Faststart universal master mix

Total RNA was extracted from tissue samples and BCSCs using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. To assess miR-376c-3p expression levels, the TaqMan MicroRNA Assay kit (cat. no. 4427975; Assay ID: 002122; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for reverse transcription and qPCR according to the manufacturer's protocol (primer sequences were withheld by the supplier). For RT-qPCR analysis of RAB2A mRNA, total RNA was reversed transcribed into cDNA using the GoScript Reverse Transcription system (Promega Corporation). Subsequently, qPCR was performed using FastStart Universal Master Mix (Roche) and a StepOnePlus real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 40 cycles of denaturation at 94˚C for 30 sec, annealing at 58˚C for 30 sec and extension at 72˚C for 60 sec. The following primers were used for qPCR: RAB2A forward, 5'-AGTTCGGTGCTCGAATGATAAC-3' and reverse, 5'-AATACGACCTTGTGATGGAACG-3'; GAPDH forward, 5'-TGCACCACCAACTGCTTAGC-3' and reverse, 5'-GGCATGGACTGTGGTCATGAG-3'; and U6 forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'. Samples were quantified using the 2^-∆∆Cq method (32 (link)). miRNA and mRNA expression levels were normalized to the internal reference genes U6 and GAPDH, respectively.

Paeonol was provided by the National Institutes for Food and Drug Control (Beijing, China). DNCB (cat. no. 237329; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in acetone and olive oil (3:1 v/v). Paeonol was dissolved in normal saline to achieve various concentrations for oral administration. Prednisolone (Pred; cat. no. BP464; Sigma-Aldrich; Merck KGaA) was dissolved in normal saline to a concentration of 10 mg. TRIzol^® was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The NucleoSpin^® RNA Clean-up kit was obtained from Macherey-Nagel GmbH & Co. KG (Düren, Germany). The real-time polymerase chain reaction (RT-PCR) FastStart Universal Master mix was purchased from Roche Diagnostics (Indianapolis, IN, USA) and the AffinityScript Multiple Temperature cDNA Synthesis kit was purchased from Agilent Technologies, Inc. (Santa Clara, CA, USA).

RT-qPCR was conducted to detect the mRNA expression levels of various cytokines, according to the manufacturers' protocols, and the conditions were similar to those described in a previous study (16 (link)). Total RNA was isolated from the excised skin using TRIzol^® and purified using the NucleoSpin^® RNA Clean-up kit. Following generation of cDNA using the AffinityScript Multiple Temperature cDNA Synthesis kit according to the manufacturer's protocol. The real-time PCR FastStart Universal Master Mix (Roche Diagnostics) was used to determine the relative expression levels of genes with an ABI 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The gene-specific primers are listed in Table I. The cycle parameters were as follows: 95°C for 10 min, followed by 50 cycles at 95°C for 15 sec and 60°C for 60 sec. β-actin was used as a reference gene to normalize the data, which were quantitatively analyzed using the 2^−ΔΔCq method (19 (link)).