Klotho sirna

Human anagen hair follicles were transfected with klotho siRNA (Invitrogen) or control siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using transfection reagent (Santa Cruz Biotechnology) at day 0. After transfection, hair follicles were maintained in a 24-well plate with 500 µl William's E medium (Biochrom, Cambridge, UK) supplemented with 10 µg/ml insulin (Sigma-Aldrich Inc.), 10 ng/ml hydrocortisone (Sigma-Aldrich Inc.), 2 mM L-glutamine (Gibco BRL), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco BRL). The efficiency of the blockage was evaluated by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis.

PPARγ siRNA, Klotho siRNA and control scrambled siRNA (Invitrogen; Thermo Fisher Scientific, Inc.) were transfected with Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were seeded in 24-well plates at 5×10⁴/well and were grown overnight to reach ~80% confluence. The cells were transfected with 30 pmol siRNA and incubated for 48 h, and used for subsequent experiments after the transfection efficiency was confirmed by western blot analysis.

This study was approved by the Institutional Ethics Committee of the Yonsei University Wonju College of Medicine (IRB no. YWMR-13-6-053), and all subjects provided were written informed consents. Scalp samples were taken from the occipital areas of healthy subjects who had not prior or current scalp diseases. Hair follicles were separated and cultured as described previously14 (link). Each of the anagen hair follicles was carefully transferred to a 24-well plate (Corning, New York, NY, USA) containing 500 µl of William's E medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10 µg/ml insulin (Sigma-Aldrich Inc., St. Louis, MO, USA), 10 ng/ml hydrocortisone (Sigma-Aldrich Inc.), 2 mM L-glutamine (Gibco BRL), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco BRL). Test groups were treated with or klotho siRNA (Invitrogen, Carlsbad, CA, USA) or serial dose of klotho protein (R&D Systems, Minneapolis, MN, USA).