2.1 Cell culture
The human embryonic kidney 293T (HEK293T) cells (ATCC Cat#CRL‐3216; RRID: CVCL-0063), the human keratinocyte (HaCaT) cells (ATCC Cat#CRL-2309; RRID: CVCL-0038), and the mouse embryonic fibroblasts (NIH3T3) cells (ATCC Cat# CRL-1658, RRID:CVCL_0594) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA) and sustained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, Cat#C11995500BT, Waltham, MA, USA) accompanied with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Cat# 10270-106, Shanghai, China). Cells were cultured with 120 µg/mL penicillin along with 200 µg/mL streptomycin (Thermo Fisher Scientific, Cat# 15-140-122, Shanghai, China) at 37 °C in an incubator with 5% CO2. ISX9 was dissolved in dimethyl sulfoxide (DMSO) for provision of a stock solution at a final concentration of 20 mM. For treating cells, 20 mM stock solution was diluted with the specific media with respect to cells, and the final DMSO concentration was <0.1%.
2.2 Plasmids and screening of drug library
Following the manufacturer’s instruction, the transfection of HEK293T and HaCat cells were performed using Lipofectamine 2000 transfection reagent (Roche Diagnostics GmbH, Cat#11668-019, Mannheim, Germany). The reporter plasmids SuperTOPFlash was modestly gift of Karl Willert (University of California, San Diego, CA, USA) while SuperFOPFlash was bought from Beyotime (Beyotime Biotechnology, Cat# D2503, Shenzhen, China). For the construction of C-terminally V5-tagged LRP6 expression plasmid, the cDNA encoding LRP6 was amplified by RT-PCR and cloned into the pcDNA 3.1/V5-His mammalian expression vector. The N-terminally tagged Flag-Axin1 expression plasmid was built by inserting the conforming cDNA into the pFlag-CMV2 expression vector.
For drugs screening, HEK293T cells were fully-fledged in 10 cm plates. After 24 h, achieving 50% convergence, cells were transfected with 5 µg of SuperTOPFlash or SuperFOPFlash reporters, hauler DNA pcDNA3 and 1 µg of control-plasmid pCMX-β-gal (β-galactosidase, β-gal) amassing a total of 10 µg/plate. After 24 h-transfection, cells were placid and disseminated in 96-well microtiter plates. Finally, cells were treated with different drugs for the preliminary screen. Exploiting a microtiter plate luminometer, the 24 h-incubated cells were lysed and activities of luciferase were detected using a luciferase assay kit (Promega Cat# E1501, Madison, WI, USA). Making use of the β-galactosidase as an internal control, the values from luciferase assay were normalized for distinctions in transfection efficiency.