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1 Supplemental Experimental Procedures: Cell Culture Hepg2

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1 Supplemental Experimental Procedures: Cell Culture Hepg2 1 Supplemental Experimental Procedures: Cell culture HepG2 cells were maintained at 37°C in DMEM high glucose medium without phenol red (BI #01-053-1) supplemented with 2mM L-glutamine (BI #03-020-1B) and antibiotics (BI #03-031-1B). To this was added 10% FBS (BI #04-007-1)(NM) or 10% charcoal-stripped FBS (BI #04-201-1A)(CDM). For sterol depletion experiments, HepG2 cells were grown for at least two passages in SDM before transfection, lysis or fixation. AcLDL supplementary cholesterol (Life Technologies L-35354; 100µg/ml for 17 hours) was added where indicated. For siRNA knockdown experiments, cells were transfected with the relevant siGENOME SMART pool (Thermo Scientific) (Table S3) and transfection reagent (Dharmacon). For overexpression, cells were transfected with vector control of GFP-tagged human LATS2 (generous gift of H. Nojima) using JetPEI reagent (PolyPlus Transfection). Animals All mouse experiments were approved by the institutional animal care and use committee (IACUC) of the Weizmann Institute (application number: 08190114-2). For diet experiments, eight week old male mice were fed for 9 weeks either normal chow diet (ND) containing no cholesterol (Harlan #2018) or Paigen high cholesterol diet (HCD) containing 1.25% cholesterol (Harlan #TD.90221)(Paigen et al. 1985). For recovery experiments, eight week old male mice were fed HCD for 18 weeks, and then returned to ND for 4 weeks. Numbers of mice in each experimental group are illustrated in Table S4. All measurements were performed on all mice in each group, unless otherwise noted. 2 Mass spectrometry HepG2 cell lysates were incubated with ectopically expressed GFP-LATS2 immunoprecipitated from HEK-293T cells using GFP-Trap agarose beads (Chromotek #gta-100). Immunoprecipitated proteins were digested with trypsin and analyzed by LC-MS/MS on Orbitrap XL (Thermo). Identification was done by Discoverer Software Version 1.4 against the mouse and human sections of the UniProt database and against decoy databases, using the Sequest and Mascot search engines. Putative LATS2 interacting proteins were filtered to possess at least 3 different identifying peptides and at least three fold area ratio of LATS2/vector in each of two biological replicates. Proteins were filtered for published metabolic function. Western blot and co-IP analysis HepG2 cells were grown in normal medium (NM) or sterol-depleted medium (SDM). The monolayer was gently washed twice with ice-cold PBS and lysed on ice for 30 minutes with NP-40 lysis buffer (50mM Tris·HCl pH8.0, 150mM NaCl, 1.0% NP-40) supplemented with protease inhibitor mix (Sigma) and phosphatase inhibitor cocktail 2+3 (Sigma P5726 and P0044). Protein A Sepharose beads (Repligen), pre-blocked with BSA, were incubated with appropriate antibodies. Lysates were incubated with the bound antibodies for 4 hours at 4oC, washed with NP-40 lysis buffer, released from the beads by boiling and resolved by SDS-PAGE. “beads” indicates antibody- bound beads, as a negative control. “HA” indicates Co-IP with irrelevant antibody (anti-HA, Santa Cruz, sc-805), as a negative control. 2.5% of whole cell lysate (WCL) input is presented, with GAPDH as a loading control. For nuclear fractionation, trypsinized cells were lysed with RSB buffer (10mM NaCl, 10mM Tris pH 7.4, 3 15mM MgCl2 supplemented with protease inhibitor mix [Sigma] and phosphatase inhibitor cocktail 2+3 [Sigma P5726 and P0044]). Nuclei were lysed by addition of 10% triton and 10% deoxycholate. Antibodies used included: anti-GFP (Roche #11814460001), anti-MYC-tag (9E10 MoAb), anti-SREBP2 (Abcam ab30682), anti- SREBP1 (Abcam ab3259), anti-YAP (Santa Cruz sc-15407), anti-p-YAP (Cell Signaling #4911), anti-LATS2 (Novus NB200-199), anti-LATS1 (Cell Signaling #3477), anti-p53 (Leica NCL-p53-CM5p), anti-H2B (Upstate 07-371) and anti- GAPDH (Millipore MAB374). Quantification and imaging of Western blots was performed using a ChemiDoc MP imaging system (BioRad) with Image Lab 4.1 software (BioRad). Immunofluorescence (IF) staining Cells grown on 12mm coverslips were fixed with 3%PFA for 20 min at RT, washed with PBS, blocked with 3% BSA in PBS, incubated for 1 h with primary SREBP2 (Abnova ab30682) antibody, washed and then incubated with secondary antibody and DAPI (0.5µg/ml final) for 40 min in the dark. Multispectral imaging flow cytometry (ImageStreamX) analysis Cells were fixed, stained with antibodies against SREBP2, PDI or p115 (see below), and imaged using multispectral Imaging Flow Cytometry (ImageStreamX markII flow cytometer; Amnis Corp, part of EMD millipore, Seattle, WA). At least 104 cells were collected from each sample and data were analyzed using image analysis software (IDEAS 6.2; Amnis Corp). Images were compensated for fluorescent dye overlap by using single-stain controls, gated for single cells using the area and aspect ratio features, and for focused cells using the Gradient RMS feature (George et al. 4 2006). Cells were gated for either positive or negative staining of GFP-LATS2. For each gated population, the nuclear localization of SREBP2 was quantified using the Similarity feature on the nuclear mask of the DAPI staining and the SREBP2 staining (the log transformed Pearson’s correlation coefficient in the two input images). Localization of SREBP2 to Golgi or ER was quantified using the bright detail similarity feature of the staining (Golgi: p115, kindly provided by Sima Lev, Weizmann Institute Israel; ER marker: PDI, Abcam ab2792) and SREBP2 (the log transformed Pearson’s correlation coefficient of the localized bright spots in the two input images) (George et al. 2006). The distribution of SREBP2 in the different compartments was quantified by measuring the intensity of the protein in the nuclear mask, the ER/Golgi mask and the rest of the cell (total intensity – nuclear intensity – ER intensity). Biochemistry analysis Liver function: GOT, GPT, albumin, glucose, triglycerides, total cholesterol and high density lipoprotein (HDL) levels in mouse serum were measured by SpotChem EZ Chemistry Analyzer (Arkray, Japan); strip catalog numbers: PANEL-1-ARK77184, HDL-CD-ARK77181, TG-ARK77163, ALB-ARK77168. Body mass composition measurement Measurements of total mouse body fat and lean mass was performed using an EchoMRI-100TM (Echo Medical Systems, Houston, TX) with a magnetic frequency of 2.2MHz. Measurements were done without sedation using the 1T4 program without water composition. 5 Isolation of primary hepatocytes Mice were anesthetized with isoflurane and perfused with 0.05% collagenase. Cell suspensions were filtered, washed and plated on collagen (Sigma C8919) coated wells. Cells were maintained in DMEM with no glucose (Gibco #11966), 0.2% BSA (Fraction V, Millipore #160069), 2mM sodium pyruvate (BI #03-042-1B), 2% antibiotics (BI #03-031-1B), 0.1µM dexamethasone (Sigma D-2915) and 1nM insulin (Sigma I-6634). Immunohistochemistry (IHC) Tissue samples were fixed overnight in 37% formaldehyde/PBS. Following fixation, samples were transferred to 70% ethanol, embedded in paraffin, sectioned, and stained with H&E (Sigma HHS332 and Sigma HT110332), PAS (Sigma P7875 and Sigma 3952016) and Sirius Red (Sigma 365548). The amount of fibrotic tissue was calculated relative to the total analyzed liver area. For immunohistofluorescence analysis, paraffin sections were rehydrated followed by antigen retrieval with boiling citric acid. Quenching of endogenous peroxidase and protein block were performed prior to blocking with normal horse serum and overnight incubation with antibody (MAC2, Cedarline CL8942AP). Staining was visualized with DAB (Sigma D4168). For TUNEL staining, 5µm tissue sections were stained using ApopTag Red Apoptosis detection kit (Millipore S7165) according to the manufacturer’s instructions. For Oil Red O (ORO) staining, OCT-fixated (Tissue-Tek 4583) liver tissues were cut into 8- 12µm-thick cryo-sections. After fixation in 4% PFA for 10 minutes, samples were washed three times with PSB and incubated for 5 min with 0.38% ORO (Sigma #00625), then washed with distilled water. Nuclei were stained using DAPI (1:200 in 2X SSC) for 20 minutes. Cell boundaries (F-actin) staining were visualized with 6 phalloidin (1:500 in 2X SSC). Slides were washed with distilled water and mounted with water-soluble mounting medium (Sigma #GG1). For senescence-associated beta- galactosidase (β-gal) staining, OCT-fixed (Tissue-Tek 4583) liver tissues were cut into 8-12µm-thick cryo-sections. Cells were washed once with PBS (pH 5.5), fixed with 0.5% glutaraldehyde (pH 7.4), and washed in PBS (pH 5.5) supplemented with 1mM MgCl2. Cells were stained in X-gal solution (1 mg/ml X-gal [Sigma D4254], 0.12 mM K3Fe[CN]6, 0.12 mM K4Fe[CN]6, 1 mM MgCl2 in PBS at pH 6.0) for several hours at 37°C and then washed with PBS. Slides were imaged using a Nikon eclipse Ti-E microscope and Nikon`s digital sight DS-U3 camera and Nikon intensilight C-HGFI illuminator for florescence. Isolation of total RNA and expression array analysis Total RNA was isolated using a NucleoSpin RNA II kit (Macherey-Nagel). A 1.5µg aliquot of the total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) and random hexamer primers (Amersham). Real-time qPCR was performed using SYBR Green Master Mix (Applied Biosystems) and a Step One instrument (Applied Biosystems). Expression profiling was performed on Affymetrix GeneChip® Mouse Gene 2.0 ST arrays. CEL files underwent RMA background correction and quantile normalization. Lists of differentially-expressed genes were generated following a two-way ANOVA on log2 transformed values, with genotype (WT vs. Lats2-CKO) and diet (normal vs. HCD) as independent categorical factors. The cut-off for entering the gene lists was a fold- change >1.5 or <-1.5 and a corrected (FDR step-up) p-value <0.05. Hierarchical clustering, based on Pearson’s dissimilarity and complete linkage, was used to create the heatmaps from the gene lists.
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