Journal: Journal of Experimental Pharmacology

Article Title: Artemisia annua Extract Ameliorates Atopic Dermatitis: Evidence from 3D Epidermal Model and Complementary in vitro Assays

doi: 10.2147/JEP.S550568

Figure Lengend Snippet: GO and KEGG pathway enrichment analyses of the putative targets of different components of Artemisia annua extract (AAE) for AD treatment ( a-e are Arteannuin B, Chlorogenic Acid, Scopolin, Vitexicarpin and Chrysoplenol D, respectively).

Article Snippet: Protein concentration was determined by BCA assay, and cell lysates were resolved by SDS-PAGE on 4–12% gradient Bis-Tris gels (GenScript, M000138), transferred to PVDF membranes, and probed with antibodies against filaggrin (ThermoFisher, PA5-115235), IL-1α and TSLP (Cell Signaling Technology, 34819S and 97630S), p38 MAPK and p-p38 MAPK (Proteintech, 66234-1-Ig and 28796-1-AP), GAPDH (Cell Signaling Technology, 14C10), β-actin (Servicebio, GB15001-100), anti-rabbit IgG and anti-mouse IgG HRP-linked Antibody (Cell SignalingTechnology, 7074P2 and 7076P2).

Techniques:

Journal: Journal of Experimental Pharmacology

Article Title: Artemisia annua Extract Ameliorates Atopic Dermatitis: Evidence from 3D Epidermal Model and Complementary in vitro Assays

doi: 10.2147/JEP.S550568

Figure Lengend Snippet: Cytotoxicity of different AAE concentrations in keratinocytes cells. ( a ) Cell viabilities of keratinocytes after AAE (0.1–3%) treatment for 24 h; 0 group, treated with DMEM; AAE group, treated with AAE (0.1–3%); DMSO (positive control) group, treated with 10% DMSO (n=5). ( b ) Cell viabilities of keratinocytes after AAE (0.1–3%) treatment for 24 h in MβCD//IL-4/IL-13/IL-25-induced AD-like models (n=5) (* p < 0.05, significant difference; ** p < 0.01, highly significant difference; *** p < 0.001, extremely significant difference; **** p < 0.0001, ultra-significant difference).

Article Snippet: Protein concentration was determined by BCA assay, and cell lysates were resolved by SDS-PAGE on 4–12% gradient Bis-Tris gels (GenScript, M000138), transferred to PVDF membranes, and probed with antibodies against filaggrin (ThermoFisher, PA5-115235), IL-1α and TSLP (Cell Signaling Technology, 34819S and 97630S), p38 MAPK and p-p38 MAPK (Proteintech, 66234-1-Ig and 28796-1-AP), GAPDH (Cell Signaling Technology, 14C10), β-actin (Servicebio, GB15001-100), anti-rabbit IgG and anti-mouse IgG HRP-linked Antibody (Cell SignalingTechnology, 7074P2 and 7076P2).

Techniques: Positive Control

Journal: Journal of Experimental Pharmacology

Article Title: Artemisia annua Extract Ameliorates Atopic Dermatitis: Evidence from 3D Epidermal Model and Complementary in vitro Assays

doi: 10.2147/JEP.S550568

Figure Lengend Snippet: MβCD/IL-mix-induced AD-like phenotypes and expression of related genes in 3D epidermal models. ( a ) Histological sections of 3D models treated with different concentrations of MβCD/IL-4/IL-13/IL-25. 25: MβCD (7.5 mM), IL-4 (25 ng/mL), IL-13 (25 ng/mL), IL-25 (10 ng/mL); 50: MβCD (7.5 mM), IL-4 (50 ng/mL), IL-13 (50 ng/mL), IL-25 (20 ng/mL); 100: MβCD (7.5 mM), IL-4 (100 ng/mL), IL-13 (100 ng/mL), IL-25 (40 ng/mL). Scale bar 50 µm (n=3). ( b ) qPCR analysis (n=4) (* p < 0.05, ** p < 0.01, **** p < 0.0001, n.s., not significant). ( c ) Cytokine array TSLP and IL-1α analysis (n=3) (** p < 0.01, **** p < 0.0001).

Article Snippet: Protein concentration was determined by BCA assay, and cell lysates were resolved by SDS-PAGE on 4–12% gradient Bis-Tris gels (GenScript, M000138), transferred to PVDF membranes, and probed with antibodies against filaggrin (ThermoFisher, PA5-115235), IL-1α and TSLP (Cell Signaling Technology, 34819S and 97630S), p38 MAPK and p-p38 MAPK (Proteintech, 66234-1-Ig and 28796-1-AP), GAPDH (Cell Signaling Technology, 14C10), β-actin (Servicebio, GB15001-100), anti-rabbit IgG and anti-mouse IgG HRP-linked Antibody (Cell SignalingTechnology, 7074P2 and 7076P2).

Techniques: Expressing

Journal: Journal of Experimental Pharmacology

Article Title: Artemisia annua Extract Ameliorates Atopic Dermatitis: Evidence from 3D Epidermal Model and Complementary in vitro Assays

doi: 10.2147/JEP.S550568

Figure Lengend Snippet: AAE partially improved the AD-related cytokine expression in AD-like skin equivalent model. ( a ) Cytokine array TSLP, IL-1α, IL-6 and IL-8 analysis (n=3) (** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s., not significant). ( b and c ) Protein expressions of IL-1α and TSLP determined by Western blot (n=4) (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Protein concentration was determined by BCA assay, and cell lysates were resolved by SDS-PAGE on 4–12% gradient Bis-Tris gels (GenScript, M000138), transferred to PVDF membranes, and probed with antibodies against filaggrin (ThermoFisher, PA5-115235), IL-1α and TSLP (Cell Signaling Technology, 34819S and 97630S), p38 MAPK and p-p38 MAPK (Proteintech, 66234-1-Ig and 28796-1-AP), GAPDH (Cell Signaling Technology, 14C10), β-actin (Servicebio, GB15001-100), anti-rabbit IgG and anti-mouse IgG HRP-linked Antibody (Cell SignalingTechnology, 7074P2 and 7076P2).

Techniques: Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 2 PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- mice show reduced levels of circulating cytokines. (A) Volcano plot of proteins regulated in Olink mouse exploratory panel comparing serum protein levels of mice fed a NC vs. PCSK9-WT mice. Red data points indicate the difference of the NPX mean between PCSK9-WT and NC for each protein. The dashed line intersecting the y-axis indicates the significance of p<0.05. (B) Shiny GO (19) (Version 0.77) enrichment analysis annotating the significantly regulated proteins to the Gene Ontology (GO) Biological Process (false detection rate (FDR) cutoff: 0.1). The top 10 regulated pathways are presented as a barplot with the colors indicating the -log10 FDR, with red as the highest and blue as the lowest. (C) Heatmap depicting the dynamics of proteins significantly regulated in Il1a-/- compared to PCSK9-WT. Data is presented as fold change of PCSK9-WT, PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- to NC. (D) Venn Diagram representing serum proteins specifically regulated in only NC or PCSK9-Il1a-/- animals compared to PCSK9-WT. Shared regulated proteins are displayed at the intersection of both areas. (E) Bargraphs of serum IL-1a levels measured in the Olink 96 mouse exploratory panel. The dashed line indicates the limit of detection of the assay. Data is presented with the Olink NPX value as mean ± SD, *p<0.05. PCSK9-WT (n=5), PCSK9-Il1a-/- (n=6), PCSK9-Nlrp3-/- (n=5), and PCSK9-Il1b-/- (n=4).

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques:

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 3 Cell surface translocation of IL-1a is not influenced by NLRP3 and IL-1b in murine BMDC. (A) Schematic overview of the experimental setup. Bone marrow-derived dendritic cells (BMDCs) were replated after 7 days of differentiation and stimulated with 100 ng/ml ultrapure Lipopolysaccharide (upLPS) overnight. Afterward, cells were harvested, and fractions were isolated using a detergent-based method. (B) Representative immunoblot of the membrane and cytoplasmic fraction of BMDCs with or without upLPS stimulation. Protein levels of IL-1a, GAPDH (glycerinaldehyde-3- phosphate-dehydrogenase, cytoplasmatic marker), and NaK ATPase (sodium–potassium ATPase, membrane marker) are presented. (C) Densiometric quantification of IL-1a in cytoplasmic fraction normalized to GAPDH and IL-1a in membrane fraction normalized to NaK ATPase. (D) Representative flow cytometry scatter plot of staining for IL-1a on stimulated BMDCs after gating for viable, non-fixated (7AAD-) cells. Total IL-1a was measured on fixed cells. (E) Barplot depicting the percentage of total IL-1a positive monocytes (7AAD+) with or without upLPS stimulation. (F) Barplot depicting the percentage of csIL-1a positive monocytes (7AAD-) with or without upLPS stimulation. (G) Barplot representing the percentage of non-permeable monocytes with or without upLPS stimulation. (H) Representative immunoblot of membrane and cytosolic fraction from upLPS-stimulated BMDCs of PCSK9-WT, PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- animals. Protein levels of IL-1a, GAPDH (cytoplasmatic marker), and NaK ATPase (membrane marker) are presented with corresponding densiometric quantification (I) of log-transformed IL-1a expression. All data are presented as mean ± SEM, *p<0.05.

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Translocation Assay, Derivative Assay, Isolation, Western Blot, Membrane, Marker, Cytometry, Staining, Transformation Assay, Expressing

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 5 Myristoylation regulates csIL-1a in murine bone marrow cells and human monocytes. (A) Barplot depicting the percentage of murine bone marrow cells with myristoylated proteins. Bone marrow cells were cultured and myristoylated proteins were labeled overnight. Data are presented as mean ± SEM of NC (n=3) and PCSK9-WT (n=3), *p<0.05. (B) Mean fluorescence intensity of human primary monocytes under culture conditions (con) or with overnight incubation of N-myristoyltransferase inhibitor IMP-1088 [1 µM]. Data are presented as mean ± SEM of three independent experiments. One-sided paired t-test was performed, *p<0.05. (C) Percentage of csIL-1a presenting monocytes stimulated with 100 ng/ml upLPS and 1 µM IMP-1088 as indicated. Data are presented as mean ± SEM of four independent experiments, *p<0.05.

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Cell Culture, Labeling, Incubation

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 4 IL-1a surface expression on human monocytes induces VCAM1 expression and leads to increased adhesion on endothelial cells. (A) Schematic principle of proximity ligation assay (PLA) and representative picture of PLA. Direct binding of csIL-1a to the Interleukin-1 receptor 1 (IL1R1) leads to a fluorescence signal detectable at 594 nm. Human umbilical vein endothelial cells (HUVECs) were treated with monocytes for 6h, stimulated with and without upLPS (100 ng/ml). Cells were imaged at a 40× magnification (scale bar 50µm). HUVECs and monocytes are presented in blue, IL-1a/IL1R1 PLA signal is visible as red dots. (B) Representative flow cytometry histogram of vascular cell adhesion molecule–1 (VCAM1) stained HUVECs after 4h treatment with upLPS-stimulated monocytes. 10 µg/ml neutralizing IL1R1 (nIL1R1) antibody was added 1h before HUVEC-monocyte co-incubation. (C) Bar graph depicting the percentage of VCAM1- positive HUVECs after treatment with unstimulated and upLPS-stimulated monocytes. HUVECs were incubated with and without nIL1R1 antibody (10 µg/ml) for 1h before co-incubation. Data are presented as mean ± SEM of seven independent experiments; *p< 0.05. (D): Schematic experimental setup of monocyte adhesion assay. Primary monocytes were treated as indicated, labeled with Calcein and 4x washings. HUVECs were treated with and without 1 µg/ml neutralizing IL-1a antibody (nIL-1a) 1h before co-incubation. Then, HUVECs were treated with labeled monocytes for 4h. The initial fluorescence of adhering monocytes was measured as well as after two washes. Cells were imaged (4× magnification), scalebar 100 µM. (E) Quantification of adhering monocytes to HUVECs presented as mean ± SEM of four independent experiments. Repeated measure ANOVA was performed, followed by Sidak’s multiple comparison test (*p< 0.05).

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Expressing, Proximity Ligation Assay, Binding Assay, Cytometry, Staining, Incubation, Cell Adhesion Assay, Labeling, Comparison

Journal: Scientific Reports

Article Title: Metapanax delavayi extract as a neurocutaneous modulator via CRHR1/POMC/MC1R signaling

doi: 10.1038/s41598-026-39343-4

Figure Lengend Snippet: MDE suppressed substance P-induced release of inflammatory factors in HaCaT cells. ( A ) CCK-8 assay, HaCaT cells were supplemented with 15, 30, 60, 120, 240, 480 μg/mL MDE for 24 h ( N = 3). ( B - D ) RT-qPCR was used to determine the mRNA expression levels of proinflammatory cytokines ( IL-6 , IL-8 , and IL-1α ) ( N = 4). ( E - G ) The content of IL-6, IL-8 and IL-1α in different groups ( N = 5). Data are presented as mean ± SEM. p value using the one-way ANOVA. * p ≤ 0.05, ** p ≤ 0.01 versus control; # p ≤ 0.05, ## p ≤ 0.01 versus substance P-induced.

Article Snippet: The expression of cortisol (Catalogue Numbers: CEA806Mu) (Cloud-Clone Corp, China), β-endorphin (Catalogue Numbers: CSB-E06821h), IL-6 (Catalogue Numbers: CSB-E04638h), IL-8 (Catalogue Numbers: CSB-E04641h), and IL-1α (Catalogue Numbers: CSB-E04620h) (all from Cusabio, China) were also quantified using ELISA kits according to the manufacturer’s instructions.

Techniques: CCK-8 Assay, Quantitative RT-PCR, Expressing, Control