level of FXR mRNA in prostate tissue from the three groups. (H) IHC of IL-17A in prostate tissue. (I) Serum concentrations of IL-17A, GM-CSF, and IFN- γ in the EAP, EAP + ABX and EAP + ABX + DCA groups were measured via ELISA. (J) Western blot detection of FXR expression in prostate from EAP, EAP + ABX and EAP + ABX + DCA mice. (K) The percentages of CD4+ IL-17A + cells in the media and DCA groups. (L) Medium concentrations of IL-17A, GM-CSF, and IFN- γ in the media and DCA groups, as determined by ELISA. (M) Predicted outcomes of the KEGG pathway analysis via proteomics. (N) The percentage of CD4+ RORγt + cells in the media and DCA groups. (O) FXR mRNA levels in sorted naïve CD4+ T cells differentiated from Th17 cells between the media and DCA groups. (P) FXR mRNA levels in sorted naïve CD4+ T cells differentiated from Th17 cells between the media and DCA group. ( n = 3–5; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The sections were incubated with mouse anti-IL-17A antibodies (Proteintech, 66148−1-lg), rabbit anti-FXR antibodies (Santa Cruz, sc−25309), rabbit anti-caspase-1β antibodies (Affinity, AF5418), rabbit anti-ASC antibodies (Affinity, DF6304), rabbit anti-NLRP3 antibodies (Santa Cruz, sc−134306), rabbit anti-cleaved caspase−1 antibodies (Affinity, AF4005), mouse anti-IL-1β antibodies (Affinity, BF8021) and cleaved-IL-1β antibodies (Affinity, AF4006) overnight at 4 °C, and appropriate biotinylated goat anti-rabbit/mouse IgG (Servicebio, China).

Techniques: Cell Differentiation, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Gut Microbes

Article Title: Antibiotic cocktail-induced changes in gut microbiota drive alteration of bile acid metabolism to restrain Th17 differentiation through the FXR–NLRP3 axis

doi: 10.1080/19490976.2025.2582944

Figure Lengend Snippet: DCA initiates the NLRP3‒IL17A pathway to promote Th17 differentiation (A) Feeding schedule for EAP mice treated with ABX, ABX + DCA or ABX + DCA + MCC950. (B) Assessment of pelvic pain in mice using von Frey filaments. (C) H&E staining revealing alterations in prostate tissue. The red arrow indicates the invasion of inflammatory cells. Inflammation score assessment in the ABX, ABX + DCA and ABX + DCA + MCC950 groups. (D) Relative level of RORγt mRNA in prostate tissue from ABX, ABX + DCA and ABX + DCA + MCC950 mice. (E, F) The percentages of CD4+ IL-17A + and CD4+ RORγt + cells in the spleen of ABX, ABX + DCA and ABX + DCA + MCC950 mice. (G, H) Sorted naïve CD4+ T cells were activated for 5 days under Th17 cell differentiation conditions without DCA (media group), with DCA (media + DCA group) or with DCA + MCC950 (media + DCA + MCC950 group). Representative images and analysis of FCM staining for CD4+ IL-17A + cells and CD4+ RORγt + cells in the three groups. (I) Western blot analysis of NLRP3-IL17A-related proteins in the media, media + DCA, and media + DCA + MCC950 groups ( n = 3). (J) IHC of NLRP3-IL17A-related proteins in prostate tissue from ABX, ABX + DCA and ABX + DCA + MCC950 mice. (K) ChIP‒qPCR was used to determine the binding status of FXR at three sites in the NLRP3 promoter. (L) Dual-luciferase assay showing the interaction between FXR and NLRP3. ( n = 3–5; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The sections were incubated with mouse anti-IL-17A antibodies (Proteintech, 66148−1-lg), rabbit anti-FXR antibodies (Santa Cruz, sc−25309), rabbit anti-caspase-1β antibodies (Affinity, AF5418), rabbit anti-ASC antibodies (Affinity, DF6304), rabbit anti-NLRP3 antibodies (Santa Cruz, sc−134306), rabbit anti-cleaved caspase−1 antibodies (Affinity, AF4005), mouse anti-IL-1β antibodies (Affinity, BF8021) and cleaved-IL-1β antibodies (Affinity, AF4006) overnight at 4 °C, and appropriate biotinylated goat anti-rabbit/mouse IgG (Servicebio, China).

Techniques: Staining, Cell Differentiation, Western Blot, Binding Assay, Luciferase

Journal: Gut Microbes

Article Title: Antibiotic cocktail-induced changes in gut microbiota drive alteration of bile acid metabolism to restrain Th17 differentiation through the FXR–NLRP3 axis

doi: 10.1080/19490976.2025.2582944

Figure Lengend Snippet: DCA inhibits FXR activation of the NLRP3-IL17A signaling pathway to promote Th17 cell differentiation (A) Feeding schedule for EAP mice treated with ABX + DCA, ABX + DCA + GW4064, ABX + DCA + MCC950, or ABX + DCA + GW4064 + MCC950. (B) Assessment of pelvic pain using von Frey filaments. (C) H&E staining revealing alterations in prostate tissue. The red arrow indicates the invasion of inflammatory cells. Inflammation score assessment in ABX + DCA, ABX + DCA + GW4064, ABX + DCA + MCC950 and ABX + DCA + GW4064 + MCC950 mice. (D, E) Relative levels of RORγt and FXR mRNA in prostate from the four groups. (F, G) The percentages of CD4+ IL-17A + and CD4+ RORγt + cells in the spleens of ABX + DCA, ABX + DCA + GW4064, ABX + DCA + MCC950 and ABX + DCA + GW4064 + MCC950 mice. (H) Serum concentrations of IL-17A, GM-CSF, and IFN- γ in the four groups detected by ELISA.Sorted naïve CD4+ T cells were activated for 5 days under Th17 cell differentiation conditions without AAV-FXR (media group), with AAV-FXR (media + AAV-FXR group), with AAV-FXR + GW4064 (media + AAV-FXR + GW4064 group), or with AAV-FXR + MCC950 (media + AAV-FXR + MCC950 group). (I) Medium concentrations of IL-17A, GM-CSF, and IFN- γ in the media, media + AAV-FXR, media + AAV-FXR + GW4064 and media + AAV-FXR + MCC950 groups detected by ELISA. (J, K) Flow cytometric analysis of the proportions of CD4+ IL−17 + and CD4+ RORγt + cells in the four groups. (L) Relative mRNA expression of IL-17A, NLRP3, FXR and ASC in the four groups determined via RT‒qPCR. (M) Western blot analysis of NLRP3-IL17A-related proteins and FXR in the media, media + AAV-FXR, media + AAV-FXR + GW4064 and media + AAV-FXR + MCC950 groups. ( N ) IHC of NLRP3-IL17A-related proteins and FXR in prostate tissue from ABX + DCA, ABX + DCA + GW4064, ABX + DCA + MCC950 and ABX + DCA + GW4064 + MCC950 mice. (O) IHC of NLRP3-IL17A-related proteins and FXR in prostate tissue from EAP and EAP + ABX mice. ( P ) IHC of NLRP3-IL17A-related proteins and FXR in the prostate tissue of recipient EAP mice treated with FMT from EAP or EAP + ABX mice ( n = 3–5; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The sections were incubated with mouse anti-IL-17A antibodies (Proteintech, 66148−1-lg), rabbit anti-FXR antibodies (Santa Cruz, sc−25309), rabbit anti-caspase-1β antibodies (Affinity, AF5418), rabbit anti-ASC antibodies (Affinity, DF6304), rabbit anti-NLRP3 antibodies (Santa Cruz, sc−134306), rabbit anti-cleaved caspase−1 antibodies (Affinity, AF4005), mouse anti-IL-1β antibodies (Affinity, BF8021) and cleaved-IL-1β antibodies (Affinity, AF4006) overnight at 4 °C, and appropriate biotinylated goat anti-rabbit/mouse IgG (Servicebio, China).

Techniques: Activation Assay, Cell Differentiation, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Integrative bioinformatics and experimental validation unveil CRISP3 as a hypoxia-, epithelial mesenchymal transition-, and immune-related prognostic biomarker and therapeutic target in breast cancer

doi: 10.3389/fimmu.2025.1634399

Figure Lengend Snippet: CRISP3 knockdown attenuates hypoxia-induced TNBC cell proliferation, migration, and EMT. MDA-MB-231 and MDA-MB-468 cells were transduced with pLKO.1-TRC shRNA as control (Con) or pLKO.1-CRISP3 shRNA to silence CRISP3 (shRNA CRISP3). qRT-PCR analysis of the mRNA expression of (A) HIF-1α and (B) CRISP3 under hypoxia conditions in MDA-MB-231 and MDA-MB-468 cells. (C) qRT-PCR analysis of CRISP3 silencing efficiency in MDA-MB-231 and MDA-MB-468 cells. (D) Immunoblotting analysis of EMT markers. (E) The CCK8 assay, and (F–G) colony formation assay were employed to evaluate cell proliferation. (H, I) Wound healing assay, and (J–K) transwell assay were conducted for assessment of migrative ability. (L) ELISA was performed to measure the level of IL-17 in the conditioned medium of MDA-MB-231 and MDA-MB-468 cells. Statistical significance was determined using one-way ANOVA. Data are presented as mean ± SEM (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 0.1 mm.

Article Snippet: Reagents were purchased or obtained from the following sources: rabbit anti-E-Cadherin antibody (A20798, ABclonal, China), rabbit anti-Vimentin antibody (A2584, ABclonal, China), rabbit anti-Snail antibody (A5243, ABclonal, China), rabbit anti-phospho-AKT (Ser 473) (#4060s, CST, USA), mouse anti-AKT antibody (60203-2-Ig, Proteintech Wuhan, China), mouse anti-Beta Actin antibody (66009-1-Ig, Proteintech Wuhan, China), NeutraKine ® IL-17A Mouse McAb (69021-1-Ig, Proteintech Wuhan, China), and goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor ® 488 conjugate (ABclonal, China).

Techniques: Knockdown, Migration, Transduction, shRNA, Control, Quantitative RT-PCR, Expressing, Western Blot, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Integrative bioinformatics and experimental validation unveil CRISP3 as a hypoxia-, epithelial mesenchymal transition-, and immune-related prognostic biomarker and therapeutic target in breast cancer

doi: 10.3389/fimmu.2025.1634399

Figure Lengend Snippet: Blocking IL-17 prevents hypoxia-induced pro-carcinogenic progression in BC cells. MDA-MB-231 and MDA-MB-468 cells were exposed to hypoxic conditions (1% O 2 , 5% CO 2 , and 94% N 2 ) and subsequently treated with IL-17 neutralizing antibody (nAb) at a final concentration of 10 μg/mL. (A) CCK8 assay. (B) Colony formation assay. (C, D) Wound healing assay. (E) The transwell assay. (F) Immunoblotting analysis of EMT markers, AKT, and p-AKT. Statistical significance was determined using one-way ANOVA. Data are presented as mean ± SEM (n = 4). ** p < 0.01, *** p < 0.001. Scale bar = 0.1 mm.

Article Snippet: Reagents were purchased or obtained from the following sources: rabbit anti-E-Cadherin antibody (A20798, ABclonal, China), rabbit anti-Vimentin antibody (A2584, ABclonal, China), rabbit anti-Snail antibody (A5243, ABclonal, China), rabbit anti-phospho-AKT (Ser 473) (#4060s, CST, USA), mouse anti-AKT antibody (60203-2-Ig, Proteintech Wuhan, China), mouse anti-Beta Actin antibody (66009-1-Ig, Proteintech Wuhan, China), NeutraKine ® IL-17A Mouse McAb (69021-1-Ig, Proteintech Wuhan, China), and goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor ® 488 conjugate (ABclonal, China).

Techniques: Blocking Assay, Concentration Assay, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Assay, Western Blot

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