Figure 1
Figure 1. The role of mTOR in leukemia-cell proliferation and survival. (A) Jurkat cells were cultured with vehicle (Ctrl) only or with 100nM rapamycin (Rapa) or 25μM LY294002 as indicated. The total number of live cells at 0, 24, 48, and 72 hours was determined by a trypan blue exclusion cell-viability assay. Each data point shown is the average of triplicate samples from 1 of 3 independent experiments. (B) Jurkat cells from panel A were stained with PI and analyzed by flow cytometry at 24 hours. The number of viable cells in each drug-treated group relative to the untreated control (Ctrl) group (set as 100%) is shown. The data presented are the average of triplicate samples with SD and are representative of 3 independent experiments. (C) Wild-type p210 BCR-Abl–transformed mouse pre-B leukemia cells were cultured with vehicle (Ctrl) or with 100nM rapamycin (Rapa), 10μM imatinib, or 25μM LY294002 for the indicated times. The total number of live cells at 0, 24, 48, and 72 hours was determined as described in panel A. Each data point shown is the average of triplicate samples from 1 of 3 independent experiments. (D) Cells in panel C were stained with PI and analyzed by flow cytometry at 24 hours. The number of live cells in the drug-treated groups relative to the control (Ctrl) group (100%) is shown. The data shown are the average of duplicate samples and are representative of 3 independent experiments. (E) Total cellular proteins from Sin1+/+ (wild-type; WT) or Sin1−/− (knockout; KO) p210 BCR-Abl leukemia cells were analyzed by immunoblotting for the indicated proteins and phosphoproteins. (F) Jurkat or Sin1+/+ p210 BCR-Abl pre-B cells were cultured with the indicated doses of pp242 for 18 hours and then analyzed by immunoblotting for the indicated proteins. (G) Jurkat cells were cultured with (+pp242) or without (Ctrl) 400nM pp242 for the indicated periods of time and live cells were counted by a trypan blue exclusion assay. Fresh cell-culture medium with or without pp242 was added to the cells every 24 hours. The data shown are the average of triplicate samples from 1 of 2 independent experiments. (H) Cells from panel G were stained with PI and annexin V and analyzed by flow cytometry at the 24-hour time point to determine cell viability. The percentage of live cells (annexin V−PI−) is indicated. (I) Sin1 WT and KO p210 BCR-Abl cells were cultured with (+pp242) or without 400nM pp242 for the indicated periods of time and live cells were counted by a trypan blue exclusion cell-viability assay. Fresh cell-culture medium with or without pp242 was added to the cells every 24 hours. The data shown are the average of triplicate samples from 1 of 3 independent experiments. (J) Sin1 WT and KO cells from panel I were stained with PI and annexin V and analyzed by flow cytometry at the 24-hour time point to determine cell viability. The percentage of live cells (annexin V−PI−) is indicated.

The role of mTOR in leukemia-cell proliferation and survival. (A) Jurkat cells were cultured with vehicle (Ctrl) only or with 100nM rapamycin (Rapa) or 25μM LY294002 as indicated. The total number of live cells at 0, 24, 48, and 72 hours was determined by a trypan blue exclusion cell-viability assay. Each data point shown is the average of triplicate samples from 1 of 3 independent experiments. (B) Jurkat cells from panel A were stained with PI and analyzed by flow cytometry at 24 hours. The number of viable cells in each drug-treated group relative to the untreated control (Ctrl) group (set as 100%) is shown. The data presented are the average of triplicate samples with SD and are representative of 3 independent experiments. (C) Wild-type p210 BCR-Abl–transformed mouse pre-B leukemia cells were cultured with vehicle (Ctrl) or with 100nM rapamycin (Rapa), 10μM imatinib, or 25μM LY294002 for the indicated times. The total number of live cells at 0, 24, 48, and 72 hours was determined as described in panel A. Each data point shown is the average of triplicate samples from 1 of 3 independent experiments. (D) Cells in panel C were stained with PI and analyzed by flow cytometry at 24 hours. The number of live cells in the drug-treated groups relative to the control (Ctrl) group (100%) is shown. The data shown are the average of duplicate samples and are representative of 3 independent experiments. (E) Total cellular proteins from Sin1+/+ (wild-type; WT) or Sin1−/− (knockout; KO) p210 BCR-Abl leukemia cells were analyzed by immunoblotting for the indicated proteins and phosphoproteins. (F) Jurkat or Sin1+/+ p210 BCR-Abl pre-B cells were cultured with the indicated doses of pp242 for 18 hours and then analyzed by immunoblotting for the indicated proteins. (G) Jurkat cells were cultured with (+pp242) or without (Ctrl) 400nM pp242 for the indicated periods of time and live cells were counted by a trypan blue exclusion assay. Fresh cell-culture medium with or without pp242 was added to the cells every 24 hours. The data shown are the average of triplicate samples from 1 of 2 independent experiments. (H) Cells from panel G were stained with PI and annexin V and analyzed by flow cytometry at the 24-hour time point to determine cell viability. The percentage of live cells (annexin V−PI−) is indicated. (I) Sin1 WT and KO p210 BCR-Abl cells were cultured with (+pp242) or without 400nM pp242 for the indicated periods of time and live cells were counted by a trypan blue exclusion cell-viability assay. Fresh cell-culture medium with or without pp242 was added to the cells every 24 hours. The data shown are the average of triplicate samples from 1 of 3 independent experiments. (J) Sin1 WT and KO cells from panel I were stained with PI and annexin V and analyzed by flow cytometry at the 24-hour time point to determine cell viability. The percentage of live cells (annexin V−PI−) is indicated.

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