Fig. 6.

Rapamycin-sensitive mTORC1 signaling contributes to Tsc1-deficient DC defects. (A) BM cells from WT or Tsc1^CreER mice were cultured with FLT3L and 4-OHT, followed by immunoblot analysis of p-S6, p-S6K, p-4EBP1, and p-AKT (Ser473) in purified CD11c⁺ cells. (B) BM cells from WT or Tsc1^CreER mice were cultured with FLT3L and 4-OHT, followed by flow cytometry analysis of p-S6 at the indicated time points of DC differentiation. (C and D) Phosphorylation of S6 was determined in splenic DCs from tamoxifen-treated WT or Tsc1^CreER mice (C) or from WT or Tsc1^CreER donor BM-derived cells (Left) and spike BM cells (Right) in the mixed chimeras (generated as described in Fig. 1F) (D). (E–J) BM cells from WT or Tsc1^CreER mice were cultured with FLT3L and 4-OHT in the presence of rapamycin or control, followed by analyses of phosphorylation of S6 (E), CD11c⁺ DC percentage (Left) and number (Right) (input BM cells, 2 × 10⁶) (F), maturation marker expression (G), F4/80 and Ly6G expression (H), Annexin V staining, cell size, ROS production, and mitochondrial mass (I), and Myc expression (the numbers below the lanes indicate the band intensity relative to that of the loading control β-actin) (J). Error bars indicate SEM. Data are representative of two to four independent experiments.