Extended exposure to ATP-competitive mTOR inhibition reduces SOCS3 expression, resulting in sustained STAT3 activation. CD11c⁺ cells were isolated from 7-day BM cell cultures and pretreated with DMSO, RAPA (10 ng/mL), or Torin1 (100 nM) for 2 hours before LPS stimulation for 30 minutes (A) or 1 to 3 hours (B). Total cell lysates were immunoblotted for the indicated protein. (C) P-STAT3 signal was quantified relative to total STAT3 signal at 3 hours following LPS stimulation and normalized to control. (D) DCs were differentiated in the presence of DMSO (vehicle) or Torin1 (100 nM) from day 2 to day 8. DCs were isolated by CD11c immunomagnetic purification, and P-STAT3 signal determined by flow cytometric analysis following LPS stimulation for 3 hours. Gray histogram depicts samples stained with secondary Ab only. MFI for P-STAT3 signal and secondary Ab only are indicated in the top left corner, and percent positive cells is indicated in the upper right corner. (E) RAPA- or Torin1-conditioned DCs were purified as described in panel D, stimulated with LPS for 0 to 3 hours, and total cell lysates immunoblotted for the indicated protein. (F) Quantification of P-STAT3/STAT3 signal 3 hours after LPS stimulation. (G) SOCS protein was assessed in RAPA- or Torin1-conditioned DCs. WT or rictor^−/− DC were stimulated with LPS for 30 minutes (H) or 0 to 3 hours (I) and probed as indicated. (J) P-STAT3/STAT3 signal was quantified at 3 hours post-LPS. Data are representative of n = 2 to 3 independent experiments. *, # P < .05 when compared with DMSO or WT and RAPA, respectively.