Journal: Frontiers in Network Physiology

Article Title: Modulation of the expression of connexins 37, 40, and 43 in endothelial cells in a culture

doi: 10.3389/fnetp.2024.1199198

Figure Lengend Snippet: HMEC-1 Cx40 mRNA expression in response to IFNγ in the absence and presence of JAK/STAT inhibitors, EGCG and AG490. EGCG inhibited the enhanced Cx40 gene expression by IFNγ 1 or 3 ng/mL at concentrations over 100 μM. Every concentration of AG490 inhibited the elevated Cx40 expression induced by IFNγ in a dose of 1 or 3 ng/mL. The highest dosages of the EGCG and AG490 inhibition resulted in a decreased Cx40 gene expression versus control. All data were derived from three independent experiments per experimental setting and three replicates in each experiment. A total of 95% confidence intervals were derived from the curve fitting. * p <0.05, ** p <0.01, *** p <0.001, and + p < 0.0001. Significance is indicated as compared to the bars pointed at with a line with a circle.

Article Snippet: ANG II, IL-6, TNFα, IFNγ, hydrocortisone, and the JAK/STAT inhibitors AG490 and epigallocatechin-3-gallate (EGCG) were purchased from MilliporeSigma (Darmstadt, Germany).

Techniques: Expressing, Gene Expression, Concentration Assay, Inhibition, Control, Derivative Assay

Journal: Frontiers in Network Physiology

Article Title: Modulation of the expression of connexins 37, 40, and 43 in endothelial cells in a culture

doi: 10.3389/fnetp.2024.1199198

Figure Lengend Snippet: Normalized impedance of HMEC-1 at 4 kHz and 64 kHz throughout 60 h of treatment. Panels A1 and A2 show impedance changes in response to IFNγ at 4 and 64 kHz, respectively. After 1 h of recording, the medium was replaced with the growth medium containing 10 ng/mL IFNγ with DPBS serving as the control. IFNγ resulted in a higher impedance level compared to the control after 30 h at both frequencies. Panels B1 and B2 show impedance time series at 4 kHz and 64 kHz, respectively, where the HMEC-1 medium was replaced with the growth medium containing AG490 5 μM and 10 uM, with DPBS as the control. At 4 kHz, impedance plateaued at similar levels to the control in the presence of 5 μM AG490; yet, the plateau in the presence of 10 uM AG490 was lower compared to the control. At 64 kHz, both doses of AG490 resulted in a lower impedance plateau compared to the control. In panels C1 and C2, the response at 4 kHz and 64 kHz is shown where HMEC-1 cells were pretreated with AG490 and then exposed to 10 ng/mL IFNγ. Both 5 μM and 10 μM AG490 diminished the enhanced impedance in response to 10 ng/mL IFNγ at both 4 kHz and 64 kHz. Data were derived from 5–12 wells in the ECIS arrays in 5–7 independent experiments.

Article Snippet: ANG II, IL-6, TNFα, IFNγ, hydrocortisone, and the JAK/STAT inhibitors AG490 and epigallocatechin-3-gallate (EGCG) were purchased from MilliporeSigma (Darmstadt, Germany).

Techniques: Control, Derivative Assay

Journal: Frontiers in Network Physiology

Article Title: Modulation of the expression of connexins 37, 40, and 43 in endothelial cells in a culture

doi: 10.3389/fnetp.2024.1199198

Figure Lengend Snippet: Comparison of the average impedance level between 40 and 50 h at 4 and 64 kHz. At 4 kHz between 40 and 50 h, IFNγ significantly increased impedance compared to the control. Pre-treatment with 10 μM AG490 decreased the impedance level below the control level. Pre-treatment with both doses of AG490 diminished the enhancement of impedance by IFNγ. At 64 kHz between 40 and 50 h, changes were similar but less pronounced. Data were obtained from 5–7 independent experiments. **: p <0.005; +: p <0.0001. Significance is indicated as compared to the bars pointed at with a line with a circle.

Article Snippet: ANG II, IL-6, TNFα, IFNγ, hydrocortisone, and the JAK/STAT inhibitors AG490 and epigallocatechin-3-gallate (EGCG) were purchased from MilliporeSigma (Darmstadt, Germany).

Techniques: Comparison, Control

Journal: Molecular medicine reports

Article Title: Astaxanthin inhibits integrin α5 expression by suppressing activation of JAK1/STAT3 in Helicobacter pylori‑stimulated gastric epithelial cells.

doi: 10.3892/mmr.2023.13014

Figure Lengend Snippet: Figure 4. AG490 and K34C suppress Helicobacter pylori‑induced integrin α5 expression, cell adhesion and migration. (A) Cells were pretreated with 40 µM AG490 for 2 h, and subsequently stimulated with H. pylori for 6 h. Protein levels of integrin α5 and β1 were determined using western blot analysis, with β‑actin as the loading control. (B) Cells were pretreated with 40 µM AG490 or 20 µM K34C for 2 h, and subsequently stimulated with H. pylori for 24 h. Adherent cells were stained and absorbance was detected at 570 nm. Results are presented as the mean ± standard error (the total number for each group was 12). The percentage of adherent cells in the ‘None’ group was set as 100%. (C) Cells were pretreated with 40 µM AG490 or 20 µM K34C for 2 h, and subsequently stimulated with H. pylori for 20 h. The wound healing assay was performed to detect cell migration. Representative images of wounds in AGS cells were captured at baseline (0 h) and at 20 h (magnification, x100). Wound closure was evaluated by measuring the remaining cell‑free area and expressed as a percentage of the initial cell‑free area. The bar graph indicates wound closure rate (%). Results are presented as the mean ± SE (the total number for each group was 12). (D) Cells were pretreated with ASX (5 µM), AG490 (40 µM) and K34C (20 µM), and then stimulated with H. pylori for 6 h. The pretreatment period for ASX was 3 h, while that of AG490 or K34C was 2 h. Protein levels of integrin α5 and β1 were determined using western blot analysis, with β‑actin as the loading control. The densitometry data are presented as the mean ± standard error from three immunoblots, and are shown as the relative density of protein bands normalized to β‑actin. *P<0.05 vs. ‘None’ (unstimulated cells without treatment of AG490, K34C or ASX). #P<0.05 vs. ‘Control’ (H. pylori‑stimulated cells without treatment of AG490, K34C or ASX). ASX, astaxanthin.

Article Snippet: The JAK/STaT inhibitor aG490 (cat. no. T3434; r&d Systems, inc.) and integrin α5β1 antago‐ nist K34c (N‐(2,6‐dimethylbenzoyl)‐O‐[3‐(2‐pyridinylamino) propyl]‐l‐tyrosine; cat. no. 5114; r&d Systems, inc.) were dissolved in dMSo at 50 mM.

Techniques: Expressing, Migration, Western Blot, Control, Staining, Wound Healing Assay

Journal: Journal of Neuroinflammation

Article Title: Activation of the inflammatory transcription factor nuclear factor interleukin-6 during inflammatory and psychological stress in the brain

doi: 10.1186/1742-2094-10-140

Figure Lengend Snippet: LPS-induced nuclear NF-IL6-IR is linked to ACTH and increased TNFα in the rat pituitary. (A, a) Representative microphotographs show that nuclear (DAPI, blue) NF-IL6-IR (red) co-localized with some ACTH-positive cells (green) 6 h after LPS (100 μg/mL, (a) but not PBS treatment (A) in primary cell cultures of the anterior pituitary lobe. (B, b) Moreover, LPS (100 μg/mL) -induced TNFα-IR (green) in S100 protein-positive folliculostellate cells (red) 6 h after stimulation (b) , compared to PBS controls (B) . (C, c and D, d) In a different set of experiments, pretreatment (30 min, 100 μM) with the JAK-STAT inhibitor AG490 reduced LPS (100 μg/mL) -induced NF-IL6-IR partly in ACTH-IR cells (green) of the anterior pituitary lobe 6 h after stimulation (d) , compared to solvent (cremophor, Cre) -treated controls (c) . Brightness, contrast, and color balance were adjusted for better representation of the actual data. Scale bars in A (applies to A, a, B and b ) and C (applies to C, c, D and d ) represent 10 μm. (E) Relative TNFα concentration in cell culture supernatants, in PBS- or LPS-stimulated and cremophor (Cre) or AG490 preincubated primary cell cultures are presented as percentage of LPS- and Cre-stimulated wells (100%) as calculated for each experiment. Means of two to three wells of the means (of each independent experiment, n = 3) were calculated and revealed a dramatic LPS- and Cre-induced TNFα-increase that was abolished by AG490 preincubation. (F) Counting of ACTH-IR cells as percentage of all cells in anterior pituitary cell cultures showed a significant increase of ACTH-positive cells after AG490 pretreatment in LPS-stimulated cultures (means of three independent experiments). AG490 did not show any significant effect on the amount of ACTH-positive cells in PBS-treated controls. * LPS & Cre vs . LPS & AG490; *** P <0.001, ** P <0.01.

Article Snippet: In another set of experiments (3 independent experiments, 2 to 3 wells for each treatment), the JAK-STAT-inhibitor AG490 (N-Benzyl-3,4-dihydroxybenzylidenecyano-acetamide; 1,5 μg/μL, 5 mM; Enzo Life Sciences International Inc., PA, USA) or its diluent (25% Cremophor®EL, Polyoxyethylenglyceroltriricinoleat 35, DAC in PBS; into serum-free medium 1:50; Sigma-Aldrich, Munich, Germany) were used to preincubate cells 30 min before control or LPS stimulation.

Techniques: Concentration Assay, Cell Culture

Journal: BioMed Research International

Article Title: TNF α Mediated IL-6 Secretion Is Regulated by JAK/STAT Pathway but Not by MEK Phosphorylation and AKT Phosphorylation in U266 Multiple Myeloma Cells

doi: 10.1155/2013/580135

Figure Lengend Snippet: IL-6 secretion by TNF α regulated by JAK/STAT pathway. U266 cells were serum starved for 8 h and treated with indicated JAK/STAT inhibitors for 1 h or transfected with TNFR siRNA. The cells were then stimulated with TNF α to induce IL-6 secretion and harvested for immunoblotting with indicated antibodies (a). Cell supernatants were collected to determine IL-6 levels using ELISA kit (b). TNF receptor targeted siRNA was used as a positive control. Data shown are the means ± SEM of three independent experiments. c: control; 1: TNF α only; 2: TNF α plus 20 μ M AG490; 3: TNF α plus 10 μ M JAK inhibitor I; 4: TNF α plus 10 μ M JAK inhibitor II; 5: TNF α plus TNFR siRNA.

Article Snippet: Bay 11-7082((E)3-[(4-merhylphenyl)sulfonyl]-2-propenenitrile), TPCK (N α -Tosyl-Phe Chloromethyl Ketone), PDTC (10 Pyrrolidinecarbodithioic Acid, Ammonium Salt), used as NF- κ B inhibitors, PI3K inhibitor (LY294002), JNK inhibitor II, MEK1/2 inhibitor (PD98059), p38 MAPK inhibitor (SB203580), JAK inhibitor I/II, and JAK/STAT inhibitor (AG490) were purchased from Calbiochem corp. (San Diego, CA, USA).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control