Quantification of ER Stress Markers in HepG2 Cells

The RNA extraction obtained from HepG2 cells was accomplished using 1 mL of Trizol (Beijing Lan Y Science & Technology) per well in 6-well plates. Reverse transcription was performed using the PrimeScript^TMRT Master Mix kit (TaKaRa, Otsu, Japan); real-time PCR was conducted using the TB Green^® Premix Ex Taq^TM Ⅱ kit (TaKaRa, Otsu, Japan) and tested with the ABI 7500 Real-Time PCR System. The mRNA expressions of the target genes included CHOP, 78 kDa glucose-regulated protein (GRP78), activating transcription factor 6 (ATF6), protein disulfide isomerase family A member 6 (PDIA6), BAX, BCL2, and CASP3. Primers were designed with Primer Premier 6 (Premier Biosoft, CA, USA) and the primer sequences are illustrated in Table 2. The results were normalized to the β-actin gene expression, and the mRNA expression was calibrated with the CON value. Fluorescence results were calculated in relation to the β-actin CT value using the 2ΔΔCT method [41 (link),42 (link),43 (link)].