*2.14. mRNA Expression Rate*

Total RNA was isolated from cells at specific times using TRIzol reagent (Invitrogen, Waltham, MA, USA) with DNase treated with RNase-free DNase (35 U/mL) for 50 min at 37 ◦C according to the method described by Liu et al. [43] and Matsubara et al. [44]. The total RNA present in each sample was quantified by measuring the absorbance at 260 nm using a spectrophotometer (Eppendorf AG 22331; Hamburg, Germany). Gene expression was measured by adding cDNA to a PCR mixture containing EXPRESS SYBR Green qPCR Supermix (Bio Prince, Seongsu, Seoul, Republic of Korea). Real-time PCR was performed using the Rotor-Gene Q (Qiagen, Düsseldorf, Germany). The reaction was carried out at 95 ◦C for 20 s, 60 ◦C for 20 s, and 72 ◦C for 25 s for 40 cycles of amplification. Relative

mRNA expression of specific genes was standardized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and sequencing was performed using PCR primer sequences (Table 1).


**Table 1.** Forward and reverse primers sequences used in this study.
