Tofacitinib citrate

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).

Tofacitinib citrate (MW:504.49) and hydrocortisone (MW:362.46) as an internal standard for high-performance liquid chromatography (HPLC) were obtained from Sigma Aldrich (St. Louis, MO, USA). ISN was donated by Professor Hyungsu Kim (Ajou University, Suwon, Republic of Korea). Ethyl acetate and acetonitrile for the quantification of tofacitinib by HPLC analysis were purchased from J.T. Baker (Phillipsburg, NJ, USA). DMN was supplied by Tokyo Chemical Industry (Tokyo, Japan), and β-cyclodextrin was obtained from Wako (Osaka, Japan). The NaCl-injectable solution and heparin were purchased from JW Pharmaceutical Corporation (Seoul, Republic of Korea). Antibodies against CYP3A1/2, CYP2E1, CYP2C11, CYP2D1, CYP2B1/2, and CYP1A1/2 were donated by Detroit R&D Inc. (Detroit, MI, USA), and primary antibodies against P-gp, pregnane X receptor (PXR), and constitutive androstane receptor (CAR) were purchased from Abcam (Cambridge, UK). β-Actin, used as a loading control, was supplied by Cell Signaling Technology (Beverly, MA, USA). Goat, rabbit, and mouse secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA). All the reagents and other chemicals were of HPLC or analytical grade and were used without further purification.

Tofacitinib citrate and hydrocortisone (an internal standard) were obtained from Sigma Aldrich (St. Louis, MO, USA), and ethyl acetate for high-performance liquid chromatography (HPLC) analysis was purchased from J.T. Baker (Phillipsburg, NJ, USA). Gentamicin and cisplatin were obtained from Shin Poong Pharmaceutical (Seoul, Korea) and Tokyo Chemical Industry (Tokyo, Japan), respectively. Heparin and 0.9% NaCl-injectable solution were purchased from JW Pharmaceutical Corporation (Seoul, Korea), and β-cyclodextrin is a product of Wako (Osaka, Japan). Primary antibodies to CYP2B1/2, CYP1A1/2, CYP2D1, CYP2C11, CYP2E1, and CYP3A1/2 were produced by Detroit R&D Inc. (Detroit, MI, USA). β-actin was purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary goat, rabbit, and mouse antibodies were purchased from Bio-Rad (Hercules, CA, USA). All other chemicals and reagents were analysis- or HPLC-grade and used without further purification.

Tofacitinib citrate and hydrocortisone, the internal standard for high-performance liquid chromatography (HPLC) analysis, were obtained from Sigma-Aldrich (St. Louis, MO, USA), and ethyl acetate was from J.T. Baker (Phillipsburg, NJ, USA). Heparin and 0.9% NaCl-injectable solution were purchased from JW Pharmaceutical Corporation (Seoul, Korea), and β-cyclodextrin was from Wako (Osaka, Japan). All other chemicals were HPLC grade and were used without further purification.

All reagents were obtained in the highest purity available. These included Stattic, Tempol, Tofacitinib citrate, PEG-Catalase, Angiotensin II and Ebselen from Sigma, DETA-NONOate from Cayman Chemical Company, and the anti-IL-6 neutralizing antibody was from BioLegend (clone MQ2-13A5). l-Nitroarginine methyl ester (l-NAME) was from Abcam.