Exicycletm 96

Total RNA from the tissues was isolated using a rapid extraction method (TRI-Reagent, Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on cDNA samples using the iQTM SYBR Green Supermix system (Bio-Rad, Hercules, CA, USA). The protocols used are as follows: denaturation at 95 °C for 2 min, followed by 40 cycles of amplification at 95 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s, and an acquisition temperature for 15 s (Bioneer ExicycleTM 96, Daejeon, Korea). The analysis was conducted using the sequence detection software supplied with the instrument. For each sample, the delta-delta cycle threshold cycle (ddCT) (crossing point) values were calculated as the Ct of the target gene minus the Ct of the β-actin gene. Gene expression was derived according to the equation 2-ddCt; changes in gene expression are expressed relative to basal levels. The primer sequences are as follows: m-FoxO6, as-TTC AGC ATC CAC CAT GAA CT, ss-GAA GAG CTC CCG ACG GAA CG; and m-b-actin, as-GGC TGT ATT CCC CTC CAT CG, ss-CCA GTT GGT AAC AAT GCC ATG T.

RNA was extracted from liver and spleen tissues of mice. RNA concentration was determined under ultraviolet spectrophotometer NANO 2000 (Thermo Fisher Scientific, Pittsburgh, PA, USA). CDNA
was obtained by beyoRT II M-MLV reverse transcriptase (Beyotime). Finally, cDNA was quantified under ExicycleTM96 fluorescence quantification instrument (Bioneer, Daejeon, Korea). The
quantitative results were calculated by 2^-ΔΔCt method. The primer sequences used in this study were as follows: MUS IL-17A-F: 5′-AAACACTGAGGCCAAGGAC-3′; MUS
IL-17A-R: 5′-CGTGGAACGGTTGAGGTAG-3′; MUS IL-10-F: 5′-TTAAGGGTTACTTGGGTTGC-3′; MUS IL-10-R: 5′-GAGGGTCTTCAGCTTCTCAC-3′.