COX-2 and EP-1R mRNA measurements

The remaining half of the collected samples were used for reverse transcription–polymerase chain reaction (RT-PCR). Briefly, the sample was homogenized in RNAiso Plus (Takara Bio Inc., Otsu, Japan) and RNA was extracted from homogenate supernatants according to the manufacturer's instructions. After RNA concentrations were determined spectrophotometrically, 1 μg of total RNA was reverse transcribed using oligo dT primer and PrimeScript RT Enzyme Mix I (Takara Bio Inc., Otsu, Japan). PCR was performed using SYBR Premix Ex Taq™. The primers used for PCR and the detail PCR amplification are described in Supplementary File 2. The relative expression of COX-2 mRNA and EP-1R mRNA were analysed using the 2^–ΔΔCT method as previously described.25, 26