Quantity one analysis software version 4

The total RNA was isolated using RNA fast 2000 kit (Fastagen, Shanghai, China) according to the manufacturer's protocols. The RNA was subsequently reverse transcribed into cDNA using Prime Script RT Master Mix Perfect Real-Time kit (DRR036A; Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols. The primers sequences used (Sangon Biotech Co., Ltd., Shanghai, China) were: β3, forward 5′-GCCAGCACCATCTCTTTACC-3′ and reverse 5′-GCACTCTCTCCCTTTGAGGA-3′, with a length of 112 bp; β-actin, forward 5′-TGACGTGGACATCCGCAAAG-3′ and reverse 5′-CTGGAAGGTGGACAGCGAGG-3′, with a length of 205 bp. The cycling protocol for PCR involved incubating the samples at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, with a final cycle of incubation at 72°C for 2 min. The amplification products were analyzed by electrophoresis (Beijing Junyi, Beijing, China) in agarose gels and detected under UV illumination (Bio-Rad Laboratories, Inc., Hercules, CA, USA) after staining with nucleic acid dye (DuRed; FanBo Biochemicals, Beijing, China). Images were analyzed using a quantitative analysis system (Quantity One Analysis Software, version 4.6.2; Bio-Rad Laboratories, Inc.).