Figure 6
Figure 6. Low spare reserve capacity renders AML cells sensitive to oxidative metabolic stress by palmitate, and this sensitivity can be rescued by genetically inhibiting fatty acid oxidation pathway. (A) Leukemic cells and MCF-7 cells were treated with increasing concentrations of palmitate for 72 hours. Cell viability and growth were measured by Cell Titer Fluor viability assay. (B) OCI-AML-2 and HL-60 cells were treated with increasing concentrations of palmitate for 24 hours. ROS production was measured by staining with MitoSOX and flow cytometry. (C) OCI-AML-2 cells were treated with increasing concentrations of palmitate for 72 hours in the presence and absence of N-acetylcysteine. Cell growth and viability was measured by Cell Titer Fluor viability assay. (D-F) OCI-AML-2 cells were infected with lentiviral vectors containing shRNAs targeting CPT1a or noncellular targets (control). A total of 6 days postinfection, CPT1a mRNA expression relative to 18s RNA was analyzed by qRT-PCR (D) and CPT1a protein expression was determined by immunoblotting (E). Cell growth and viability were measured by Cell Titer Flo after treating cells with palmitate for 72 hours (F). (G) Infected OCI-AML-2 cells were treated with increasing concentrations of palmitate for 24 hours. ROS production was measured by staining with MitoSOX and flow cytometry. (H-K) OCI-AML-2 cells were infected with lentiviral vectors containing shRNAs targeting PPARα or noncellular targets (control). Four days postinfection, PPARα mRNA expression relative to 18s RNA was analyzed by qRT-PCR (H). Cell growth and viability were measured by Cell Titer Flo after treating cells with palmitate for 72 hours (I). Infected OCI-AML-2 cells were treated with increasing concentrations of palmitate. 72 hours after treatment, cell viability was measured by Annexin V/PI staining (J). Infected OCI-AML-2 cells were treated with increasing concentrations of palmitate for 24 hours. ROS production was measured by staining with MitoSOX and flow cytometry (K). In all panels, error bars represent mean ± SD of independent/representative experiments. *P < .05; **P < .001 as determined by Tukey’s test after 1-way analysis of variance, comparing to controls. CPT1a and PPARα knockdown experiments were repeated twice.

Low spare reserve capacity renders AML cells sensitive to oxidative metabolic stress by palmitate, and this sensitivity can be rescued by genetically inhibiting fatty acid oxidation pathway. (A) Leukemic cells and MCF-7 cells were treated with increasing concentrations of palmitate for 72 hours. Cell viability and growth were measured by Cell Titer Fluor viability assay. (B) OCI-AML-2 and HL-60 cells were treated with increasing concentrations of palmitate for 24 hours. ROS production was measured by staining with MitoSOX and flow cytometry. (C) OCI-AML-2 cells were treated with increasing concentrations of palmitate for 72 hours in the presence and absence of N-acetylcysteine. Cell growth and viability was measured by Cell Titer Fluor viability assay. (D-F) OCI-AML-2 cells were infected with lentiviral vectors containing shRNAs targeting CPT1a or noncellular targets (control). A total of 6 days postinfection, CPT1a mRNA expression relative to 18s RNA was analyzed by qRT-PCR (D) and CPT1a protein expression was determined by immunoblotting (E). Cell growth and viability were measured by Cell Titer Flo after treating cells with palmitate for 72 hours (F). (G) Infected OCI-AML-2 cells were treated with increasing concentrations of palmitate for 24 hours. ROS production was measured by staining with MitoSOX and flow cytometry. (H-K) OCI-AML-2 cells were infected with lentiviral vectors containing shRNAs targeting PPARα or noncellular targets (control). Four days postinfection, PPARα mRNA expression relative to 18s RNA was analyzed by qRT-PCR (H). Cell growth and viability were measured by Cell Titer Flo after treating cells with palmitate for 72 hours (I). Infected OCI-AML-2 cells were treated with increasing concentrations of palmitate. 72 hours after treatment, cell viability was measured by Annexin V/PI staining (J). Infected OCI-AML-2 cells were treated with increasing concentrations of palmitate for 24 hours. ROS production was measured by staining with MitoSOX and flow cytometry (K). In all panels, error bars represent mean ± SD of independent/representative experiments. *P < .05; **P < .001 as determined by Tukey’s test after 1-way analysis of variance, comparing to controls. CPT1a and PPARα knockdown experiments were repeated twice.

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