Fig. 4: ACSS2 is activated to facilitate fatty acid synthesis by c-SRC-mediated phosphorylation.

a, b Plasmids expressing ACSS2, c-SRC-WT and c-SRC-KD were transfected in U87 cells as indicated. After 6 h of transfection, cells were shifted to a complete medium containing 5 mM [2-¹³C]-acetate for 24 h of cell culture. The levels of acetate-derived acetyl-CoA (M + 1) (a) and malonyl-CoA (M + 1) (b) were analyzed by LC–MS (n = 3 independent experiments). KD: kinase dead. c, d U87 cells were transfected with ACSS2, c-SRC-WT, and c-SRC-KD as indicated (d) and incubated with 5 mM [2-¹³C]-acetate for 3 days. The levels of acetate-derived fatty acids (C16:0 and C18:0, M + 1) were analyzed by GC–MS (n = 3 independent experiments) (c). The samples derive from the same experiment but different gels for Flag (d). e U87 cells were transfected with c-SRC and different mutants of ACSS2 as indicated. After 24 h of transfection, cells were cultured in a fresh complete medium containing 5 mM [2-¹³C]-acetate for another 24 h. [2-¹³C]-acetate-derived acetyl-CoA (M + 1) was detected by LC–MS (n = 3 independent experiments). f U87 cells were transfected with ACSS2-WT, ACSS2-Dmut and c-SRC and incubated with 5 mM [2-¹³C]-acetate for 3 days. C16:0 (M + 1) and C18:0 (M + 1) were analyzed by GC–MS (n = 3 independent experiments). g ACSS2 knockdown of CHG-5 cells were constructed and re-expressed for ACSS2-WT (re-ACSS2-WT) or its double mutation (re-ACSS2-DMut). The same samples derived from same experiment but different gels for p-Tyr530, p-Tyr562, and ACSS2. h, i CHG-5 cells after reconstitution with ACSS2-WT or ACSS2-DMut were incubated with medium containing 5 mM [2-¹³C]-acetate for 24 h. The acetyl-CoA (M + 1) (h) and malonyl-CoA (i) were analyzed by LC–MS (n = 3 independent experiments). j CHG-5 cells as indicated in (g) were incubated with a complete medium containing 5 mM [2-¹³C]-acetate for 3 days. Then [2-¹³C]-acetate-derived fatty acids (C14:0, C16:0, and C18:0) (M + 1) were determined by GC–MS (n = 3 independent experiments). k U87 cells were transfected with ACSS2. After 24 h of transfection, cells were cultured in a fresh medium containing 5 mM [2-¹³C]-acetate for another 24 h. The levels of acetate-derived acetyl-CoA (M + 1) (left) and malonyl-CoA (M + 1) (right) for de novo fatty acid synthesis were then analyzed by LC–MS (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.