Minoxidil

Thirty-six-week-old C57BL/6J mice (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) were randomly assigned to 5 groups (n = 6): Normal group, normal mice without any treatment; Vehicle group, depilated mice treated with normal saline; TSG group, depilated mice treated with 200 μM TSG (National Institute for the Control of Pharmaceutical and Biological Products, China); p53 inhibitor group, which received 200 mM Pifithrin-α (Selleck Chemicals, USA); Minoxidil group, which was treated with 2% Minoxidil (Sigma, USA). All medicines were performed topically on the upper back once per day for 2 weeks. Afterwards, mice were sacrificed and dorsal skins were fixed in 4% paraformaldehyde (PFA) (Sigma) or frozen in liquid nitrogen for further study. All animal experiment protocols were approved by the Animal Experiment and Care Committee of Shanghai Jiao Tong University School of Medicine.
Five groups of mice were all treated with the same depilated model, which was induced as previously described [19 (link)]. Briefly, a melted wax/rosin mixture (1 : 1) under general anesthesia was used on the dorsal skin which could peel off all hair shafts and immediately induce all follicles to turn into homogeneous growth phase [19 (link)].

Ranolazine was obtained from Sigma-Aldrich (Dorset, UK). Minoxidil was obtained from Alfa Aesar TM (Thermo Fisher Scientific, UK). Four concentrations of Ranolazine were used: 0.625, 1.25, 2.5 and 5 μM; three concentrations of Minoxidil were used: 2.5, 5 and 50 μM. Stock solutions of Ranolazine (2 mM) and Minoxidil (31 mM) were prepared by dissolving the drugs in DMEM and 100% dimethyl sulfoxide (DMSO), respectively (Sigma-Aldrich). The stocks were kept frozen at − 20 °C until use. Control solutions for Minoxidil and combined treatment contained the final concentration of DMSO. Fresh solutions were made at desired concentrations by dilution in DMEM and warming to 37 °C prior to each experiment. Treatments were either short‐term/acute (electrophysiology) or long‐term/48 h (electrophysiology and functional assays).

HDPCs (6 × 10⁵ cells per well) were seeded on Aggrewell 800 plates (Stemcell Technologies Inc. Seattle, WA, USA) and incubated for 24 h. Next, the HaCaTs (1.2 × 10⁶ cells per well) were seeded in an HDPC spheroid in the upper compartment and incubated overnight. Five μM NO–PBS, which was diluted from a NO solution (1.9 mM), or 1 μM minoxidil (Sigma-Aldrich) was added to the medium of co-cultured HaCaT + HDPC (Co-HaCAt + HDPC) and incubated for 3 days. Each experiment was conducted in triplicate.
After harvesting the cells, the total RNA was isolated using a Qiazol lysis reagent (Qiagen, Valencia, CA, USA) and 1 μg of the isolated RNA was reverse transcribed into complementary DNA (cDNA) and used in quantitative (qPCR) assays using a SYBR Green PCR Master Mix (Thermo Fisher Scientific, Seoul, Korea) in a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Seoul, Korea). The reactions (20 μL) comprised 2 μL of diluted cDNA, 2 μL of each primer, 10 μL of 2× SYBR Green PCR Master Mix, and 6 μL of RNase-free water. The sequences of the oligonucleotide primers used in the qPCR assays are shown in Table 1. Thermocycling conditions were 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. All samples were assayed in triplicate, and mRNA levels were calculated using the 2^−ΔΔCt method.