RNA extraction, first-strand cDNA synthesis and the design of primers were as described previously (7 (link)). The primers used for qPCR were as follows: GAPDH (107 bp product; internal control), 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ (forward) and 5′-TCCTTGGAGGCCATGTGGGCCAT-3′ (reverse); PAR4 (147 bp product), 5′-CCTTCATCTACTACTACTACGTGTCG-3′ (forward) and 5′-ACTGGAGCAAAGAGGAGTGG-3′ (reverse). qPCR for PAR4 and GAPDH were performed using an SYBR Green Real-Time PCR kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the conditions as follows: Initial denaturation at 95°C for 1 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each sample was run three times. Products were analyzed using a continuous fluorescence detector with Opticon Monitor 3.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). No-template controls (no cDNA in PCR) were included to detect non-specific or genomic amplification and primer dimerization. Relative quantitative evaluation of PAR4 was performed using the E-method (6 (link),7 (link)) and expressed as a ratio of the transcript of PAR4 to GAPDH in the different cell lines. The identities of qPCR products were confirmed by DNA sequencing.