Fig. 6: c-SRC phosphorylation of ACLY inhibits its activity and facilitates NADPH production for fatty acid synthesis. | Nature Communications

Fig. 6: c-SRC phosphorylation of ACLY inhibits its activity and facilitates NADPH production for fatty acid synthesis.

From: The proto-oncogene tyrosine kinase c-SRC facilitates glioblastoma progression by remodeling fatty acid synthesis

Fig. 6

a Flag-ACLY was co-transfected with HA-tagged c-SRC-WT, c-SRC-CA and c-SRC-KD in U87 cells. After 24 h of transfection, cells were lysed and subjected to malate dehydrogenase (MDH)-coupled ACLY activity assay (n = 3 independent experiments). b U87 cells with c-SRC knockdown were analyzed for ACLY activity (n = 3 independent experiments). c ACLY-WT or ACLY-Y682F were co-transfected with HA-c-SRC in U87 cells, followed by ACLY activity analysis (n = 3 independent experiments). d ACLY activity assay with various concentrations of citrate was performed in U87 cells to analyze velocities of ACLY after c-SRC-mediated phosphorylation (n = 3 independent experiments). e The double-reciprocal plot of ACLY after phosphorylation by c-SRC is calculated based on the result in (d) (n = 3 independent experiments). f Primary glioma cells with c-SRC knocked down by two independent shRNAs were analyzed for NADPH levels using LC–MS (n = 3 independent experiments). g NADPH level was determined by LC–MS after c-SRC knockdown in U87 and CHG-5 cells (n = 3 independent experiments). h ACLY knockdown U87 and CHG-5 cells were reconstituted with ACLY-WT (re-ACLY-WT) and ACLY-Y682F (re-ACLY-Y682F). Cells were then analyzed for NADPH level by LC–MS (n = 3 independent experiments). i The levels of acetyl-CoA and malonyl-CoA in re-ACLY-WT and re-ACLY-Y682F U87 cells were analyzed by LC–MS (n = 3 independent experiments). j U87 cells reconstituted with ACLY-WT (re-ACLY-WT) or ACLY-Y682F (re-ACLY-Y682F) were incubated in fresh medium containing 20 mM [U-13C]-glucose for 3 days. The [U-13C]-glucose-derived fatty acids were quantified by GC–MS (n = 3 independent experiments). k The abundance of metabolites in the TCA cycle was determined after reconstitution with ACLY-WT or ACLY-Y682F in U87 cells (n = 3 independent experiments). Statistical data are presented as the mean ± SD (n = 3). p-values were calculated using unpaired and two-sided Student’s t-test.

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