Gene Expression Analysis Protocol

Total RNA extraction was performed as previously described [38 (link)]. First-strand cDNA was synthesized with the PrimeScript first-strand cDNA Synthesis Kit (TaKaRa Bio Inc., Shiga, Japan) as previously described [35 (link)]. qPCR was performed as previously described [39 (link)]. The following primer sequences were used: human p21, 5′-GATTTCTACCACTCCAAACGCC-3′ (forward) and 5′-AGAAGATGTAGAGCGGGC-3′ (reverse) [40 (link)]; human Sestrin2, 5′-GACCATGGCTACTCGCTGAT-3′ (forward) and 5′-GCTGCCTGGAACTTCTCATC-3′ (reverse) [41 (link)]; human HPRT1, 5′-TTTGCTTTCCTTGGTCAGGC-3′ (forward) and 5′-GCTTGCGACCTTGACCATCT-3′ (reverse) [40 (link)]. The specificities of the detected signals were confirmed by a dissociation curve, which consisted of a single peak. Values were normalized by HPRT1.