Figure 4.

Analysis of the transmaternal DEHP effect on Igf2r expression and function in splenic cDC subsets. (A) Splenic cDCs from F1 or F3 pups were assessed for Igf2r mRNA expression by qPCR analysis. Naïve F1 mice: n = 6 in vehicle or 4 in DEHP group (both from two dams); Immunized F1 mice: n = 4 in vehicle or 6 in DEHP group (both from two dams); Immunized F3 mice: n = 6 in each group (both from two dams). (B, C) The frequency of Igf2r⁺ cells (M1) and expression levels of Igf2r (MFI) in gated splenic CD11c^high cDCs from immunized F1 offspring. Gray area in (B) (right panel): isotype control. MFI: mean fluorescence intensity. n = 6 or 4 in each group (both from two dams). One representative data set from two independent experiments. (D, E) Purified F1 cDCs treated with or without Leu²⁷-IGF2 in response to anti-mouse CD40 mAb or ODN 1826 for 24 h, and assessed for apoptotic cell percentages (all Annexin V⁺ cells and ViViD⁺ cells) in CD11c^hi (D) or CD8α⁺CD11c^hi (E)-gated cells using flow cytometry. Y axis represents the increase of dead cell percentages from Leu²⁷-IGF2-treated cells compared to non-treated cells. n = 6 or 4 in each group (both from two dams). Results are shown as mean ± SEM. *p-value < 0.05; **p-value < 0.01 by Mann-Whitney U test. The number of offspring (n) are pooled from two independent breeding.