![Figure 4. DNR altered the control of AML cell protein translation. (A) Total protein synthesis rate of HL60 cells as a function of incubation with DNR (8 μmol/L) or CHX (3.6 μmol/L) relative to vehicle-treated cells. Cells were pulse-labeled with [35S]methionine during the last 15 minutes of the incubation. Synthesis is the ratio between the labeling, determined by autoradiography of SDS-PAGE gels of extracted proteins, and the protein staining of the same gels (see right inset). The data represent the means (± SEM) of 4 separate experiments. (B) HL60 cells were treated for 2 hours with CHX (3.6 or 36 μmol/L) or rapamycin (100 nmol/L), the last 0.5 hours of which was in the presence of [35S]methionine. Autoradiograms of SDS-gels (right panel) show that CHX was a far more efficient protein synthesis inhibitor than rapamycin in these cells. The left panel shows protein staining of the same gels. (C) Autoradiogram of 2DE gels (pI 4-5) of extract from HL60 cells treated with vehicle or 8 μmol/L DNR for 5 hours. A pulse of [35S]methionine was given during the last 30 minutes of the incubation. (D) Autoradiograms of proteins translated in vitro in the presence [35S]methionine with RNA isolated from HL60 cells incubated with vehicle (left subpanel) or 8 μmol/L DNR for 4.5 hours (right subpanel) as template. (E) Autoradiograms of proteins from HL60 cells prelabeled with [35S]methionine for 90 minutes and chased with unlabeled methionine for 6 hours in the absence and presence of 8 μmol/L DNR. (F) HL60 cells were treated with DNR (1.6 or 8 μmol/L) or rapamycin (100 nmol/L) for the periods indicated; cell extracts were immunoblotted and probed for P-Ser371-p70S6K, p70S6K, P-Thr37/46-4E-BP1, 4E-BP1, as well as P-Ser2448-mTOR, mTOR, and β-actin. (G) IPC-81 cells were treated for 6.5 hours with various concentrations of DNR (10-300 nmol/L), and extracts were immunoblotted and probed as for (F). For panels C-E: The circled spots correspond to proteins in Table 1. These proteins were subjected to comparative quantitative analysis. Spots found in all gels are circled with closed lines; spots found only in panel B and E are circled with dashed lines. The gels shown are representative of 3 to 5 experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/111/5/10.1182_blood-2007-07-103242/2/zh80060816500004.jpeg?Expires=1724246732&Signature=pXipVBzu0qk9OHGwgoWXsV8OZymjwnnhr2CmZsxzVtiIHFG5bR9KcmqNAYfb486a~H2Mpn-uhTbOWxBEqOgayZ3sulVcTfGKIuJCFbc6q--oHMHyRvUWTVnu6ITjishDmXr0t8kzd0NCcYumBb88Kgt-adlpuqPHBt7y-BvICqxXhjdjL2KHzl~MCrRTAEsPKyWWiBHlx53sGNmxcTJEuCu50UwPWvGCpG9j3c5vVa0eFKW5ZI6WfNC2oIRxsiuobqM~lKGxmAsZtF8CsZZOkOjN8jixEc9x-9SWpPNK4Oxh40ZWKc1AtR6eDaDX~mgTtlFtZ6OlLht11z14o6-n-g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
DNR altered the control of AML cell protein translation. (A) Total protein synthesis rate of HL60 cells as a function of incubation with DNR (8 μmol/L) or CHX (3.6 μmol/L) relative to vehicle-treated cells. Cells were pulse-labeled with [35S]methionine during the last 15 minutes of the incubation. Synthesis is the ratio between the labeling, determined by autoradiography of SDS-PAGE gels of extracted proteins, and the protein staining of the same gels (see right inset). The data represent the means (± SEM) of 4 separate experiments. (B) HL60 cells were treated for 2 hours with CHX (3.6 or 36 μmol/L) or rapamycin (100 nmol/L), the last 0.5 hours of which was in the presence of [35S]methionine. Autoradiograms of SDS-gels (right panel) show that CHX was a far more efficient protein synthesis inhibitor than rapamycin in these cells. The left panel shows protein staining of the same gels. (C) Autoradiogram of 2DE gels (pI 4-5) of extract from HL60 cells treated with vehicle or 8 μmol/L DNR for 5 hours. A pulse of [35S]methionine was given during the last 30 minutes of the incubation. (D) Autoradiograms of proteins translated in vitro in the presence [35S]methionine with RNA isolated from HL60 cells incubated with vehicle (left subpanel) or 8 μmol/L DNR for 4.5 hours (right subpanel) as template. (E) Autoradiograms of proteins from HL60 cells prelabeled with [35S]methionine for 90 minutes and chased with unlabeled methionine for 6 hours in the absence and presence of 8 μmol/L DNR. (F) HL60 cells were treated with DNR (1.6 or 8 μmol/L) or rapamycin (100 nmol/L) for the periods indicated; cell extracts were immunoblotted and probed for P-Ser371-p70S6K, p70S6K, P-Thr37/46-4E-BP1, 4E-BP1, as well as P-Ser2448-mTOR, mTOR, and β-actin. (G) IPC-81 cells were treated for 6.5 hours with various concentrations of DNR (10-300 nmol/L), and extracts were immunoblotted and probed as for (F). For panels C-E: The circled spots correspond to proteins in Table 1. These proteins were subjected to comparative quantitative analysis. Spots found in all gels are circled with closed lines; spots found only in panel B and E are circled with dashed lines. The gels shown are representative of 3 to 5 experiments.
