Skip to main content
. 2020 Jun 23;31(12):107806. doi: 10.1016/j.celrep.2020.107806

Figure 3.

Figure 3

Inhibition of mTOR Reduces FADS2 Transcription, Protein Expression, and Sapienate Metabolism

(A and B) Relative FADS2 mRNA expression upon treatment with DMSO (n = 10), rapamycin (A) (20 nM, 72 h, n = 9), or Torin1 (B) (40 nM, 72 h, n = 9) in HUH7 cells. Bar graphs are presented as mean ± SD from biological independent samples. Statistical testing was performed by a two-sided unpaired Student’s t test.

(C and D) Sapienate to palmitate ratio upon treatment with DMSO (C: n = 9; D: n = 12), rapamycin (C) (20 nM, 72 h, n = 9), or Torin1 (D) (40 nM, 72 h, n = 12) in HUH7 cells. Bar graphs are presented as mean ± SD from biological independent samples. Statistical testing was performed by a two-sided unpaired Student’s t test.

(E and F) Relative sapienate level upon treatment with DMSO (E: n = 9; F: n = 12), rapamycin (E) (20 nM, 72 h, n = 9), or Torin1 (F) (40 nM, 72 h, n = 12) in HUH7 cells. Bar graphs are presented as mean ± SD from biological independent samples. Statistical testing was performed by a two-sided unpaired Student’s t test.

(G) Relative FADS2 protein expression upon treatment with DMSO or Torin1 (40 nM, 72 h, n = 3) in HUH7 cells. Bar graphs are presented as mean ± SD from biological independent samples. Statistical testing was performed by a two-sided unpaired Student’s t test.

(H–J) Fraction of newly synthesized sapienate (H), palmitoleate (I), and palmitate (J) based on isotopomer spectral analysis in the HUH7 cultured in the presence of DMSO or Torin1 with 13C6 glucose. Cells were grown in DMEM (4.5 g/L glucose, 10% FBS), after which cells were grown for 72 h in 10% dialyzed FBS DMEM containing 4.5 g/L 13C6 glucose supplemented with DMSO or 40 nM Torin1 (n = 3). Bar graphs are presented as lower and upper 95% confidence intervals. Statistical testing was performed by a two-sided unpaired Student’s t test.

(K–M) 13C6 glucose incorporation into sapienate (K), palmitoleate (L), and palmitate (M) in HUH7 in control condition (DMSO, black) or upon Torin1 treatment (white). Cells were grown in DMEM (4.5 g/L glucose, 10% FBS) after which cells were grown for 72 h in 10% dialyzed FBS DMEM containing 4.5 g/ L 13C6 glucose supplemented with DMSO or 40 nM Torin1 (n = 3). Bar graphs are presented as mean ± SD from biological independent samples. Statistical testing was performed by a two-sided unpaired Student’s t test.