Figure 1
Figure 1. ST1326 induces FAO decrease, palmitate accumulation, and apoptosis in leukemia cell lines. (A) CPT1a expression in isolated mitochondria of leukemia cell lines. Cytochrome c oxidase subunit IV (COXIV) was used as housekeeping. Densitometry values of CPT1a/COXIV ratio are reported for each cell line as mean ± standard deviation (SD) of 3 independent experiments. (B) ST1326 induces FAO decrease in cell line and primary AML cells. Time course of a representative FAO experiment on U937 (i), MOLT4 (ii), and MEC-2 (iii) cell lines exposed to increasing concentrations of ST1326. BSA, bovine serum albumin; OCR, oxygen consumption rate. (C) ST1326 induces cell growth arrest in dose- and time-dependent fashion in hematopoietic cell lines with different ontogenesis. Dose-response growth curves: several hematopoietic cell lines were exposed to increasing concentrations of ST1326 for the indicated periods of time. Cell counts and viability were then assessed by trypan blue exclusion counting. (D) ST1326 induces a dose- and time-dependent cell cycle modulation and apoptosis in U937 cells. U937 cells were exposed to the indicated concentrations of ST1326 or vehicle control up to 72 h. Distribution of cells in the different phases of cell cycle and in the sub-G0/1 peak was assessed by acridine orange DNA/RNA staining as described.16,17 DNA histograms show results of 1 representative experiment performed on U937 cells. (E) ST1326 induces intracellular palmitate accumulation. Representative images of U937 cells stained with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY-C16) with or without ST1326 treatment as described in “Study design.” Images were acquired with laser scanning confocal microscope (TCS SP2 AOBS; Leica Microsystems, Mannheim, Germany) using a ×40 (numerical aperture = 1.25) oil immersion lens with optical pinhole at 1 AU. Scale bar indicates 20 μm. (F) Effects of ST1326 on palmitate accumulation. Relative BODIPY-C16 uptake is expressed as fluorescence intensities (mean ± SD of 3 independent experiments) in leukemia cell lines exposed to ST1326 as compared with the respective untreated controls. The increase of fluorescence intensities in ST1326-exposed cells is proportional to the intracellular palmitate accumulation (details in supplemental Materials and methods).

ST1326 induces FAO decrease, palmitate accumulation, and apoptosis in leukemia cell lines. (A) CPT1a expression in isolated mitochondria of leukemia cell lines. Cytochrome c oxidase subunit IV (COXIV) was used as housekeeping. Densitometry values of CPT1a/COXIV ratio are reported for each cell line as mean ± standard deviation (SD) of 3 independent experiments. (B) ST1326 induces FAO decrease in cell line and primary AML cells. Time course of a representative FAO experiment on U937 (i), MOLT4 (ii), and MEC-2 (iii) cell lines exposed to increasing concentrations of ST1326. BSA, bovine serum albumin; OCR, oxygen consumption rate. (C) ST1326 induces cell growth arrest in dose- and time-dependent fashion in hematopoietic cell lines with different ontogenesis. Dose-response growth curves: several hematopoietic cell lines were exposed to increasing concentrations of ST1326 for the indicated periods of time. Cell counts and viability were then assessed by trypan blue exclusion counting. (D) ST1326 induces a dose- and time-dependent cell cycle modulation and apoptosis in U937 cells. U937 cells were exposed to the indicated concentrations of ST1326 or vehicle control up to 72 h. Distribution of cells in the different phases of cell cycle and in the sub-G0/1 peak was assessed by acridine orange DNA/RNA staining as described.16,17  DNA histograms show results of 1 representative experiment performed on U937 cells. (E) ST1326 induces intracellular palmitate accumulation. Representative images of U937 cells stained with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY-C16) with or without ST1326 treatment as described in “Study design.” Images were acquired with laser scanning confocal microscope (TCS SP2 AOBS; Leica Microsystems, Mannheim, Germany) using a ×40 (numerical aperture = 1.25) oil immersion lens with optical pinhole at 1 AU. Scale bar indicates 20 μm. (F) Effects of ST1326 on palmitate accumulation. Relative BODIPY-C16 uptake is expressed as fluorescence intensities (mean ± SD of 3 independent experiments) in leukemia cell lines exposed to ST1326 as compared with the respective untreated controls. The increase of fluorescence intensities in ST1326-exposed cells is proportional to the intracellular palmitate accumulation (details in supplemental Materials and methods).

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