Anti glut1

Cultures in the microchips were washed once with DPBS and fixed in 4% paraformaldehyde (Fisher Scientific) for 20 min at room temperature. The chips were then washed once with DPBS, blocked/permeabilized in saponin buffer (0.1% saponin, 1% BSA, 10% FBS in DPBS) for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies (1:50 dilution) in saponin buffer without FBS. The following antibodies were used: anti-GFAP (BD Biosciences), anti-Alpha-Smooth Muscle Actin (aSMA) (Sigma), anti-Glut1 (Novus Biologicals, Toronto, Canada), anti-ZO-1 (Invitrogen). The next day, the cells were washed twice with DPBS, then incubated in Alexa-conjugated secondary antibodies of the appropriate species (goat-anti rabbit, goat-anti mouse, donkey anti-mouse; Alexa Fluor 488; Alexa Fluor 555; Thermo Fisher Scientific) and DAPI nuclear stain diluted 1:500 in saponin buffer without FBS for 1 h at room temperature. Lastly, the cells were washed three times with DPBS and stored at 4 °C.

Tumors were digested to single cell suspension enzymatically and filtered twice through 70μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with Cell Staining Buffer (BioLegend), and then stained with the fluorophore-conjugated antibodies for 30 min at 4 °C. The excess of unbound antibodies was washed out before acquisition in flow cytometry. Cells were analyzed using ID700 Spectral Cell Analyzer (SONY) and sorted using FACS-ARIA III (BD, Pharmingen). The following antibodies were for flow cytometry: anti-CD45 PAC (30-F11), anti-CD8b APC-Cy7 (YTS156.7.7), anti-CD4 PE(H129.19), anti-NK1.1 APC-Cy7 (PK136), anti-CD11b APC (M1/70), anti-Ly6c PE (HK1.4), anti F4/80 (BM8), anti-CXCR6 APC (SA051D1), anti-GranzymeB, anti-CD90 FITC (30-H12), anti-CD73 APC (TY/11.8), anti-PD-1 APC (RMP1-30) all purchased from BioLegend and used at 1:100 dilution. Anti-CD31-PE (eBioscience, 390, 1:100), anti-GLUT1 (Novus, Fgi.72, 1:100), anti-Cxcl16 (Bioss, BS-1441R, 1:50) and anti-Rabbit IgG AF488 (Invitrogen, A21245, 1:100) were used to identify CAF populations. Ghost Dye Red 710 (Tonbo, Cat #13-0871-T100) was used to exclude dead cells from the analysis.

Antibodies used were the following: anti-ACC (1:500; C83B10, Cell Signaling Technology, 3676), anti–β-actin (1:1000; LI-COR Biosciences, 926-42210), anti-NFκB p65 (1:500; Cell Signaling Technology, 8242), anti–phospho-NFκB p65 (S536) (1:500; Cell Signaling Technology, 3033), anti-ERK p42/p44 (1:1000; Cell Signaling Technology, 4696), anti–phospho-ERK p42/p44 (T202/Y204) (1:1000; Cell Signaling Technology, 4377), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; Cell Signaling Technology, 5174), anti–phospho-STAT6 (Y641) (1:1000; Cell Signaling Technology, 9361), anti-STAT6 (1:1000; Cell Signaling Technology, 9362), anti-vinculin (1:1000; Cell Signaling Technology, 13901), anti-FASN (1:1000; Cell Signaling Technology, 3180), anti-arginase (1:500; Santa Cruz Biotechnology, sc-18351), anti–acetyl-H3 (K27) (1:500; Active Motif, 39133), anti-H3 (1:5000; Abcam, ab1791), rodent OXPHOS antibody cocktail (1:250; Abcam, ab110413), and anti-GLUT1 (1:1000; Novus Biologicals, NB110-39113).