Figure 6
Figure 6. Effects of Notch on the mTOR pathway are independent of changes in Akt activity but dependent on c-Myc. (A) Notch inhibition blocks the induction of S6 RP phosphorylation in response to fetal calf serum (FCS) and insulin-like growth factor I (IGF-I) stimulation. TALL-1 cells were treated with GSI (DAPT) or vehicle (DMSO) for 6 days. The cells were then serum starved for 24 hours and subsequently stimulated with FCS (10%) or IGF-I (20 ng/mL) for an additional 24 hours. Wortmannin (50 nM) was added to the cells during the last hour of incubation. Cell lysates were prepared and equivalent amounts of protein were loaded per lane on a gel for Western blot analysis using the 2 antibodies shown. Expression of Hes-1 mRNA normalized to GAPDH in each sample was measured using quantitative reverse-transcription–polymerase chain reaction (RT-PCR) to confirm inhibition of Notch activity. The values shown indicate the amount of Hes-1 mRNA relative to serum-starved mock-treated cells. GSI-treated (right panel) and DMSO control (left panel) samples were processed on the same blot. (B) Protein microarray ratiometric data for phospho-Akt (Ser473) and phospho-S6 RP in GSI-sensitive cell lines. Ratios (GSI/DMSO) are logged in base 2 and median-centered for each cell line. (C) GSI treatment abolishes S6 RP phosphorylation in the absence of changes in Akt and GSK3β phosphorylation. HPB-ALL and TALL-1 cells were treated with GSI (compound E) or vehicle (DMSO) for 3 days. Whole-cell lysates were prepared and equivalent amounts of proteins were loaded per lane for Western blot analysis using the indicated antibodies on the left. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples. (D) GSI-induced dephosphorylation of S6 ribosomal protein is not dependent on changes in PI3K/Akt activity. HPB-ALL and T-ALL cells were exposed to either DMSO or 1 μM GSI (compound E) for 3 days. At the end of the 3-day period, cells were intracellularly stained with an antibody specific for either phospho-S6 RP or phospho-Akt (Ser 473) and analyzed using flow cytometry. Gates were placed around mock- and GSI-treated cell populations on forward and side scatter dot plots with equivalent levels of phospho-Akt staining. Phospho-S6 RP staining was compared between the 2 groups using the same gated populations. Each bar graph represents background-subtracted mean fluorescent intensity of the indicated phospho-epitope normalized to the corresponding value in mock-treated control cells. Error bars represent standard deviation. (E) Similar experiment as in Figure 3 except the cell lines were stably transduced with retroviruses expressing c-Myc. Blots were probed with the antibodies indicated on the left panel. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples.

Effects of Notch on the mTOR pathway are independent of changes in Akt activity but dependent on c-Myc. (A) Notch inhibition blocks the induction of S6 RP phosphorylation in response to fetal calf serum (FCS) and insulin-like growth factor I (IGF-I) stimulation. TALL-1 cells were treated with GSI (DAPT) or vehicle (DMSO) for 6 days. The cells were then serum starved for 24 hours and subsequently stimulated with FCS (10%) or IGF-I (20 ng/mL) for an additional 24 hours. Wortmannin (50 nM) was added to the cells during the last hour of incubation. Cell lysates were prepared and equivalent amounts of protein were loaded per lane on a gel for Western blot analysis using the 2 antibodies shown. Expression of Hes-1 mRNA normalized to GAPDH in each sample was measured using quantitative reverse-transcription–polymerase chain reaction (RT-PCR) to confirm inhibition of Notch activity. The values shown indicate the amount of Hes-1 mRNA relative to serum-starved mock-treated cells. GSI-treated (right panel) and DMSO control (left panel) samples were processed on the same blot. (B) Protein microarray ratiometric data for phospho-Akt (Ser473) and phospho-S6 RP in GSI-sensitive cell lines. Ratios (GSI/DMSO) are logged in base 2 and median-centered for each cell line. (C) GSI treatment abolishes S6 RP phosphorylation in the absence of changes in Akt and GSK3β phosphorylation. HPB-ALL and TALL-1 cells were treated with GSI (compound E) or vehicle (DMSO) for 3 days. Whole-cell lysates were prepared and equivalent amounts of proteins were loaded per lane for Western blot analysis using the indicated antibodies on the left. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples. (D) GSI-induced dephosphorylation of S6 ribosomal protein is not dependent on changes in PI3K/Akt activity. HPB-ALL and T-ALL cells were exposed to either DMSO or 1 μM GSI (compound E) for 3 days. At the end of the 3-day period, cells were intracellularly stained with an antibody specific for either phospho-S6 RP or phospho-Akt (Ser 473) and analyzed using flow cytometry. Gates were placed around mock- and GSI-treated cell populations on forward and side scatter dot plots with equivalent levels of phospho-Akt staining. Phospho-S6 RP staining was compared between the 2 groups using the same gated populations. Each bar graph represents background-subtracted mean fluorescent intensity of the indicated phospho-epitope normalized to the corresponding value in mock-treated control cells. Error bars represent standard deviation. (E) Similar experiment as in Figure 3 except the cell lines were stably transduced with retroviruses expressing c-Myc. Blots were probed with the antibodies indicated on the left panel. The values shown below each blot represent ratios (phospho/total) of calibrated densitometry readings normalized to mock-treated (DMSO) control samples.

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