ARPE-19 cells were treated with human recombinant IL-17A (Catalog No. 7955-IL/CF, R&D Systems, Abingdon, UK) for 6 days of treatment. Overall, 50 ng/mL IL-17A was sufficient for tight junction dysmorphia and barrier dysfunction [48 (link)]. Then 30 min treatment with the same dose was used to examine JAK1 phosphorylation in response to IL-17A.
The bEnd.3 cells were treated with murine recombinant IL-17A (R&D Systems, Catalog No. 7956-ML-025/CF), at a concentration of 100 ng/mL. Three days of treatment with 100 ng/mL IL-17A was sufficient for tight junction dysmorphia and barrier dysfunction [49 (link)]. Thirty minutes of treatment with the same dose was used to examine JAK1 phosphorylation in response to IL-17A. Tofacitinib Citrate (Catalogue No. PZ0017, Sigma-Aldrich, St. Louis, MO, USA) (25 mg) was dissolved in 100 µL DMSO and further diluted in PBS, immediately before use, to 2.5 ug/mL (4.955 µM). Vehicle control for Tofacitinib was DMSO diluted 1:100,000 in PBS, i.e., 0.00001% DMSO.
Cell viability was assessed by using AlamarBlue™ Cell Viability Reagent (Thermo Fisher, Catalog No. DAL1100, Thermo Fisher, Waltham, MA, USA). Cells were regularly screened for mycoplasma, using LookOut® Mycoplasma PCR Detection Kit (Catalog No. MP0035, Sigma-Aldrich).
The bEnd.3 cells were treated with murine recombinant IL-17A (R&D Systems, Catalog No. 7956-ML-025/CF), at a concentration of 100 ng/mL. Three days of treatment with 100 ng/mL IL-17A was sufficient for tight junction dysmorphia and barrier dysfunction [49 (link)]. Thirty minutes of treatment with the same dose was used to examine JAK1 phosphorylation in response to IL-17A. Tofacitinib Citrate (Catalogue No. PZ0017, Sigma-Aldrich, St. Louis, MO, USA) (25 mg) was dissolved in 100 µL DMSO and further diluted in PBS, immediately before use, to 2.5 ug/mL (4.955 µM). Vehicle control for Tofacitinib was DMSO diluted 1:100,000 in PBS, i.e., 0.00001% DMSO.
Cell viability was assessed by using AlamarBlue™ Cell Viability Reagent (Thermo Fisher, Catalog No. DAL1100, Thermo Fisher, Waltham, MA, USA). Cells were regularly screened for mycoplasma, using LookOut® Mycoplasma PCR Detection Kit (Catalog No. MP0035, Sigma-Aldrich).
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