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. 2018 Nov 22;12(2):336–349. doi: 10.1016/j.tranon.2018.11.001

Figure 4.

Figure 4

Bortezomib induces REDD1 expression to downregulate the mTORC1 pathway and subsequently the FLT3-ITD expression. (A) MV4-11 cells were treated with indicated concentrations (ng/ml) of bortezomib (BZM) for 6 hours and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: S6K-PT, phospho-T389-S6K; 4EBP1-P, phospho-T37/46-4EBP1; 4EBP1-nonP, non-phospho-T46-4EBP1. (B) Primary AML cells from FLT3-ITD–positive patients (ITD+ AML #1) were treated with indicated concentrations (ng/ml) of BZM for 6 hours and lysed. Cell lysates were run on duplicate gels and analyzed. Relative expression levels of REDD1 analyzed by densitometry and normalized by that of HSP90 (RED/HSP) are indicated. S6RP-SP: phospho-S240/244-S6RP. (C) BaF3/ITD cells inducibly overexpressing REDD1 (REDD1) or vector control cells (Cont.) cultured with or without doxycycline (DOX), as indicated, were treated with indicated concentrations (ng/ml) of BZM for 6 hours and analyzed. Akt-PT: phospho-T308-Akt. (D) Cells indicated were treated with 2 ng/ml BZM for 48 hours and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (E) MV4-11 cells were treated with or without 5 ng/ml BZM and 50 μM chloroquine (CQ), as indicated, for 8 hours and analyzed. Positions of LC3B-I and -II are indicated. Cl.Casp-3: cleaved Caspase-3. (F) MV4-11 cells were treated with indicated concentrations (ng/ml) of BZM for 8 hours simultaneously with 100 μM Boc-d-FMK (B-d-F) or with 5 μM MG132 added for the last 2 hours, as indicated, and analyzed. (G) MV4-11 cells were left untreated as control (Cont.) or treated with 5 ng/ml BZM, as indicated, for 5 hours. Cells were then treated with 50 μg/ml cycloheximide (CHX) for indicated times along with 100 μM CQ where indicated or treated with 5 μg/ml actinomycin D (ACD) for 4 hours, as indicated, and lysed for Western blot analysis. (H) MV4-11 cells were cultured for 4 hours with 50 μg/ml CHX in the absence (Cont.) or presence of 5 ng/ml of BZM. Cells were then washed three times and cultured with or without BZM, as indicated, for indicated times and analyzed. (I) MV4-11 cells were treated for 6 hours with 4 ng/ml BZM or left untreated as control (Cont.), as indicated, and subjected to flow cytometric analysis with an antibody against FLT3 or phospho-S240/244-S6RP (S6RP-SP), as indicated. Gray lines represent isotype controls. MFIR: mean fluorescent intensity ratio. (J) MV4-11 cells were treated with indicated concentrations of PP242 for 8 hours and analyzed.