The AKT–mTOR pathway controls PD-L1 protein expression. The AKT–mTOR pathway controls PD-L1 protein expression. A, CL13 and H1299 cell lines were treated with 10 ng/mL IFNγ or 5 ng/mL EGF in 1% serum for the indicated times. An early time point (30 m) was included after EGF addition to confirm pathway activation. B, cells were treated for 24 hours with 100 nmol/L rapamycin alone, for 23 hours with 5 ng/mL EGF or 10 ng/mL IFNγ alone, or the combination by treating with rapamycin for 1 hour, then adding EGF or IFNγ to culture media and harvesting 23 hours later. C, CL13 cells treated as in B and RNA was harvested for RT-PCR. D, cells were treated with 100 μg/mL cycloheximide for the indicated time points. E, cells were treated with 5 μg/mL actinomycin D for the indicated time points. Immunoblots shown are representative of three independent experiments. F, cells were treated for 6 hours with 300 μmol/L chloroquine (CLQ) alone, for 4 hours with 100 nmol/L rapamycin alone, or the combination by treating with CLQ for 2 hours, then adding rapamycin to culture media and harvesting 4 hours later. G, cells were treated for 6 hours with 100 nmol/L PS-341 alone, for 5 hours with 100 nmol/L rapamycin alone, or the combination by treating with PS-341 for 2 hours, then adding rapamycin to culture media and harvesting 5 hours later. Kristin J. Lastwika et al. Cancer Res 2016;76:227-238 ©2016 by American Association for Cancer Research