Figure 2.
Knockdown of VLCAD decreases leukemic proliferation, clonogenic growth, and intact cellular and palmitate (C16)-supported ETF respiration. (A) Western blot showing knockdown of VLCAD in TEX cells, where D6 and D8 are knockdown constructs. (B) Densitometry of VLCAD knockdown in TEX cells. Proliferation counts after 1, 3, and 5 days (C) and colony count after 2 weeks (D) for TEX wild-type and VLCAD-knockdown cells. (E) Representative oxygraph quantifying basal and maximal respiration of intact leukemia cells (left panel). In brief, following injection into the high-resolution respirometer, cells demonstrate a basal rate of respiration. ATP synthesis is uncoupled by addition of oligomycin (OLI). Maximal respiration is stimulated with an injection of a chemical uncoupler (FCCP) and pyruvate (PYR). All mitochondrial respiration is inhibited by addition of antimycin A (ANTIA). Intact cell basal and maximal respiration of TEX wild-type and VLCAD knockdown cells (right panel). (F) Schematic diagram showing the flow of electrons following the oxidation of fats of different carbon lengths to support respiration of the ETC. Electrons supplied by VLCAD (for very long fats; 14-18 carbons long), medium chain acyl-CoA dehydrogenase (MCAD; for medium-length fats; 6-12 carbons long), and short chain acyl-CoA dehydrogenase (SCAD; for short-length fats; 4 carbons long) reduce the FAD cofactor attached to the ETF (ETF-FAD). Reduced ETF-FADH2 is oxidized by ETF dehydrogenase (ETFDH), passing electrons to ubiquinone (UQ) and then complex III (III). Malonate (MALON) and rotenone (ROT) inhibit electron flow from complexes I (I) and II (II), respectively; residual oxygen flux measured by the respirometer is exclusively ETF supported respiration, an indirect measure of VLCAD activity when palmitate is supplied. (G) Representative oxygraph quantifying C16-supported ETF respiration (left panel). In brief, permeabilized cells are injected into the respirometer. C16 in the form of palmitoyl-carnitine (PC), malate (MAL), and adenosine diphosphate (ADP) stimulate C16-supported FAO respiration. MALON and ROT inhibit any contribution of electrons from complexes I and II, allowing ETF respiration to be exclusively assessed. C16-supported (middle panel) and C8-supported (right panel) ETF respiration of TEX wild-type and VLCAD-knockdown cells. Data in (B-D, E [right panel] and G [middle and right panels]) are mean ± standard deviation. Panel F created in Biorender. *P ≤ .05, **P ≤ .002, ***P ≤ .001, 1-way ANOVA with Tukey’s post-hoc test.

Knockdown of VLCAD decreases leukemic proliferation, clonogenic growth, and intact cellular and palmitate (C16)-supported ETF respiration. (A) Western blot showing knockdown of VLCAD in TEX cells, where D6 and D8 are knockdown constructs. (B) Densitometry of VLCAD knockdown in TEX cells. Proliferation counts after 1, 3, and 5 days (C) and colony count after 2 weeks (D) for TEX wild-type and VLCAD-knockdown cells. (E) Representative oxygraph quantifying basal and maximal respiration of intact leukemia cells (left panel). In brief, following injection into the high-resolution respirometer, cells demonstrate a basal rate of respiration. ATP synthesis is uncoupled by addition of oligomycin (OLI). Maximal respiration is stimulated with an injection of a chemical uncoupler (FCCP) and pyruvate (PYR). All mitochondrial respiration is inhibited by addition of antimycin A (ANTIA). Intact cell basal and maximal respiration of TEX wild-type and VLCAD knockdown cells (right panel). (F) Schematic diagram showing the flow of electrons following the oxidation of fats of different carbon lengths to support respiration of the ETC. Electrons supplied by VLCAD (for very long fats; 14-18 carbons long), medium chain acyl-CoA dehydrogenase (MCAD; for medium-length fats; 6-12 carbons long), and short chain acyl-CoA dehydrogenase (SCAD; for short-length fats; 4 carbons long) reduce the FAD cofactor attached to the ETF (ETF-FAD). Reduced ETF-FADH2 is oxidized by ETF dehydrogenase (ETFDH), passing electrons to ubiquinone (UQ) and then complex III (III). Malonate (MALON) and rotenone (ROT) inhibit electron flow from complexes I (I) and II (II), respectively; residual oxygen flux measured by the respirometer is exclusively ETF supported respiration, an indirect measure of VLCAD activity when palmitate is supplied. (G) Representative oxygraph quantifying C16-supported ETF respiration (left panel). In brief, permeabilized cells are injected into the respirometer. C16 in the form of palmitoyl-carnitine (PC), malate (MAL), and adenosine diphosphate (ADP) stimulate C16-supported FAO respiration. MALON and ROT inhibit any contribution of electrons from complexes I and II, allowing ETF respiration to be exclusively assessed. C16-supported (middle panel) and C8-supported (right panel) ETF respiration of TEX wild-type and VLCAD-knockdown cells. Data in (B-D, E [right panel] and G [middle and right panels]) are mean ± standard deviation. Panel F created in Biorender. *P ≤ .05, **P ≤ .002, ***P ≤ .001, 1-way ANOVA with Tukey’s post-hoc test.

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