Ab137043

Cells and spheroids were harvested as previously described [11 (link)]. Whole cell lysates were generated using Tris Glycine SDS sample buffer (Gradipore) by shaking at room temperature for 1 h and further processed via SDS-PAGE as described previously [16 (link)]. The following primary antibodies were used: anti-AKR1C3 (clone NP6.G6.A6, 1:500, Sigma), anti-HMGCS2 (mitochondrial) (ab137043, 1:300, Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:50000, Millipore). Visualization and quantification of protein bands were performed with Image Studio software Version 5.2 (LI-COR Biosciences).

Tissue proteins were extracted according to kit instructions (Sigma Aldrich, R0278, St. Louis, MO, USA). Quantifying protein concentrations and samples was done routinely with a BCA kit (Beyotime Biotechnology, P0010S, Beijing, China) from Louis, MO, USA. SDS-PAGE of 30 μg was conducted on each sample using either 8% or 12% SDS-PAGE, and then the semi-dry transfer was made to PVDF membranes. After blocking with 5% skim milk prepared with TBST for 1 h at room temperature, the membranes were incubated overnight at 4°C with pre-diluted primary antibodies. The secondary antibody was then incubated with the membrane for 1 hour at room temperature after it had been washed. The dilution ratios of all antibodies were as follows: Hmgcs2 (1:500, Abcam, ab137043, Cambridge, London, UK), Src (1:500, Abcam, ab109381, Cambridge, London, UK), p-PI3K (1:500, CST, Cell Signaling Technology, #17366, Danvers, MA, USA), PI3K (1:500, CST, #4292), p-AKT (1:500, CST, #4060), AKT (1:500, CST, #4685), and GAPDH (1:500, CST, #97166).

The frozen tissues were homogenized in a lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, and protease inhibitor, pH 7.4) and then centrifuged for 20 min at 10,000 g to discard cell debris. The total protein concentrations were determined using a Bio-Rad kit. The proteins were subjected to Western blot analysis with the desired antibodies. Antibodies against carnitine palmitoyltransferase 1A (CPT1A) (ab234111), HMGCS2 (ab137043), FAS (ab128870), HMGCR (ab174830), liver glycogen phosphorylase (PYGL) (ab198268) and PYGL (phospho S15, ab227043) were obtained from Abcam. Antibodies against COX4 (A21348) was obtained from Invitrogen. Antibody against phosphoenolpyruvate carboxykinase 1 (PCK1) (A2036) and GCK (A6293) was purchased from ABclonal. Antibodies against AMPKα (2532), phospho-AMPKα (2535), CREB (9197), Phospho-CREB (9198), and SCD1 (2438) were purchased from Cell Signaling Technology. Antibody against G6PC (22169-1-AP) was purchased from Proteintech. Antibody against TUBULIN (T0198) was obtained from Sigma-Aldrich.

Dewaxed, rehydrated, and antigen retrieval were performed on paraffin slices. Methanol was used to inactivate endogenous peroxidase for 15 min. The slices were then sealed after being treated with 5% BSA for 1 h, followed by an overnight incubation with primary antibodies. The primary antibodies used in this study were Pdk4 (Dilution 1:50, YN5701, Immunoway), Hmgcs2 (Dilution 1:200, ab137043, abcam) Decr1 (Dilution 1:50, A13014, ABclonal) and Ivd (Dilution 1:500, 10822-1-AP, ProteinTech). The paraffin slices were cultured with secondary antibodies the next day for one hour at 37 °C. Finally, the slides were stained with a DAB Detection Kit, and the counterstained with haematoxylin.

Mouse embryos were fixed in 4% PFA in PBS at 4°C, embedded in paraffin, sectioned at 5μm, and immunofluorescence performed as described previously [39 (link)]. Primary antibodies used for this study were anti-HMGCS2 rabbit monoclonal (1:50; ab137043, Abcam), anti-SOX9 sheep polyclonal (1:100; [40 (link)]), anti-MVH goat polyclonal (1:200; AF2030, R&D systems), anti-AMH goat polyclonal (1:200; sc6886, Santa Cruz), anti-AMH goat polyclonal (1:50; AF1446 R&D systems), anti-SYCP3 mouse monoclonal (1:100; ab97672, Abcam), anti-FOXL2 rabbit polyclonal (1:300; [41 (link)]) and anti-CYP11A1 rabbit polyclonal [42 (link)]. Secondary antibodies used were donkey anti-rabbit Alexa 488, donkey anti-rabbit Alexa 568, donkey anti-goat Alexa 488, donkey anti-goat Alexa 546, donkey anti-mouse Alexa 488, and donkey anti-sheep Alexa 647 obtained from Invitrogen and used at 1:300. Images were taken with a Zeiss LSM 510 Meta confocal microscope at the Australian Cancer Research Foundation Dynamic Imaging Centre for Cancer Biology, University of Queensland and with a Zeiss LSM800 confocal microscope at the Biological Optical Microscopy Platform (BOMP) at the Department of Anatomy and Neuroscience, The University of Melbourne.

Western blot was performed as previously described (31 (link)). Briefly, total proteins of tumor tissues were isolated with RIPA buffer (#P0013C, Beyotime) and the concentrations of which were measured using a BCA detecting kit (#P0012, Beyotime). The lysates were fractionated by SDS PAGE and transferred to PVDF membranes. Primary and secondary antibodies were used to detect the targets on the membranes. The primary antibodies used were: anti-OXCT1 (#12175-1-AP, Proteintech), anti-HMGCS2 (#ab137043, Abcam), anti-ENO2 (#10149-1-AP, Proteintech), anti-PKM2 (#15822-1-AP, Proteintech), anti-HK2 (#22029-1-AP, Proteintech), anti-β-actin (#4970s, CST). The quantitative analysis was performed using Photoshop.

IHC analysis was performed as we previously described [17 (link)]. Briefly, mouse lung tissues were embedded in paraffin, sectioned, and rehydrated; these antigens were recovered by heating in relative buffer at 95 °C for 10 min following the antibody instructions (anti-HMGCS2 (1:200; Abcam ab137043)). Endogenous peroxidase was neutralized using the endogenous peroxidase-blocking solution. Afterwards, the lung sections were incubated with a primary antibody at 4 °C overnight. After biotin-labelled secondary antibodies were incubated at 37 °C for 30 min, the lung sections were developed with DAB working solution and then stained with hematoxylin and mounted with a mounting medium.