Fig. 2

Cellular stability of YY1 mRNA and protein. Influence of the transcriptional inhibitor actinomycin-D (ACT-D, 5 μg/mL) (a-d) and of the translational inhibitor cycloheximide (CHX, 100 μg/mL) (e, f) on YY1 expression in the B-NHL cell lines RAMOS (a, b and e) and U2932-R2 (c, d and f). a YY1 mRNA quantification relative to GAPDH by qRT-PCR after 0 h, 30 min, 1 h, 2 h, 3 h and 6 h of ACT-D treatment of RAMOS and (c) of U2932-R2. Error bars indicate 95% confidence interval of the mean expression. MYC levels were analyzed as a positive control for a fast turnover mRNA. b Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h of ACT-D treatment in RAMOS and (d) in U2932-R2. MYC was used as a positive control for a fast turnover protein and GAPDH as loading control. The arrow indicates the caspase-cleaved YY1 form. e Western blot analysis of YY1 protein after 0 h, 6 h, 10 h, 24 h, 34 h and 48 h CHX treatment of RAMOS and (f) of U2932-R2. MYC expression was determined as a positive control for a fast turnover protein and GAPDH and histone 3 (H3) served as loading controls. The arrow indicates the caspase-cleaved YY1 form. Numbers underneath the blots refer to the relative amount of YY1 normalized to GAPDH levels according to densitometric analyses of the blots. The 0 h sample was set to 1 at each time point, treatment and cell line