DMSO served as a solvent control. To investigate whether inhibition of HDAC8 might be counteracted by concomitant upregulation of other class I-HDACs (HDAC1, HDAC2 and HDAC3) their

expression levels were compared by real-time PCR and western blot analysis (Figures 11 and 12). In brief, HDAC1, HDAC2 and HDAC3 mRNA levels exhibited variable changes after siRNA-mediated knockdown of HDAC8. Both significant up-and downregulation of specific HDACs were observed. In LDN-193189 supplier particular, either HDAC1 or HDAC2 seems to become PCI-32765 mw upregulated after HDAC8 knockdown (Figure 11A). Western blot analysis shown in Figure 11B revealed a decrease of HDAC2 protein in RT-112 cells and HDAC3 protein in UM-UC-3 cells after siRNA mediated HDAC8 knockdown. No significant AS1842856 deregulation of other class I-HDACs took place (Figure 11B). Figure 11 Compensation mechanism after HDAC8 knockdown in RT-112, VM-CUB1,

SW-1710, 639-V and UM-UC-3 cells. Effects of siRNA mediated HDAC8 knockdown on (A) mRNA and (B) protein expression levels of the class I histone deacetylase HDAC8, HDAC1, HDAC2 and HDAC3 (72 h) in comparison to untreated and irrelevant control. The mRNA expression values were measured by quantitative RT-PCR analysis and were normalized to TBP as a reference gene. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. Protein expression levels were analyzed by western blotting, and α-tubulin was stained on each blot as a loading control. Figure 12 Compensation mechanism after specific HDAC8 inhibition in RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells. Effects of HDAC8 inhibitor treatment on (A) mRNA and (B) protein expression of the class I histone deacetylases HDAC8, HDAC1, HDAC2 and HDAC3, compared

to DMSO solvent control (compound 2, compound 5, compound 6; IC50, 72 h). The mRNA expression values were measured by quantitative RT-PCR analysis and were normalized to TBP as a reference gene. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances of the treated Benzatropine value refer to the DMSO solvent control. Protein expression levels were analyzed by western blotting, and α-tubulin was stained on each blot as a loading control. Measurements of mRNA expression after pharmacological inhibition of HDAC8 showed significant, but overall slight decreases or increases of the expression of several HDACs in the UCC (Figure 12A). Apart from a slightly reduced expression of HDAC1 and HDAC2/3 in SW-1710 and VM-CUB1 cells, no changes of protein expression were observed after c5 and c6 treatment (Figure 12B).