AhR retains HuR in the nucleus in response to CSE but does not contribute to HuR expression.

<p>(A) HuR and CUGPB2 are constitutively expressed and are unaffected by AhR expression or CSE exposure. Note that there was an increase in Cox-2 protein in CSE-exposed <i>AhR^−/−</i> fibroblasts but not <i>AhR^+/+</i> cells. (B) <i>AhR^−/−</i> and <i>AhR^+/−</i> lung fibroblasts were exposed to 1% CSE for 4 hours and IF performed for CUGBP2. Nuclei are visualized by Hoechst (blue) and the merged images are shown. CUGBP2 was localized predominantly in the nucleus in <i>AhR^−/−</i> and <i>AhR^+/−</i> fibroblasts (<i>arrowheads</i>), although cytoplasmic expression was detectable (<i>arrows</i>). Cytoplasmic CUGBP2 increased in both <i>AhR^−/−</i> and <i>AhR^+/−</i> fibroblasts exposed to 1% CSE (<i>arrows</i>). (C) In cells treated with media, HuR is predominantly localized in the nucleus both in <i>AhR^−/−</i> and <i>AhR^+/−</i> fibroblasts (<i>arrowheads</i>). CSE exposure (1%) for 4 hours in absence of AhR expression (<i>AhR^−/−</i>) induces HuR shuttling from the nucleus to the cytoplasm (<i>arrows</i>). When <i>AhR^+/−</i> fibroblasts are challenged with 1% CSE, HuR remains in the nucleus. Results are representative of three independent experiments. (D) There was an increase in cytoplasmic HuR only in the <i>AhR^−/−</i> cells beginning at one hour of exposure and continuing through 4 hours. The purity of the extraction was determined by Tubulin, which was not detectable in the nuclear fraction. Representative western blot is shown. (E) Densitometric analysis of cytoplasmic and nuclear extracts following exposure to CSE: there was a significant increase in cytoplasmic HuR in response to CSE in only <i>AhR^−/−</i> cells (2.5±0.3; *p<0.05 compared to media only; ^¶p<0.05 compared to respective <i>AhR^+/−</i> fibroblasts exposed to CSE at the indicated time-point). Results are expressed as mean ± SEM of three independent experiments. (F) Classic AhR ligands do not cause cellular HuR redistribution in mouse lung fibroblasts. HuR remained within the nucleus upon exposure to B[<i>a</i>]P. Images are representative of two independent experiments. Magnification = 40×. (G) There was no increase in cytoplasmic HuR in <i>AhR^+/+</i> or <i>AhR^−/−</i> cells exposed to B[<i>a</i>]P for 4 hours. Representative western blot is shown.