Figure 3

Figure 3

Mitochondrial FAO inhibition activates mTORC1 activity dependent on Raptor acetylation.A, immunoprecipitation of Raptor followed by detection of Raptor acetylation levels with antilysine acetylation antibody in CN- and MD-treated ZFL cells after 48 h. N = 3. B, immunoprecipitation of Raptor followed by detection of Raptor acetylation levels with antilysine acetylation antibody. The NC, MD, acly siRNA, and MD with acly siRNA–treated cells were lysed and Western blots for Raptor acetylation. N = 3. C, subcellular localization of Raptor and lysine-acetylated proteins in NC, MD, acly siRNA, and MD with acly siRNA–treated cells after 48 h. Scale bars represent 5 μm. D, relative mRNA levels of Raptor in CN, negative control (NC), and raptor siRNA–treated ZFL cells. N = 5. E–I, the effect of raptor knockdown on mTORC1 signaling caused by mitochondrial FAO inhibition. NC and MD cells were treated with raptor siRNA for 48 h. Relative protein quantification of Raptor/tubulin (F), p-mTOR/tubulin (G), p-S6k/tubulin (H), and p-S6/S6 (I) in ZFL cells. Cells were lysed, and Western blots for Raptor, p-mTOR^Ser2448, p-S6k^Thr389, p-S6^Ser235/236, and S6 are shown (E). N = 3. Data represent mean ± SD. ∗p < 0.05 and ∗∗p < 0.01. Ac-Raptor, lysine acetylation of Raptor; CN, control; FAO, fatty acid oxidation; MD, mildronate; mTORC1, mechanistic target of rapamycin complex 1; NC, negative control; T-Raptor, total Raptor; ZFL, zebrafish liver.