Ab47010

White adipose tissue was excised, fixed in formalin overnight, embedded in paraffin and sectioned. The immunofluorescence analysis of F4/80 (MCA497GA; Abd Serotec, NC, USA), lactate dehydrogenase (ab47010, Abcam)  and HIF-1α (NB100-479; Novus Biologicals, CO, USA) was conducted after deparaffinization as described previously^{23 (link)}. Sections were mounted and visualized using a Nikon Eclipse microscope.

The protein extraction protocol from cancer cells was previously reported [30 (link)]. Briefly, cells were collected and lysed using Radio Immunoprecipitation Assay (RIPA) protein extraction reagent (Beyotime) with a protease inhibitor cocktail (Roche, IN, USA). Equal amounts of protein lysate were quantified using BCA kit (Thermo Fisher, USA), separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then sealed with 5% non-fat milk for 1 hour, and incubated with primary antibodies (Abcam, Cambridge, UK) against: FUS (ab84078, 1/1000), SLC10A1 (ab131084, 1/1000), β-actin (ab8227, 1/1000), GLUT1 (ab14683, 1/2500), HK2 (ab227198, 1/5000), LDHA (ab47010, 1/1000), PGK1 (ab154613, 1/1000) overnight at 4°C, with GAPDH (ab9485, 1/2500) as control. After washing, the membrane was incubated with goat anti-rabbit secondary antibody (ab96899, 1/1000, Abcam, Cambridge, UK) at 37°C for 1 hour. The protein bands were finally detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions.

H1975 and H1299 were seeded at 5.0 × 10³ cells/well into 24-well plates. Cells were then fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Subsequently, cells were incubated with appropriate concentrations of primary antibody of GLUT1 (ab115730, 1:200, abcam, USA) and LDHA (ab47010, 1 µg/ml, abcam, USA) at 4 °C overnight, and then probed with ProteinFind® Goat Anti-Rabbit IgG (H + L), AF488 Conjugate (HS131-01, 1:100, TransGen BioTech, China) for 60 min at room temperature away from light. Next, the secondary antibody solution was removed and the cells were washed thrice by PBS and then incubated with Hoechst for 15 min at room temperature in the dark. Immunofluorescence was visualized and photographed under a fluorescence microscope (OLYMPUS, Tokyo, Japan).

Western blot analysis was done following a previously described procedure [43 (link)]. Antibodies against lactate dehydrogenase monomers and beta-actin were purchased from Abcam (Cambridge, United Kingdom) and used in the following concentrations: LDHA (1 µg/mL; ab47010), LDHB (0.1 µg/mL; ab85319), and β-actin (0.5 µg/mL; ab8226). Prior to loading onto the gel, nine samples from each group were pooled by three to obtain three final samples per group (Figure 2 shows two representative bands for each group cut from the same blot, for full blots, see Supplementary Information Figure S2). ImageJ software (National Institute of Health, Bethesda, Maryland, United States) was used for densitometric analyses of immunoreactive bands. Original band density was calculated as the sum of pixel intensities within a band. Final band density was obtained as the ratio of dots per band for the target protein averaged against β-actin (gel loading control). Protein expression is expressed in arbitrary units (AU) from three independent experiments.

Cells were firstly lysed in RIPA buffer supplemented with a cocktail of protease inhibitors. Cell lysates were centrifuged at 13000 g for 10 minutes at 4°C before aliquots of 20 μg proteins were resolved on SDS-PAGE, transferred onto polyvinylidenedifluoride transfer membrane(PVDF), and blotted for targeting antibodies:MPC1(HPA045119, Sigma-Aldrich, St. Louis, MO,USA, dilution 1:1000), MPC2(ab111380, abcam, Cambridge, UK, dilution 1:1000), GLUT1(Rabbit mAb#12939, cell signaling technology, Leiden, Netherlands, dilution 1:1000), HK2 (ab104836, abcam, Cambridge, UK, dilution 1:1000), LDHA (ab47010, abcam, Cambridge, UK, dilution 1:1000) and α-Tubulin antibody(T9026, Sigma-Aldrich, St. Louis, MO, USA dilution 1:3000).