FIGURE 4. TCDD activates the polymorphic hu-hs1,2 enhancer in a concentration-dependent manner.

A, Schematic of human V_H promoter alone and α₁ hs1,2 allelic constructs containing the invariant sequence (IS, denoted by a star). All luciferase reporter plasmids originated from the pGL3 Basic vector (Promega) and the polymorphic α₁ hu-hs1,2 enhancers were inserted 3’ of the luciferase gene. B and C, CH12.LX cells were transiently transfected with V_H, α_1A, α_1B, or α_1C reporter constructs. Transfected cells were cultured for 24 h with either media alone (NA, naïve) or with varying concentrations of TCDD (0 – 10.0 nM) in the absence or presence of LPS (1.0 µg/mL) stimulation. B, Luciferase enzyme activity (mean ± SEM, n=3) is represented on the y-axis as relative light units (RLUs) normalized to transfection efficiency and relative to the naïve control (set to 1 RLU) of the V_H reporter. “C” denotes LPS stimulation alone. “>” denotes a synergistic activation by a TCDD and LPS co-treatment compared to the activation induced by either treatment alone. LPS-stimulated samples were significantly (at least p<0.01) elevated compared to their corresponding unstimulated samples as determined by a 1-way ANOVA followed by a Bonferroni’s post-hoc test (not represented on graph). Results are representative of three separate experiments. C, TCDD-induced activation is represented on the y-axis as fold-change relative to the respective vehicle control (0.01% DMSO, 0.0 nM TCDD) and was generated from 3 to 11 separate experiments; each symbol (square, triangle, inverted triangle, and diamond signify the V_H, α_1A, α_1B, and α_1C reporter constructs, respectively) represents the mean (n=3) from a single experiment. Significance compared to either the corresponding vehicle control (0.01% DMSO, denoted 0 nM TCDD) (B) or the respective V_H reporter (C) was determined by a 1-way ANOVA followed by a Dunnett’s post-hoc test: “*”, “**”, significance at p<0.05 and p<0.01, respectively.