Figure 1
Enhanced induction of the CAMP gene by HO53 and HO56 in BCi cells. (a) Structure of the two novel aroylated phenylenediamine (APD) compounds investigated in this study, HO53 and HO56. (b) Induction of the CAMP gene with increasing concentration of Entinostat, HO53 and HO56 in BCi cells after 24 h post treatment. DMSO (final concentration lower than 1%) was used as a solvent control (Solvent Ctrl). Each bar represents mean value of 3 independent experiments ± SEM; statistical significance was calculated in comparison to control cells (Ctrl) using one-way ANOVA with Dunnett’s multiple comparisons test. Time-dependent CAMP induction by (c) Entinostat (10 μM), (d) HO53 (75 μM) and (e) HO56 (75 μM) in BCi cells, respectively. Bars for 2–8 h (for HO53 and HO56) and bars for 2–12 h (for Entinostat) represent values from two technical repeats. Bars for 12–72 h (for HO53 and HO56) and bars for 24–72 h (for Entinostat) are mean values of 3 independent experiments ± SEM; statistical significance was calculated in comparison to control cells (Ctrl) using one-way ANOVA with Sidak’s multiple comparisons test. (f) Cooperation of HO53 and HO56 (both at 75 μM) with sodium 4-phenylbutyrate (PBA; 2 mM) or 1α,25-dihyroxyvitamin D3 (VitD3; 100 nM) for the induction of the CAMP gene at 24 h and 48 h. Each bar represents mean value of 3 independent experiments ± SEM; statistical significance was calculated in comparison to HO53 (red) and HO56 (black) using two-way ANOVA with Dunnett’s multiple comparisons test. (g) Concentrated culture medium after 24 h stimulation was used for Western blot analyses using monoclonal antibody against LL-37. Positive control included 2 ng of synthetic LL-37 peptide. GAPDH analyzed in cell lysates was used as a loading control. The representative Western blot is one of 3 independent experiments. The full-length blots are presented in Supplementary Figure S10a. For all qRT-PCR experiments the CAMP gene expression was normalized to TUBB (tubulin-β) reference gene and presented as fold change of the expression compared to control cells (Ctrl) set as 1. Symbols for statistical analysis indicate p-values *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
