Ssofast evagreen supermix detection system

Chromatin immunoprecipitation (ChIP) analysis was performed using the EZ-ChIP kit according to the manufacturer’s instructions (Millipore) as described previously (Li et al., 2013 (link); Meeran et al., 2010 (link), 2012 (link)). The ChIP-validated anti-acetyl-histone H3 (AcH3), anti-acetyl-histone H4 (AcH4), anti-acetyl-histone H3K9 (AcH3K9), anti-trimethyl-histone H3K9 (H3K9me3), anti-trimethyl-histone H3K27 (H3K27me3) (Millipore) and anti-MeCP2 (Abcam, Cambridge, MA) antibodies were used at 5 μg/mL dilutions. Input DNA was used as an internal control and no specific antibody IgG control was used to check ChIP efficiency. ChIP-purified DNA was quantified by using real-time PCR using primers spanning the CpG-rich region of the distal promoter regions of p21^CIP1/WAF1 and KLOTHO. PCR amplification was performed in a Light Cycler (Roche) using SsoFast EvaGreen Supermix detection system (Bio-Rad). The sequences of primers with their respective annealing temperature (T_A) are given in Supplementary Table S3. Real-time PCR amplification was performed for 30 s at 94 °C followed by 45 cycles (5 s at 94 °C; 20 s at T_A °C; 30 s at 72 °C).

Human breast cancer MDA-MB-231 and MDA-MB-453 cells were treated with either GTPs or SFN or the combinations of both at the indicated concentrations for 12, 24, 48 and 72 h. Total RNA was extracted at approximately 70–80% confluence using the Trizol–RNA isolation protocol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Two micrograms of RNA was reverse-transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time quantitative PCR was carried out in a lightcycler-480 thermocycler (Roche, Germany) using SsoFast EvaGreen Supermix detection system (Bio-Rad). The primers specific for p21^CIP1/WAF1, KLOTHO and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were obtained from Sigma-Aldrich. The sequences of primers with their respective annealing temperatures (T_A) are given in Supplementary Table S1. The reaction conditions were 30 s at 94 °C followed by 45 cycles (5 s at 94 °C; 20 s at T_A °C; 30 s at 72 °C) and extension at 72 °C for 2 min. The relative levels of mRNA expression were calculated using the cycle threshold (C_t) method: 2^−ΔΔCt = 2^{{ΔCt (treated samples)−ΔCt (untreated control)}}, where ΔC_t = C_t (Gene of interest) − C_t (GAPDH).