Quantitative Analysis of STAT3 Target Genes

MDA-MB-468 cancer cells were treated with 0, 50, or 100 μM BENDA for 8 h. Total RNA was extracted with RNeasy kits (Qiagen, Valencia, CA, USA) and reverse-transcribed to cDNA with the One-Step SYBR PrimeScript RT-PCR Kit II (Takara bio, Shiga, Japan). PCR amplification was performed under the following conditions: 5 min at 42°C, 10 s at 95°C, 40 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, and a final 5-min extension at 72°C. The DNA sequences of the primers for the STAT3 downstream target genes used for RT-PCR analysis were c-myc (forward: 5’-TACCCTCTCA ACGACAGCAG-3’, reverse: 5’-TCTTGACATT CTCCTCGGTG-3’) [29 (link)], cyclin D1 (forward: 5’-GCTGGAGCCC GTGAAAAAGA-3’, reverse: 5’-CTCCGCCTCT GGCATTTTG-3’), survivin (forward: 5’-ACCAGGTGAG AAGTGAGGGA-3’, reverse: 5’-AACAGTAGAG GAGCCAGGGA-3’), Bcl-2 (forward: 5’-TCTTTGAGTT CGGTGGGGTC-3’, reverse: 5’-TGCATATTTG TTTGGGGCAGG-3’), and GAPDH (forward: 5’-TGATGACATC AAGAAGGTGG TGAAG-3’, reverse: 5’-TCCTTGGAGG CCATGTGGGC AT-3’) [30 (link)]. The mRNA expression levels of the STAT3 target genes in cells treated with BENDA or HP2 were evaluated by quantitative RT-PCR (PikoReal, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Primers were obtained from Sigma-Aldrich (St. Louis, MO, USA).