Nonidet p 40

Prostate tissues were harvested from Ing3-LacZ heterozygous and wild-type control male mice, placed in PBS, and immediately proceeded with the staining procedure.
Tissues were transferred to 0.2% (w/v) glutaraldehyde-fixative (0.2% glutaraldehyde, 2 mM MgCl₂·6H₂O, 5 mM EGTA (Sigma-Aldrich, Saint Louis, MO, USA; cat# E4378-10G) in sodium phosphate buffer (SPB; 5.3 mL of solution A (200 mM NaH₂PO₄·2H₂O (Sigma-Aldrich, Saint Louis, MO, USA; cat# 71500-250G)) and 94.7 mL of solution B (200 mM Na₂HPO₄·2H₂O diluted with Milli-Q water to 200 mL, pH 8.0))) for 90 min at 4 °C. Afterwards, the samples were washed three times in basis staining buffer (BSB; 2 mM MgCl₂·6H₂O, 0.01% (w/v) Na-deoxycholate (Sigma-Aldrich, Saint Louis, MO, USA; cat# D6750-10G), 0.02% (v/v) Nonidet P-40 (Sigma-Aldrich, Saint Louis, MO, USA; cat# I3021), 5 mM EGTA in SPB, pH 8.0) for 30 min and submersed in X-gal staining solution (80 µL of X-gal stock solution in 2 mL of BSB containing 10 mM ferricyanide and 10 mM ferrocyanide) for five to eight hours (depending on the staining solution, which was re-used up to four times with a clear reduction in staining intensity). The samples were covered with aluminium foil and placed in a 37 °C shaking incubator. Stained samples were washed two times in basis staining buffer for 30 min and submerged into 4% formaldehyde (buffered aqueous solution, 0.5–1.5% methanol; VWR, Radnor, PA, USA; cat# 9713.1000) for nine to ten hours at 4 °C. Fixed samples were washed briefly in 70% ethanol and placed into fresh 70% ethanol for storage at 4 °C. Images were taken and evaluated with the Leica MZ 16 FA microscope (Leica Microsystems, Wetzlar, Germany) and a Leica DFC310 FX camera utilizing the Leica LAS AF software (version 3.3; Leica Microsystems, Wetzlar, Germany).

Cell supernatants (500 µL) were incubated with anti-preS1 antibodies and 10 µL of protein G- Sepharose beads (Invitrogen), overnight, at 4 °C, to precipitate enveloped HBV particles. After washing five times with PBS, the bound virions were eluted with 50 mM Tris–HCl buffer, pH 8, containing 1 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) and 1% Nonidet P-40 (Sigma-Aldrich). The encapsidated viral DNA was quantified as described [14 (link)].

Hypotonic propidium iodide staining solution [50 µg/ml propidium iodide (Sigma-Aldrich, #P4170), 0.1% trisodium citrate dihydrate (HIMEDIA^®, #GRM1415), 0.3 µl/ml Nonidet P-40 (Sigma-Aldrich, #56741) in distilled water] was added to iPSCs and scraped using a cell scraper to harvest the nuclei. The harvested suspension was centrifuged at 850 g for 5 min. The obtained pellet was resuspended and incubated in 1 ml staining solution. The nuclei were analysed using a BD LSR II flow cytometer (BD Biosciences) with BD FACSDiva™ software (BD Biosciences), and the data were processed using FCS Express 6 software (https://denovosoftware.com/). Per experimental condition, 10,000 events were recorded.

Diethylnitrosamine (DEN), dimethylsulfoxide (DMSO), paraformaldehyde, formaldehyde, glycine, TRI Reagent, Bromodeoxyuridine (BrdU), 2-deoxyglucose (2-DG), 3-bromopyruvate (3-BrP), doxorubicin (DOXO), etoposide (ETO), MG-132, C646, Nutlin-3, Oil Red-O (ORO), Propidium Iodide (PI), Triton X-100, Nonidet P-40 (NP-40), Adenosine 5′-triphosphate sodium salt (ATP), glucose, nicotinamide adenine dinucleotide phosphate sodium salt (NADP⁺), nicotinamide adenine dinucleotide (NAD⁺), and glucose-6-phosphate dehydrogenase were from Sigma-Aldrich. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG), MitoTracker^™ Green FM (MTG), Hoechst 33342 and Alexa Fluor™ 568 donkey anti-mouse IgG (H + L) were from Thermo Fisher Scientific. Trypan blue 0.4% solution was from Lonza. (H + L)-horseradish peroxidase conjugated goat anti-mouse and anti-rabbit IgG were from Bio-Rad Laboratories. l-lactate dehydrogenase (LDH) was from Roche Applied Science. Polyethylenimine (PEI) was from Polysciences. GW-7647 and GW-6471 were from Cayman Chemical. Choline deficient diet (CDD) was from Research Diets.
Human HCC samples were kindly provided by Prof. Grazi from the Hepato-Pancreato-Biliary Surgery Unit, Department of Clinical and Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy. Analyses were performed after approval from the Regina Elena Cancer Institute ethical committee. All mouse experimentation was performed in accordance with accepted standard of human animal care and after approval by the Italian Ministry of Welfare committee and the Institutional Animal Care and Use Committee of the University of Rome “Tor Vergata” (Italy). C57BL/6 mice were purchased from Harlan Laboratories Srl (Urbino, Italy) and randomly divided in two groups; treated group (n = 7, male) was intraperitoneally injected with a 20 mg/kg DEN dose at 15 days after birth and fed a CDD at weaning. Reference group (n = 7, male) was intraperitoneally injected with saline solution and fed standard pellet, at weaning (ND). Hepatic status was periodically followed-up by analyses on hepatic clinical markers aspartate aminotransferase (GOT-AST) and alanine aminotransferase (GPT-ALT) on sera from retro orbital blood collection. Before sacrifice, mice were fasted for 4 h and euthanized. Livers were perfused with saline solution prior to all the analyses performed. Mice liver thick sections were also stained for H&E morphological evaluation and for ATGL expression by IHC.
For this study, three different liver cancer cell lines were used. HepG2 and Hep3B cell lines were purchased from Leibniz-Institut DSMZ, Braunschweig (Germany). Huh7.5 cell line was a kind gift from Dr. Carla Montesano, University of Rome “Tor Vergata” (Italy). All cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) 1 g/L glucose supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 10 U/ml penicillin/streptomycin and 1% MycoZap^™. Cells were cultured at 37 °C in a 5% CO₂ atmosphere and plated at a 2 × 10⁵ cells/ml density for all the experiments. After 24 h plating, cells were transiently transfected with pcDNA™4/HisMaxC and pcDNA™4/HisMaxC-ATGL or pEGFP, pATGL^WT-EGFP and pATGL^(Ser47Ala)-EGFP or pcDNA3.1 and pcDNA3.1-p53^WT plasmids, for the times indicated in the experiments, by the PEI reagent, according to manufacturer’s instructions. sip53 and siATGL were reversely transfected using Lipofectamine^® RNAiMAX Transfection Reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol. GW-7647 and Nutlin-3 were used at a final concentration of 1 µM for 24 h. GW-6471 was used at a final concentration of 5 µM for 24 h. C646 and MG-132 were used at a final concentration of 10 µM for 24 h and 4 h, respectively. 2-DG and 3-BrP were used for 24 h at 30 mM and 30 µM final concentrations, respectively. DOXO and ETO were used for 16 h at 2 µM and 50 µM final concentrations, respectively.
TGs content was evaluated by Triglyceride Quantification Colorimetric/Fluorometric Kit (BioVision) and total neutral lipids stored in LDs by Oil Red-O staining procedure. Briefly, cells were fixed, rinsed in 60% isopropanol and stained with the Oil Red-O working solution. Oil Red-O was eluted and relative absorbance measured at 510 nm. Oxidative metabolism was examined by RT-qPCR analysis of both mitochondrial FAs uptake and β-oxidation genes and Krebs cycle genes, also by Western blot. FAs utilization was assessed by Seahorse Bioscience XF^e96 analyzer in combination with the Seahorse XF Palmitate-BSA FAO Substrate assay kit. Mitochondrial function was assessed both using a Seahorse Bioscience XF^e96 analyzer in combination with the Seahorse Bioscience XF Cell Mito Stress Test assay kit and by a Clark-type oxygen electrode. ATP levels were detected using the ATP Bioluminescence Assay Kit CLS II (Roche Applied Science). SIRT1 activity was determined by the SIRT1/Sir2 Deacetylase Fluorometric (Human) Assay Kit (Abnova).
Glycolysis was monitored by RT-qPCR analysis of SLC2A1/GLUT1 and SLC2A2/GLUT2 glucose transporters and SLC16A1/MCT1 lactate transporter. Glucose uptake was cytofluorimetrically recorded in FL-1 channel by a FACScalibur instrument after a 1-h-incubation with 100 µM 2-NBDG. HK activity and extracellular lactate measurements were determined spectrophotometrically.
Proliferation was evaluated by Trypan blue staining, CCK-8 colorimetric assay (Cell Counting Kit-8, Dojindo Molecular Technologies) and by BrdU incorporation assay. For BrdU incorporation assay, cells were fixed and incubated with an anti-BrdU primary antibody (after DNA denaturization) and nuclei stained with 1 µg/ml Hoechst 33342. Images were digitized with a Delta Vision Restoration Microscopy System (Applied Precision) equipped with an Olympus IX70fluorescence microscope (Olympus Italia).
Nuclear fractions were obtained as previously described [64 (link)] and used for both Western blot and ChIP assays.
Cell viability was determined by Trypan Blue and Propidium Iodide staining procedures (dead cells were expressed as percentage of subG1 cells).
Data were presented as means ± SD. Statistical analyses were performed by unpaired 2-tailed Student t test and one- or two-way ANOVA. Comparisons were statistically considered significant at p ≤ 0.05 (*).
RT-qPCR, Western blot, and fluorescence microscopy procedures as well as complete and detailed description of all methods employed are reported in Supplementary information.