Fig. 5.

Contribution of the co-factor HDAC1 to the TBX2 mediated repression of ADAM10 gene expression. a Overexpression of HDAC1 and TBX2 in SH-SY5Y cells was verified by western blot. A non-specific band in the HDAC1 detecting blot part is marked with *. Actin was detected as loading control. b TBX2 and HDAC1 expression plasmids or empty vector were transiently transfected together with ADAM10-promoter luciferase plasmid. Relative light units (RLU) obtained from cell lysates were normalized to protein amount and values for controls set to 100%. Values are given as mean ± SD from three independently conducted experiments in triplicate (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, One-way ANOVA, Bonferroni post-test). c Inhibition of HDACs was achieved by pharmacological intervention using 2.5 µM or 0.5 µM entinostat respectively. Functionality of inhibitors was demonstrated by detection of acetylated histone H3 (Lys9) as compared to respective total histone or Actin via western blot (20 µg of proteins per lane). DMSO served as solvent control. d Cells were transiently transfected with TBX2 expression vector or empty vector (mock) together with ADAM10-promoter reporter construct and subsequently incubated with entinostat in indicated concentrations or solvent (DMSO) for 24 h. Obtained luminescence values were normalized to protein content of cell lysates and set in relation to empty vector transfected cells. Results are represented as mean ± SD from three independent experiments performed in triplicate. (ns p > 0.05, ***p < 0.001, One-way ANOVA, Bonferroni post-test)