cDNA synthesis and qPCR assay
For samples from experiment II, total RNA was extracted as described above and cDNA synthesis was conducted using the SuperScript IV VILO Master Mix (Invitrogen, USA). Synthesized cDNA was subsequently subjected to a qPCR assay comprising 10 stress genes (FAS, CASP8, FGFR2, FGF2, RHO, MMP19, MMP24, JNK, NFKB1, and XIAP; Table 2) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA). A β-tubulin gene (TUBB) was selected as the internal control for the qPCR analysis. For each qPCR reaction, 200 μM of each primer and 1 μL of synthesized cDNA (concentration undetermined) were mixed with iQ SYBR Green Supermix (Bio-Rad, USA) to a total volume of 10 μL. qPCR conditions included an initiation step at 95°C for 3 min and 40 cycles of PCR amplification at 95°C for 15 sec and 60°C for 30 sec. A melting curve analysis (MCA) was conducted after qPCR to confirm the specificity of each primer pair. For each target stress gene, the mean ∆CT value (CTcontrol gene – CTtarget stress gene) from two technical replicates was used to calculate relative gene expression (log2-transformed). ∆CT values of the treatment group were then normalized to those of the control group (on average of three replicates) and to the initial conditions (0 h) to determine expression changes during experiments (∆∆∆CT = ∆∆CTgiven time point– ∆∆CT0 h; ∆∆CT = ∆CTtreatment group – ∆CTcontrol group). Data are presented as means ± SD (standard deviation) for each gene, and statistical significance between time points was tested using Welch’s ANOVA with a Games-Howell post-hoc test.