Figure 3.

Butyrate increases SELENBP1 promoter activity and mRNA levels in parallel with the induction of differentiation in proliferating Caco-2 cells. The cells were cultured for one day and then incubated with sodium butyrate for 48 h at the indicated concentrations. (A) Cell viability was assessed by Neutral Red assay (untreated control set to 100%). (B) To monitor successful differentiation, enzymatic activity of ALPI was assessed photometrically in cell lysates. (C) Cells were co-transfected with hSELENBP1-luc and pRL-SV40 plasmids the day after seeding and then incubated with butyrate for another 48 h. SELENBP1 promoter activity was determined as relative luciferase activity (the untreated control was set as reference), with normalization against renilla luciferase activity. (D) Relative mRNA levels (with the untreated control set as the reference point) of the H₂S-modulating enzymes and the differentiation marker ALPI in cells treated for 48 h with butyrate were determined as described in Figure 2. Three independent experiments were performed; the data represent means ± SD. Statistical analysis was done using the Friedman test and Dunn’s post-hoc test, with statistical significance at p < 0.05 (*) and p < 0.01 (**).