Indirubin

Normal human adult keratinocytes (Lonza) were seeded into 48-well culture plates and cultured to passage three with an approximate density of 70–80%. Keratinocytes were stimulated with IL-17A (100 ng/mL) (R&D Systems, Inc., Minneapolis, MN) and treated with varying concentrations of isatin, tryptanthrin, and indirubin (Sigma-Aldrich, St. Louis, MO), and a control compound, PD 0325901 [33 (link)]. PD 0325901 is a mitogen-activated protein kinase kinase (MEK) inhibitor; a similar compound has been demonstrated to inhibit IL-17-induced cytokine release [34 (link)]. Cell viability was confirmed using the PrestoBlue® assay (Thermo Fisher Scientific, Waltham, MA). tryptanthrin showed no impact on cell viability at all testing concentrations. Tissue culture supernatants were analyzed for pro-inflammatory cytokines IL-6 and IL-8 using MesoScale Discovery V-plex assay (MesoScale Discovery, Rockville, MD). Data were analyzed using Graphpad Prism v5.0 (http://www.graphpad.com).

Aryl-hydrocarbon receptor activity was measured using a stably transfected gene reporter human cell line AZ-AhR30 (link). Briefly, 2 × 10⁴ cells/well were seeded in 96-well plates and incubated for 18 hours at 37 °C and 5% CO_2, before cells were stimulated 18 h in triplets with acrolein, cinnamaldehyde, crotonaldehyde, propanal and methacrolein (all from Sigma) in increasing concentrations (0.018/0.18/1.8/9/18/45/90/180 μM) was used as negative control, whereas indirubin (0.03 μg/ml, Sigma) was used as a positive control and as AhR-antagonists resveratrol 100 μM (Sigma) and 3′-methoxy-4′-nitroflavone 5 μM (Sigma). After adding lysis buffer and a single freeze-thaw cycle, 20 μl/well of lysates were transferred into a black 96-well flat-bottom plate (Thermo Scientific) and bioluminescent reaction were started with addition of 100 μl/well of luciferase assay reagent (Promega). Chemiluminscence was measured (10 sec/well) using a spectrophotometer Tecan InfiniteM200 PRO.

The chemical compounds derived from CMAP screening were purchased from Chemical Biology Technology Platform of Chinese Academy of Sciences (shanghai, china) and applied in a dose of 10 μM (final concentration) for each drug within cell experiments. Indirubin used in animal treatment was purchased from MedChemExpress (Catalog No.: HY-N117; CAS No.: 479–41-4; Purity: >98%, Monmouth Junction, NJ). Indirubin was first dissolved in dimethyl sulfoxide (DMSO) and stored in the dark at − 20 °C. During our experiments, Indirubin was diluted into corresponding concentration with the Corn oil (Sigma-Aldrich). All the other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified. The primary antibodies included anti-UCP1 (Abcam, ab10983 and ab23841), anti-PGC1α (Santa Cruze Biotechnology, sc-13,067), anti-OXPHOS (Abcam, ab110413), anti- phospho-(Ser/Thr) PKA Substrate (Cell Signaling Technology, #9621), anti-phospho-CREB (Cell Signaling Technology, #4276S, San Antonio, TX, USA), anti-VDAC1 (Cell Signaling Technology, #4661), anti-β-Tubulin (Cell Signaling Technology, #2146).