Validated specific primers

Total RNA from the K562 cell line was extracted using an Aurum Total RNAMini Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. 0.5 mg of total RNA was then reverse-transcribed to generate cDNA for PCR by using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). Semi-quantitative determination of mRNA levels was performed by real-time qRT-PCR using SYBR Green (Bio-Rad, Hercules, CA, USA). Reactions were performed in duplicate for each sample. Multiple reactions were performed in a volume of 20 μL containing 10 μL of 2 × PCR master mix, 1X of validated specific primers (Qiagen, Hilden, Germany) (see Table 1), and 2 μL of cDNA template. Amplifications were performed in the Stratagene MX3000P Real-Time PCR Detection System (Stratagene, San Diego, CA, USA), using the following cycle program: denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 1 min, and extension at 72 °C for 30 s. The relative mRNA expression levels were calculated using the comparative CT method (2^-ΔΔCT). Quantitative normalization for each sample was performed by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Qiagen, Hilden, Germany) as an internal control.

The purification of total RNA from fibroblasts was carried out by using Aurum Total RNAMini Kit (Bio-Rad, Hercules, California, USA) according to the manufacturer’s protocol. 0.5 microgram of total RNA was then reverse-transcribed to generate cDNA for PCR by using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, California, USA). Semi-quantitative determination of mRNA levels were performed by real-time qRT-PCR, using SYBR Green (Bio-Rad, Hercules, California, USA). Reactions were performed in duplicate for each sample. Multiple reactions were performed in a volume of 20 μL containing 10 μL of 2× PCR master mix, 1× of validated specific primers (Qiagen, Venlo, The Netherlands), and 2 μL of cDNA template. Amplifications were performed in the Stratagene MX3000P Real-Time PCR Detection System (Stratagene, San Diego, CA, USA), using the following cycle program: denaturation step at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 1 min, and extension at 72 °C for 30 s. The relative mRNA expression levels were calculated by using the comparative CT method (ΔΔCT). Quantitative normalization for each sample was performed by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Validated primers (Qiagen, Venlo, The Netherlands) for qRT-PCR are provided in Table S1.