BORIS-knockdown using lentivirus with doxycycline inducible BORIS-specific shRNA.

Four different BORIS shRNA lentiviral vectors (BORIS sh-1, sh-2, sh-3 and sh-4) and the vector carrying a scrambled sequence (CTR sh) were tested. NCCIT were transduced with the indicated lentivirus and sorted for eGFP marker expression. Then, cells were cultured with doxycycline-containing medium and after 3 days were analyzed. (A) BORIS mRNA levels were determined by qRT-PCR analysis, normalized to GAPDH and compared to that of CTR sh. Error bars represent the mean ± SD of 3 independent experiments. Asterisks indicate statistically significant difference (p<0.05) between BORIS sh-1, sh-2, sh-3 or sh-4 and CTR sh cells. (B) Representative western blot analysis. BORIS and β-actin (as a loading control) protein levels were determined by western blot. For both analyses (qRT-PCR and western blot) the knockdown was especially noticed with the BORIS sh-3 and sh-4 compared to the CTR shRNA.