Effect of SCF and/or MBCD on glucose transport and cholesterol content in M07e cells.

<p>(<b>A</b>) IL-3-starved cells were incubated (or not) in PBS at 37°C with 5 ng/mL SCF for 15 min, then assayed for glucose transport activity as described in the Materials and Methods section. To test the effect of MBCD, cells were incubated in PBS at 37°C with 10 mM MBCD for 20 min, washed, re-suspended in PBS and assayed for glucose transport activity both immediately and after 40 min incubation at 37°C (reported as “after 40′ ”). When subjected to both <i>stimuli</i>, M07e cells were incubated with MBCD, washed, treated with SCF and assayed for glucose uptake. (<b>B</b>) M07e cells were incubated with [³H]-cholesterol (0.5 µCi/mL) in cell culture medium for 16 h at 37°C, washed, re-suspended in PBS and treated (or not) with 10 mM MBCD for 20 min in the presence or absence of 5 ng/mL SCF for 15 min. When subjected to both <i>stimuli</i>, M07e cells were incubated with MBCD, washed and treated with SCF. Cell suspensions were washed with PBS, then [³H]-cholesterol content was estimated by liquid scintillation counting. Results are expressed as means ± SD of four independent experiments, each performed in triplicate. **<i>P</i><0.005, significantly different from control cells; ***<i>P</i><0.0001, significantly different from control cells; ○○<i>P</i><0.005, significantly different from the corresponding sample treated with SCF; ○○○<i>P</i><0.0001, significantly different from the corresponding sample treated with SCF; §<i>P</i><0.005, significantly different from the corresponding sample treated with MBCD.</p

Effect of cholesterol replenishment on glucose transport in MBCD-treated M07e cells.

<p>IL-3-starved cells were incubated (or not) in PBS with 10 mM MBCD for 20 min, washed and re-suspended in PBS. A third sample of IL-3-starved cells was treated with MBCD for 20 min in PBS, washed and re-suspended in PBS, then added with MBCD/cholesterol mixture (25 mM cholesterol and 10% MBCD in PBS) for 40 min at 37°C, washed and re-suspended in PBS. Glucose uptake was assayed as described in the Materials and Methods section. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. **<i>P</i><0.005, significantly different from control cells.</p

Effect of 10 mM MBCD on cholesterol content and cell viability/proliferation of M07e cells.

<p>(<b>A</b>) M07e cells were incubated with [³H]-cholesterol (0.5 µCi/mL) in cell culture medium for 16 h at 37°C, washed, re-suspended in PBS and treated with 10 mM MBCD at the times indicated. Cell suspensions were washed with PBS, then [³H]-cholesterol content was estimated by liquid scintillation counting. (<b>B</b>) Cells treated as described in Fig. 2A, were exposed to different MBCD concentrations for 20 min. Viability was evaluated by Trypan Blue exclusion test. (<b>C</b>) Cells treated as described in Fig. 2A, were exposed to different MBCD concentrations for 20 min. Proliferation was evaluated by MTT assay as described in the Materials and Methods section. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. *<i>P</i><0.05, significantly different from control cells.</p

Enrichment of plasma membrane GLUT1 content in MBCD-treated M07e cells.

<p>(<b>A</b>) M07e cells were incubated (or not) in PBS at 37°C with 10 mM MBCD for 20 min. To isolate plasma membranes, cells were treated with NHS-LC-biotin; the mixtures were then added with streptavidin-agarose beads and the samples subjected to SDS-PAGE and immunoblotting with anti-GLUT1 as described in the Materials and Methods section. CD71, a plasma membrane protein marker, was used as a control. (<b>B</b>) M07e cells incubated (or not) in PBS at 37°C with 10 mM MBCD for 20 min, were fixed in 3% (w/v) paraformaldheyde for 15 min. Cells were then immunolabelled with anti-GLUT1 (N-20) antibody (raised against an extracellular domain of GLUT1), treated with fluorescent FITC-conjugated secondary antibody and visualized using immunofluorescence microscopy.</p

Effect of MBCD on Akt, PLCγ1 and ERK phosphorylation in M07e cells.

<p>IL-3-starved cells were incubated (or not) in PBS at 37°C with 10 mM MBCD for 20 min, or with 5 ng/mL SCF for 15 min. MBCD-treated cells were also added with MBCD/cholesterol mixture (25 mM cholesterol and 10% MBCD in PBS) for 40 min at 37°C, or 5 ng/mL SCF for 15 min. Cell lysates were electrophoresed and immunoblotted with the indicated antibodies as described in the Materials and Methods section. Tubulin detection was used as a control. NC: negative control; PC: positive control; IP: immunoprecipitation; IB: immunoblotting.</p

GLUT1 and cholesterol distribution along sucrose-gradient fractionation of M07e cells treated or not with MBCD.

<p>(<b>A</b>) M07e cells treated (or not) with 10 mM MBCD for 20 min were lysed with 1% Triton X-100 at 4°C and separated by sucrose density-gradient ultracentrifugation as described in the Materials and Methods section. Equal volume aliquots of each fraction were subjected to SDS-PAGE and Western blotting. Flotillin-2 and Lyn were used as markers for DRM fractions; CD71 for non-DRM fractions. (<b>B</b>) Typical profile of protein concentrations in gradient fractions after ultracentrifugation. Protein content was determined as described in the Materials and Methods section. (<b>C</b>) M07e cells were pre-incubated at 37°C for 16 hours with [³H]-cholesterol (0.1 µCi/mL) in cell culture medium. Cells exposed (or not) to 10 mM MBCD for 20 min were lysed with 1% Triton X-100 at 4°C and subjected to sucrose density-gradient ultracentrifugation as previously described. [³H]-cholesterol content of each fraction collected was quantified by liquid scintillation counting. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. ***<i>P</i><0.001, significantly different from untreated cells.</p

Effect of phloretin and nystatin on glucose transport in M07e cells with or without MBCD.

<p>(<b>A</b>) IL-3-starved cells were incubated (or not) in PBS at 37°C with 10 mM MBCD for 20 min, washed and re-suspended in PBS prior to the measurement of glucose transport as described in the Materials and Methods section (empty bars). A second batch of IL-3-starved cells was added with 0.3 mM phloretin for 10 sec, washed and re-suspended in PBS. Cells were then incubated (or not) with 10 mM MBCD for 20 min at 37°C, washed and re-suspended in PBS prior to the measurement of glucose transport (striped bars). (<b>B</b>) IL-3-starved cells were incubated with 50 µg/mL nystatin (Nys) in cell culture medium for 3 h at 37°C, washed, re-suspended in PBS and treated or not with 10 mM MBCD for 20 min at 37°C, washed and re-suspended in PBS prior to the measurement of glucose transport as described in the Materials and Methods section. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. **<i>P</i><0.005, ***<i>P</i><0.001, significantly different from control cells; ○○<i>P</i><0.005, significantly different from the corresponding sample untreated with MBCD.</p

Effect of different MBCD concentrations on cholesterol content and cell viability of M07e cells.

<p>(<b>A</b>) M07e cells were incubated with [³H]-cholesterol (0.5 µCi/mL) in cell culture medium for 16 h at 37°C, washed, re-suspended in PBS and treated with MBCD for 40 min at the concentrations indicated. Cell suspensions were washed with PBS, then [³H]-cholesterol content was estimated by liquid scintillation counting. (<b>B</b>) The viability of the cells treated as described in Fig. 1A was evaluated by Trypan Blue exclusion test. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. **<i>P</i><0.005, significantly different from control cells; ***<i>P</i><0.0005, significantly different from control cells.</p

Additional file 4: Figure S4. of Improving nelarabine efficacy in T cell acute lymphoblastic leukemia by targeting aberrant PI3K/AKT/mTOR signaling pathway

The combination of nelarabine and ZSTK-474 is synergistic in CEM-R cells, which overexpress P-gp. Cell viability assay of CEM-R cell line treated for 48 h with increasing concentrations of nelarabine alone or combined with the pan PI3K p110 inhibitor ZSTK-474. One representative of two different experiments is shown

Additional file 5: Figure S5. of Improving nelarabine efficacy in T cell acute lymphoblastic leukemia by targeting aberrant PI3K/AKT/mTOR signaling pathway

Specific effects of nelarabine on PI3K/AKT and MEK/ERK1/2 pathways. Western blotting analyses for the expression of p-AKT and p-ERK in resistant T-ALL cell lines treated with the specific inhibitors LY294002 (PI3K inhibitor), CCI-779 (mTOR allosteric inhibitor) or trametinib (MEK1/2 inhibitor) alone or in combination with nelarabine. Thirty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. CTRL: untreated cells; Nela and N: nelarabine at 10 μM; LY: LY294002 at 10 μM; CCI: CCI-779 at 100 nM; Tram: trametinib at 1 μM. Cells were treated for 48 h