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. 2018 Sep 12;4(9):eaar3938. doi: 10.1126/sciadv.aar3938

Fig. 5. mTORC3 kinase activity is insensitive to rapamycin, but sensitive to AZD-8055.

Fig. 5

(A) Growth curves of Karpas-299, K-E7−, K-E7−–ETV7, and K-EΔ120/Δ159+60 cells each inoculated at a density of 6 × 104 cells/ml and grown in liquid spinner cultures for 72 hours. Data are means ± SEM from three independent experiments. The inset shows an ETV7 IP/Western blot of lysates of 2 × 107 Karpas-299, K-E7−, K-E7−–ETV7, and K-EΔ120/Δ159+60 cells. (B) Graph representing cell densities (percent control) of Karpas-299, K-E7−, K-E7−–ETV7, and K-EΔ120/Δ159+60 cells cultured for three population doublings with increasing amounts of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/ml), plotted as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. Thirty percent control or less indicates no proliferation. (C) Graph representing cell densities (percent control) of Karpas-299 and K-E7− cells cultured for three population doublings with increasing amounts of AZD-8055 (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/ml), plotted as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. Thirty percent control or less indicates no proliferation. (D) Immunoblots of lysates from equal numbers of Karpas-299, K-E7−, and K-E7−–ETV7 cells treated with increasing amounts of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/ml) probed for p-mTORSer2448 p-AKTSer473, p-AKTThr308, p-ERK1/2Thr202/Tyr204, p-p70S6KThr389, p–4E-BP1Thr37/46, total 4E-BP1, p-GRB10Ser501/503, total GRB-10, LC3BI/II, and SQSTM1/p62. (E) mTOR IP (mTOR IP) of Karpas-299 cells treated with increasing amounts of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, 1000 ng/ml) immunoblotted for mTOR, Rictor, Raptor, and ETV7. (F) Lysates of the AZD-8055–treated Karpas-299 and K-E7− cells immunoblotted for p-mTORSer2448, p-AKTSer473, p-AKTThr308, p-ERK1/2Thr202/Tyr204, p-P70S6KThr389, p–4E-BP1Thr37/46, total 4E-BP1, KI-67, LC3BI/II, and SQSTM1/p62.