FIGURE 3.

The effector kinase MSK1 is important for p21 gene induction by TSA/anisomycin. Proliferating T98G cells were transfected with control siRNA oligonucleotides and siRNA oligonucleotides specifically targeting MSK1 mRNA for 5 h. Next day-transfected T98G cells were arrested by serum deprivation for 48 h. Whole cells extracts, total RNA, and chromatin were gained from control (siCtrl) and knockdown cells (siMSK1) that were left untreated or were stimulated with 188.5 nm anisomycin and 165.5 nm TSA (+ or A/T) for 1 h. A, knockdown efficiency was analyzed by Western blot using an antibody against MSK1. Antibody against β-tubulin ensured equal loading. B, p21 expression levels in control and MSK1 knockdown cells were monitored by qRT PCR using GAPDH for normalization. C, qRT-ChIP analysis was performed with antibodies specific for MSK1, H3S10phK14ac, or an unspecific control antibody (IgG) and primers specific for the proximal p21 promoter. ChIP results are shown as percentage of input; for histone modifications, the values were corrected for changes in nucleosomal density.