Figure 6

AAI promotes the nuclear localization of GNMT in the liver. (a) Immunohistochemical staining for GNMT expression in the liver of the female C57BL/6 mouse intraperitoneally injected with 5 mg/kg bw/day AAI (AAI 5) or corn oil (vehicle control) for 3 weeks (original magnification ×200). (b) Immunofluorescent microscopy of fixed Huh7 Lv-GNMT cells stained for GNMT (green, Alexa 488) and DAPI. Huh7 Lv-GNMT cells were treated with 250 μM AAI, 10 μM BaP (positive control) or vehicle (0.01% DMSO, negative vehicle control) for 16 hours (original magnification, ×200). (c) Western blot analysis of nuclear and cytoplasmic (non-nuclear) fractions from AAI-, BaP- and DMSO-treated Huh7 Lv-GNMT cells. Histone H3 and α-tubulin were used as loading and purity controls for the nuclear and cytoplasmic fractions, respectively. The full-length blots are presented in Supplementary Fig. S7. (d–h) mRNA levels of (d) Nrf2, (e) CAR, (f) PXR (g) AhR, (h) PPARα in the liver from AAI- or corn oil-treated hGNMT Tg mice, as described in Fig. 2a and b, were determined by qPCR. (C: mice treated with corn oil; AA2 orAA5: 2 or 5 mg/kg BW/day AAI administration; F: female; M: male; Tg: hGNMT transgenic mice; WTL: wild-type littermates of Tg mice) All qPCR data were normalized to β-actin. Values are presented as the mean ± SEM; n = 4 in each group. p-values were calculated using Student’s t-test. *p < 0.05, **p < 0.001, ***p ≤ 0.0001.