Figure 3
Figure 3. Activation of autophagy by bortezomib in AML cells. (A-B) LC3-II accumulation after bortezomib (Bort) exposure. MV4-11 or primary AML samples were treated 24 hours with 10 or 30 nM bortezomib, respectively. Total cell lysates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. (C) Bortezomib induces an active autophagic flux. MV4-11 cells were incubated 24 hours with 10 nM bortezomib alone or in combination with 10 nM Bafilomycin A for the last 2 hours. Cells were lysed and LC3-I to LC3-II conversion was assessed by western blot analysis using the appropriate antibodies. (D) Immunofluorescence staining of LC3 positive structures. MV4-11 cells were exposed 24 hours with 10 nM bortezomib before immunofluorescence analysis using LC3-B antibody and an antimouse-Alexa 488 antibody, and labeling with 4,6 diamidino-2-phenylindole. Analyses were performed on a Zeiss Apotome microscope. (E) Quantification of LC3-positive dots. At least 100 cells per condition of 3 independent experiments were counted. (F) Inhibition of the mammalian target of rapamycin complex 1 pathway. MV4-11 cells were incubated 12 hours with 10 nM bortezomib before cell lysis and western blot analysis. (G-J) Bortezomib induces a cytotoxic autophagy. (G) MV4-11 or MOLM-14 cells were treated 24 hours with 10 nM bortezomib alone or in combination of 5 mM 3-MA before Annexin-V/7-AAD staining and flow cytometry analysis. (H) MV4-11 cells were incubated 24 hours with bortezomib and/or 5 mM 3-MA before immunoblotting analysis using the appropriate antibodies. (I-J) MV4-11 cells were infected with TRIPZ lentiviral shRNA targeting ATG5 or ATG13 and transduced cells were selected by 1 µg/mL puromycin. First, shRNA were induced by 72 hours 1 µg/mL doxycycline treatment and then next the cells were incubated for 24 hours in the presence of 10 nM bortezomib. (I) Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies (J) Apoptosis was evaluated after Annexin-V/7-AAD labeling via flow cytometry. *P < .05; **P < .01; ***P < .001.

Activation of autophagy by bortezomib in AML cells. (A-B) LC3-II accumulation after bortezomib (Bort) exposure. MV4-11 or primary AML samples were treated 24 hours with 10 or 30 nM bortezomib, respectively. Total cell lysates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. (C) Bortezomib induces an active autophagic flux. MV4-11 cells were incubated 24 hours with 10 nM bortezomib alone or in combination with 10 nM Bafilomycin A for the last 2 hours. Cells were lysed and LC3-I to LC3-II conversion was assessed by western blot analysis using the appropriate antibodies. (D) Immunofluorescence staining of LC3 positive structures. MV4-11 cells were exposed 24 hours with 10 nM bortezomib before immunofluorescence analysis using LC3-B antibody and an antimouse-Alexa 488 antibody, and labeling with 4,6 diamidino-2-phenylindole. Analyses were performed on a Zeiss Apotome microscope. (E) Quantification of LC3-positive dots. At least 100 cells per condition of 3 independent experiments were counted. (F) Inhibition of the mammalian target of rapamycin complex 1 pathway. MV4-11 cells were incubated 12 hours with 10 nM bortezomib before cell lysis and western blot analysis. (G-J) Bortezomib induces a cytotoxic autophagy. (G) MV4-11 or MOLM-14 cells were treated 24 hours with 10 nM bortezomib alone or in combination of 5 mM 3-MA before Annexin-V/7-AAD staining and flow cytometry analysis. (H) MV4-11 cells were incubated 24 hours with bortezomib and/or 5 mM 3-MA before immunoblotting analysis using the appropriate antibodies. (I-J) MV4-11 cells were infected with TRIPZ lentiviral shRNA targeting ATG5 or ATG13 and transduced cells were selected by 1 µg/mL puromycin. First, shRNA were induced by 72 hours 1 µg/mL doxycycline treatment and then next the cells were incubated for 24 hours in the presence of 10 nM bortezomib. (I) Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies (J) Apoptosis was evaluated after Annexin-V/7-AAD labeling via flow cytometry. *P < .05; **P < .01; ***P < .001.

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