Fig. 3

Fig. 3 Fig. 3. (A) and (B) Treatment with vitamin D3 in combination with salpn super-activates VDR signaling in HEK293T cells. CYP24A1 is induced upon activation of VDR signaling, and its expression was assessed in cells with native VDR or cells transfected with wild type VDR (VDR-WT). Upon transfection, HEK293T cells were treated for 24 h with vitamin D3 with or without salpn at various concentrations. Vitamin D3 at 10−7 M was used unless stated otherwise. 40x diluted 100% ethanol (EtOH) in cell growth media was used as vehicle control. The bars represent the mean value of triplicate treatments, and error bars represent standard error of the mean. (A) Treatment with 10−7 M of vitamin D3 induces CYP24A1 expression even further when cells are transfected with VDR-WT. Additionally, 10−8 M of vitamin D3 increases CYP24A1 expression, showing a dose-dependent effect. Control (CTRL) and vehicle (EtOH) treated or VDR-WT transfected cells did not increase CYP24A1 expression in the absence of vitamin D3. (B) Salpn induces CYP24A1 expression in a dose-dependent manner when vitamin D3 is present, showing an additive effect over vitamin D3 effect by itself. (C) Effect of salpn alone (orange) at 10 µM, 16 h, (D) vitamin D3 alone (blue), and (D) salpn + Vitamin D3 mixture (orange) on nuclear receptor activity in HepG2 cells. HepG2 cells were transiently transfected with optimized trans-FACTORIALTM library. Twenty-four hours after transfection cells were washed and supplied with fresh low serum (1% FBS, charcoal stripped) culture medium and treated with inducer for 16 h. Profile of the trans-FACTORIALTM activities was determined as fold of induction values versus vehicle-treated (DMSO) control cells. Graph shows fold-induction data plotted in logarithmic scale.