Figure 3
TCDD exposure induces the Nkx2-1/p53/p65/IĸBα pathway in vivo and in vitro. (a) Nuclear localization of p65 protein was assayed by immune-histochemistry in thyroids of control (CTRL) and 0,001 μg/kg/day TCDD treated-males (top panel) and TCDD-females (bottom panel) from E0.5 to PND30. Negative control was performed using purified IgG and it is shown in Fig. S3g. (b,c) Levels of IκBα (b) and Tp53 (c) transcripts were assayed by RT-qPCR in thyroid of CTRL- and TCDD-mice of both sexes (black bar for male, white bar for female). (d) Nuclear/cytosolic localization of p65 protein in FRTL-5 untreated (CTRL) and exposed to 10−9 M TCDD for 1-, 7-, 14-, 21-, 28- days was assayed by western blotting analysis. β-tubulin and Topoisomerase 1 were used as loading control of cytoplasmic and nuclear fraction, respectively. A cropped version of the image is shown (full-lenght blots are shown in Supplementary Fig. S7). Cytoplasmic and nuclear fractions were loaded on two different gels. (e,f) RT-qPCR analysis of IĸBα (e) and Tp53 (f) transcript levels in FRTL-5 cells treated with TCDD 10−9 M for 28days. In vitro data are reported as means ± SD of Gapdh normalized-mRNA levels of three independent experiments. In vivo data are reported as means ± SD of Gapdh normalized-mRNA levels; 5 mice for each group and sex were analyzed. *p-value < 0.05 and ***p-value < 0.001.
