Journal: Cells

Article Title: Okadaic Acid Activates JAK/STAT Signaling to Affect Xenobiotic Metabolism in HepaRG Cells

doi: 10.3390/cells12050770

Figure Lengend Snippet: Activation of JAK/STAT signaling in HepaRG cells after exposure to OA. Differentiated HepaRG cells were treated with 33 and 100 nM OA alone or 33 µM OA in combination with 40 µM Decernotinib, or 40 µM Tofacitinib, two JAK activation inhibitors or with the respective solvent control for 24 h. ( A ): Nuclei were stained with DAPI, and the actin cytoskeleton was stained using ActinGreen™ 488 ReadyProbes™ reagent. Immunostaining of STAT3 was carried out using a primary antibody against phosphorylated STAT3 and stained using a secondary antibody conjugated with the fluorophore Alexa Fluor™ 633. Fluorescence was detected using a confocal laser scanning microscope at ex wavelengths of 405 nm (DAPI, blue), 488 nm (ActinGreen, green), and 633 nm (phosphoSTAT3, red). Z-stacks spanning through the entire cell layer were recorded at 63× magnification. Brightness was increased by 40% for the blue and red channels in each image. ( B ): The nuclear fluorescence ratio was determined using ImageJ 1.53e. Nuclei were marked as ROI and the fluorescence intensity of the red and blue channels in each ROI was determined separately. A ratio of pSTAT3/DAPI signal intensity was then calculated and normalized to the mean of the solvent controls. The nuclei were divided into groups based on their signal intensity. Results show the number of nuclei per signal intensity group for each treatment group. Between 96 and 142 nuclei were evaluated per condition. Statistical analysis was performed against the solvent control using Wilcoxson rank-sum test (*** p < 0.001). ( C ): Western blot of lysed HepaRG cells against phosphorylated STAT3. Cells were incubated as described above with the addition of 30 µM JSH-23 and 30 µM Methysticin alone or in combination with 33 nM OA. Western blot were obtained using the wet blot method. Three biological replicates were independently analyzed and normalized against the housekeeper GAPDH. Afterwards, all samples were normalized against their respective solvent control. ( D ): Analysis of RNA levels of CYP1A1, CYP2B6, and CYP3A4 by qPCR in HepaRG cells after exposure to OA and the inhibitors. The bar charts show the resulting fold changes as mean of three independent replicates, relative to the solvent control. Statistical analysis (n = 3) was performed against the 33 nM OA sample using one-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001). ( E ): DigiWest results for JAK1 and JAK2. Three independent replicates were incubated and combined before measuring. ( F ): The nuclear fluorescence ratio was determined as in ( B ). Between 109 and 142 nuclei were evaluated for each condition. Statistical analysis was performed against the 33 nM OA sample using Wilcoxson rank-sum test (** p < 0.01; *** p < 0.001).

Article Snippet: JSH-23 and Methysticin were purchased from Merck KGaA (Darmstadt, Germany) and Decernotinib and Tofacitinib were obtained from Selleck Chemicals (Munich, Germany).

Techniques: Activation Assay, Solvent, Control, Staining, Immunostaining, Fluorescence, Laser-Scanning Microscopy, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Advances in Diabetes, Autoimmune, and Neurological Diseases

doi: 10.3390/ijms22062805

Figure Lengend Snippet: Approved and investigational biologics and small molecules for RA.

Article Snippet: SM , Decernotinib , Aclaris Therapeutics , Phase II , n/a , Oral.

Techniques:

Journal: Frontiers in Neurology

Article Title: Selective Inhibition of Janus Kinase 3 Has No Impact on Infarct Size or Neurobehavioral Outcomes in Permanent Ischemic Stroke in Mice

doi: 10.3389/fneur.2017.00363

Figure Lengend Snippet: (A) Dose–response curves measuring infarct volume in vehicle-treated and 0.03, 0.1, 0.3, 3, and 10 mg/kg of decernotinib-treated mice. There was no significant difference in infarct size across groups (vehicle, n = 8; 0.03 mg/kg, n = 6; 0.1 mg/kg, n = 8; 0.3 mg/kg, n = 8; 3 mg/kg, n = 10; and 10 mg/kg, n = 8). Data were analyzed using a one-way ANOVA with a Dunnett’s multiple comparison post-test. (B) Representative 2,3,5-triphenyl-tetrazolium chloride (TTC) images of vehicle- and 10 mg/kg decernotinib-treated mice to show extent of cortical injury in our model of permanent middle cerebral artery occlusion (MCAO). (C) Representative immunoblots of brain tissue obtained from the ipsilateral (CXI) and contralateral (CXC) cerebral cortex depicting reduced phosphorylation of Janus kinase 3 (JAK3) in the 10 mg/kg decernotinib group compared to vehicle. There is no effect of decernotinib on stroke-induced increase in total JAK3 levels. (D,E) Graphical summary of immunoblot data (* P < 0.05 compared to vehicle ipsilateral; vehicle, n = 9; 10 mg/kg decernotinib, n = 5). Data were analyzed using a two-way ANOVA. (F) Latency to remove the sticky tape from the contralateral paw, measuring sensorimotor deficits, in vehicle and decernotinib groups. Stroke induced a significant increase in the latency to remove the sticker at 24 h in the vehicle-treated group. Comparing both treatments at 24 h, there was a trend toward a better performance in the adhesive removal test in mice given decernotinib, but this effect did not reach a statistical significance (two-way ANOVA with Bonferroni post-test; vehicle n = 9; decernotinib at a dose of 10 mg/kg, n = 10).

Article Snippet: Animals were treated with vehicle or 0.03, 0.1, 0.3, 1.0, 3.0, or 10 mg/kg decernotinib (Cat # CT-DECE; ChemieTek, Indianapolis, IN, USA), a highly selective JAK3 inhibitor.

Techniques: Comparison, Western Blot, Phospho-proteomics, Adhesive

Journal: Frontiers in Neurology

Article Title: Selective Inhibition of Janus Kinase 3 Has No Impact on Infarct Size or Neurobehavioral Outcomes in Permanent Ischemic Stroke in Mice

doi: 10.3389/fneur.2017.00363

Figure Lengend Snippet: (A) IL-21 is increased in the ipsilateral cortex compared to the contralateral cortex (** P < 0.01) (B) IL-4 does not appreciably change between treatment groups or hemisphere. (C) There is a trend toward increased IL-7 in the ipsilateral cortex. (D) There is no change in IL-9 between groups. (E) There is no change in IL-15 between groups. (F) There is no significant change in IL-2 across groups. (G) Ccl2 is significantly increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (*** P < 0.01). (H) Cxcl10 is significantly increased in the ipsilateral cortex in both groups (*** P < 0.01; ** P < 0.01). (I) Cxcl1 is increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (*** P < 0.01). (J) IL-1 β is increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (*** P < 0.01). (K) IL-6 is increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (** P < 0.01; *** P < 0.01). (L) Tnfα is increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (*** P < 0.01). (M) Ptgs2 is increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (* P < 0.05; *** P < 0.01). (N) Mmp-9 is increased in the ipsilateral cortex in vehicle- and 10 mg/kg decernotinib-treated groups (** P < 0.01) Statistical analyses performed with a two-way ANOVA and Bonferroni post-test; vehicle n = 5, 10 mg/kg decernotinib, n = 7.

Article Snippet: Animals were treated with vehicle or 0.03, 0.1, 0.3, 1.0, 3.0, or 10 mg/kg decernotinib (Cat # CT-DECE; ChemieTek, Indianapolis, IN, USA), a highly selective JAK3 inhibitor.

Techniques:

Journal: Frontiers in Neurology

Article Title: Selective Inhibition of Janus Kinase 3 Has No Impact on Infarct Size or Neurobehavioral Outcomes in Permanent Ischemic Stroke in Mice

doi: 10.3389/fneur.2017.00363

Figure Lengend Snippet: (A) Representative immunoblot of mouse brain tissue. (B) Graphical summary of phosphorylated STAT3 immunoblot data normalized to actin (* P < 0.05; ** P < 0.01, vehicle n = 9; 10 mg/kg decernotinib, n = 5). Phosphorylated STAT3 is increased in the ipsilateral cortex compared to the contralateral. Phosphorylated STAT3 is decreased in the ipsilateral cortex with 10 mg/kg decernotinib compared to the vehicle-treated group. (C) Graphical summary of total STAT3 immunoblot data normalized to actin (vehicle, n = 9; 10 mg/kg decernotinib, n = 5). Total STAT3 does not vary between groups.

Article Snippet: Animals were treated with vehicle or 0.03, 0.1, 0.3, 1.0, 3.0, or 10 mg/kg decernotinib (Cat # CT-DECE; ChemieTek, Indianapolis, IN, USA), a highly selective JAK3 inhibitor.

Techniques: Western Blot