3.4.2. Phospholipid and Sphingolipid Relative Quantification

The lipid extract was dried, dissolved in 50 μL of MeOH then stored at −20 ◦C prior to analysis. Analysis were performed on an Agilent 1290 UPLC system coupled to a G6460 triple quadripole spectrometer (Agilent Technologies, Les Ulis, France)) using a Kinetex HILIC column (Phenomenex, Le Pecq, France), 50 × 4.6 mm, 2.6 μm). The column temperature was controlled at 40 ◦C. The mobile phases A and B were ACN and 10 mM AF in H2O at pH 3.2, respectively. The gradient was as follows:

from 10% to 30% B in 10 min; 10–12 min, 100% B; and, then back to 10% B at 13 min for 1 min. The flow rate of mobile phase was 0.3 mL/min and the injection volume was 5 μL. Electrospray ionization (ESI) was employed at 325 ◦C in positive (for Cer, PE, PC, and SM analysis) and negative ion mode (for PI and PS analysis). The collision gas was nitrogen. Needle voltage was set at +4000 V. SRM transitions were used for relative quantification with a precursor ion scan of 184 *m*/*z*, 241 *m*/*z,* and 264 *m*/*z* to PC/SM, PI, and Cer, respectively; and, a neutral loss scan of 141 *m*/*z* and 87 *m*/*z* to PE and PS, respectively. In each family, individual molecular species were scanned with suitable SRM scan mode, the area of each peak were measured via Mass Hunter software. The relative quantitative calculations were based on the peak area ratios relative to the internal standards [37].

#### 3.4.3. Sphingoid Bases

The lipid extract was dried and dissolved in 50 μL of MeOH then stored at −20 ◦C prior to analysis. Analysis were performed on an Agilent 1290 UPLC system coupled to a G6460 triple quadripole spectrometer (Agilent Technologies, Les Ulis, France) an Acquity UPLC BEH-C8 (Waters, Issy les Moulineaux, France), 100 × 2.1 mm, 1.7 μm) maintained at 35 ◦C. The mobile phases A and B were H2O, FA (99.9:0.1; *v*/*v*), and ACN, FA (99.9:0.1, *v*/*v*), respectively. The gradient was as follows: 50% B at 0 min, 60% B at 2 min, 60% B at 3 min, 100% B at 4 min, 100% B at 8.5 min, and 50% B at 9 min. The flow rate of mobile phase was 0.3 mL/min and the injection volume was 5 μL. ESI was performed in positive ion mode at 300 ◦C. The collision gas was nitrogen. Needle voltage was set at + 4000 V. SRM transitions in neutral loss scan were used. For quantitative analysis, calibration samples (500 to 0.976 ng) were prepared with commercial sphingolipid standards. All of the quantitative calculations were based on the peak area ratios relative to the internal standards [adapted from Sikora [52].

#### 3.4.4. Neutral Lipid Relative Quantification

The lipid extract was dried, dissolved in 30 μL of EtOAc, and then stored at −20 ◦C prior to analysis. 1 μL of the lipid extract was analyzed by gas chromatography on a FOCUS Thermo Electron system using a Zebron-1 fused silica capillary column (Phenomenex, 5 m × 0.32 mm, 0.50 μm film thickness). Oven temperature was programmed from 200 ◦C to 350 ◦C at a rate of 5 ◦C/min and the carrier gas was hydrogen (0.5 bar). The injector and the detector temperatures were at 315 ◦C and 345 ◦C, respectively. All of the quantitative calculations were based on the chromatographic peak area relative to the internal standards [39].

#### 3.4.5. Total FA Profiling

The lipid extract was hydrolysed in KOH (0.5 M in MeOH) at 50 ◦C for 30 min, and transmethylated in 1 mL of BF3-MeOH and 1 mL of heptane at 80 ◦C for 1h. After the addition of 1 mL H2O to the crude, FAs methyl esters (FAMEs) extract was extracted with 3 mL of heptane, dried, and dissolved in 20 μL of EtOAc. 1 μL of FAMEs extract was analyzed by gas chromatography (GC) on a Clarus 600 Perkin Elmer system using a Famewax RESTEK fused silica capillary column (30 m × 0.32 mm, 0.25 μm film thickness). Oven temperature was programmed from 110 ◦C to 220 ◦C at a rate of 2 ◦C/min and the carrier gas was hydrogen (0.5 bar). The injector and the detector temperatures were at 225 ◦C and 245 ◦C, respectively. All of the quantitative calculations were based on the chromatographic peak area relative to the internal standards [38].

#### *3.5. FA Isotopic Labeling Profiling*

#### 3.5.1. Sample Preparation

Cell pellet (5 M) was extracted like previously in the presence of the internal standards: glyceryl trinonadecanoate (0.2 μg) for TFA and heptadecanoate (0.2 μg) for FFA. The equivalent of 4 M of cell was collected for the direct derivatization of FFA. Concerning TFA, the equivalent of 1 M of cells was hydrolysed in KOH (0.5 M in MeOH) at 50 ◦C for 30 min. Free and total FA were derivatized in pentafluorobenzyl esters with 1% PFB-Br and 1% DIPEA in ACN (50 μL), at RT for 20 min. Samples were dried and dissolved in EtOAc (10 μL).

#### 3.5.2. GC-MS Analysis

Labelled total FA analysis were performed on a Thermo Fisher Trace GC system that was connected to a ThermoFisher TSQ8000 (Les Ulis, France)) triple quadrupole detector using a HP-5 MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness). Oven temperature was programmed, as follows: 150 ◦C for 1 min, 8 ◦C/min to 350 ◦C, then the temperature is kept constant for 2 min. The carrier gas was helium (0.8 mL/min). The injector, the transfer line, and the ion source temperature were at 250 ◦C, 330 ◦C, and 300 ◦C, respectively. The TSQ8000 was operated in negative ionization mode (Methane at 1 mL/min) in selected ion monitoring (SIM) mode and 1 μL of sample was injected in splitless mode.

#### 3.5.3. Data Processing

GC-MS analysis produced a mass spectrum for each FA, which contains the abundance of each isotopologue. For each FA, the lightest (unlabeled) isotopologue is denoted M + 0; e.g., PFB-palmitate M + 0 has a mass of 255.3, whereas the isotopologue with 1 atom [13C] PFB-palmitate (M + 1) has a mass of 256.3, etc., (Supplementary Table S1). Isotopic clusters were obtained by integrating gas chromatographic signals for each isotopologue. Isotopologue distributions were obtained from the corresponding isotopic clusters after correction for natural abundance of carbon and non-tracer elements (oxygen and hydrogen) using the software IsoCor, and purity of the tracer was corrected assuming 99% 13C-purity. Finally, the 13C-enrichment, which represents the mean content in tracer atoms (13C) within the molecule, was calculated from the corresponding IDs, as detailed in Millard et al. [61].

#### *3.6. Immunoblotting of Total Proteins*

For immunoblot assay, cells were first subjected to lysis in NuPAGE LDS Sample Buffer. Total proteins content from every samples was measure using Pierce BCA Protein Assay Kit according to the manufacturer's recommendations. Samples lysates were then loaded onto NuPAGE 4–12% Bis-Tris Protein Gels (10, 12, or 15 wells). After electrophoresis, proteins were transferred to nitrocellulose membranes. The transblotted membranes were blocked for 1 h and then probed with appropriate primary antibodies (dilution as recommended by manufacturers) overnight at 4 ◦C. Next, the membranes were washed three times for a total of 30 min and then incubated with secondary antibodies at room temperature for 1 hr. After another three washes, proteins were detected using SuperSignal West Pico PLUS chemiluminescent Substrate, PXi imager (Syngene), and GeneSys software (Syngene, Paris, France), according to the manufacturer's manual. Proteins expression were quantified using GeneTool software (Syngene) and normalized to their corresponding loading control. Antibodies for immunoblotting were purchased from the following sources: FASN (#3180S) from Cell Signaling Technology; Actin (MAB1501) from Millipore; IDH1 R132H (DIA-H09) from Dianova. HRP conjugate anti-rabbit (W4011) and anti-mouse (W4021) secondary antibodies were purchased from Promega (Charbonnières les Bains, France).

## *3.7. ChIP Assays*

To perform chip assays briefly, 10<sup>7</sup> cells were cross-linked in 1% formaldehyde/1% paraformaldehyde for 5 min, followed by addition of 125 mM Glycine to stop the reaction. Cells were then washed in PBS, resuspended in lysis buffer (10 mM Tris pH 8, 140 mM NaCl, 0.5 mM EGTA, 0.1% SDS, 0.5% Triton X-100, 0.05% NaDoc and protease inhibitors) and chromatin was shared by sonication. qChIPs were carried out by incubating cell lysates (Input) with 20 μL of protein G-Dynabeads and 5 ug of antibody. The same amount of rabbit IgGs (Santa Cruz, Boulogne Billancourt, France) was used for control ChIP experiments. After O/N incubation, washing, reverse cross-linking, and treatment

with both RNase A and Proteinase K, proteins were removed with phenol/chloroform extraction and DNA was recovered using the NucleoSpin Extract II kit. Input and immunoprecipitated DNA were then analyzed by QPCR using the SYBR Green Master mix on a LightCycler 480 SW 1.5 apparatus (Roche, Boulogne Billancourt, France). Results are represented as the mean value of at least three independent experiments of immunoprecipitated chromatin (calculated as a percentage of the input) with the indicated antibodies after normalization by a control ChIP performed with rabbit IgGs.
