Western blot and immunofluorescence.

For western blotting tissues were homogenized in radioimmunoprecipitation assay buffer (RIPA) supplemented with protease and phosphatase inhibitor cocktails (aprotinin, leupeptin, benzamidin, pepstatin A, PMSF, sodium fluoride, sodium orthovanadate and β-glycerophosphate), with an Ultra-Turrax® homogenizer. Samples were centrifuged at 15,000 x g for 15 min at 4°C. Supernatantprotein was quantified and protein extracts were mixed with a loading buffer 4X (40% glycerol, 2% SDS, 250 mM Tris pH 6.8, 0.02% bromophenol blue, and 12% β-mercaptoethanol). Western blots were performed with 20 μg of tissue extracts. Proteins were separated in 8–12% SDS-PAGE and transferred to a nitrocellulose membrane (BioRad). The membrane was blocked in 5% milk, incubated with primary antibody in TBST with 5% BSA overnight at 4°C, and then incubated with Horseradish peroxidase activity linked to secondary antibody. Protein signal was detected with ECL substrate (Pierce) using X-Ray films. Primary antibodies used were c-MYC (1472–1, Epitomics), PSAT1 (20180–1-AP, Protein Tech), PHGDH (sc292792, Santa Cruz), β-Actin (A2228 and A3854, Sigma), Cleaved CASP3 (9661, Asp175, Cell Signaling), Glucokinase (AP7901C, Abgent), GLS2 (6217, ProSci), FASN (#23180, C20G5, Cell Signaling), HK1 (#2024, C35C4, Cell Signaling), HK2 (#2867, C64G5, Cell Signaling), PCNA (#13110, D3H8P, Cell Signaling), PARP (#9542, Cell Signaling), with the GLS1-KGA specific antibody was a generous gift from Astra Zeneca.

For immunofluorescence, tissues were fixed for 24 hours in 4% paraformaldehyde, equilibrated in 30% sucrose, embedded in OCT compound (VWR international), and stored at −80°C until 7-μm-thick cryosections were obtained. Sections were permeabilized by incubating with 0.2% Triton X-100 in PBS for 10 min, and treated with the indicated primary antibody for 16 h. Primary antibodies used were: GFP (4745–1051, Bio-Rad) and PanCK (Z0622, Dako). After four washes with 0.05% Tween in PBS, cells were incubated with anti-rabbit Alexa Fluor® 488 or 555 secondary antibodies (Invitrogen) for 1 hour. Nuclear marker DAPI was included in the mounting media. Samples were examined using a confocal microscope (TCS SP5 II Leica) and LCS Lite software (Leica) was used to collect digital images.