1 Supplemental Experimental Procedures: Cell Culture Hepg2

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.

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.

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. All analyses were done with Partek Genomics Suite, 7 ver. 6.6.

Bioinformatic analysis

For tgYAP expression data, microarray data of 1 week YAP-expressing liver samples

(Yimlamai et al. 2014)(GEO accession#: GSM1339148, 1339149, 1339150) was compared to control hepatic tissue RNA samples (GSM305568, 305569, 305570) using a one-way ANOVA. The cut-off for gene lists was a fold-change >2 or <-2 and a corrected (FDR step-up) p-value <0.05. Gene functional classification and pathway enrichment were determined by RefDIC analysis (Hijikata et al.

2007)(http://refdic.rcai.riken.jp/tools/go_analysis.cgi). SubMap global transcriptome comparison between mouse and human datasets was performed using a previously published method of assessing similarity of predetermined phenotypes (Hoshida et al.

2007). Gene Set Enrichment Analysis (GSEA) (Nadler et al. 2000; Subramanian et al.

2005) of raw intensities of WT and Lats2-CKO liver data, and gene sets was compared to SREBP (this study, Table S2) and YAP signatures (Dupont et al. 2011) were examined in GEO accession GSE37031, GSE48452 and GSE49541 data sets, using public software (http://www.broadinstitute.org/gsea/index.jsp). Gene sets were considered significantly enriched at FDR < 0.05 and 1,000 permutations of gene sets.

Statistical analyses were performed using GenePattern genomic analysis tool kits

(www.broadinstitute.org/genepattern) and R statistical language (www.r-project.org).

Statistics

All value points of all line and bar graphs are mean±SEM, unless noted otherwise.

Significance of all averages presented in bar or line graphs was tested with ANOVA. 8 p-values are denoted as follows: * < 0.05; ** < 0.01; *** < 0.005; NS = not significant. 9

Supplemental Figure Legends:

Figure S1. LATS2 binds SREBP2 and SREBP1

(A) Scheme of metabolism-related LATS2-interacting proteins. Length of connecting line is proportional to the strength of protein interaction as measured by area ratio of

LATS2/vector. Bold red font denotes LATS2 and SREBP2. Blue connecting lines indicate previously published interactions (Couzens et al. 2013). Colored triangles encompass enriched metabolic pathways; asterisks denote p-value of pathway enrichment.

(B) Co-IP of MYC-tagged LATS1, LATS2 full-length (FL) or LATS2 deletion mutants (together referred to as LATS in figure) with endogenous SREBP2 from

HepG2 cells. Precursor (P-SREBP2) and nuclear SREBP2 (N-SREBP2) forms of

SREBP2 are indicated. Extracts were immunoprecipitated (IP) with anti-MYC-tag antibody (9E10) and subjected to SDS-PAGE followed by Western blot analysis with the antibodies indicated to the left of the panel. Vector only served as negative control. WCL: 2.5% of whole cell lysate; GAPDH = loading control. HC: antibody heavy chain. Values below blots were calculated by normalizing the value of the corresponding sample to the value of “vector”.

IP experiment depicted in Fig. S1B.

(D) Co-IP of GFP-tagged LATS2 with endogenous precursor SREBP1 (P-SREBP1) and nuclear SREBP1 (N-SREBP1) from HepG2 cells. Extracts were immunoprecipitated with antibody against GFP and subjected to SDS-PAGE followed by Western blot analysis with the antibodies indicated to the left of the panel. Vector only serves as a negative control. WCL: 2.5% of whole cell lysate; GAPDH = loading 10 control. HC: antibody heavy chain. Values below blots were calculated by normalizing the value of the corresponding sample to the value of “vector”.

(E) Nuclear fractionation analysis of HepG2 cells transfected with control

(siControl), LATS2 siRNA (siLATS2) or LATS1 plus LATS2 (siLATS1/2) siRNA oligos. Purity of the nuclear and cytoplasmic fractions was validated with H2B and

GAPDH antibodies. Fold N/P SREBP2 was calculated by normalizing each sample to the siControl sample. Fold nuclear YAP (nuc YAP) was calculated by normalizing each sample to the siControl nuclear sample. Note: Blots of H2B and GAPDH are the same as those presented in Figure 1B.

(F) Immunofluorescence analysis of HepG2 cells grown in SDM and transfected with

GFP-LATS2. LATS2-transfected cells (arrow) are visualized as GFP positive cells; transfected GFP-LATS2 is seen primarily in centrosomes and more diffusely in the cytoplasm and to a lower extent in the nucleus.

Figure S2. Depletion of LATS2 activates SREBP2

(A) Western blot analysis of whole cell lysates of HepG2 cells grown in sterol- depleted medium (SDM), normal medium (NM) or cholesterol-enriched medium

(NM+acLDL) and transfected with either empty vector, MYC-LATS2 expression plasmid or siControl or siLATS2 siRNA oligos. Blots were probed with antibodies against LATS2, SREBP2 and GAPDH. “fold N/P SREBP2” was calculated by normalizing the ratio of N-SREBP2/P-SREBP2 to vector-transfected cells in NM.

(B) ImageStream analysis of similarity of signal distribution between GFP-LATS2 and SREBP2 ER localization. Similarity >1 is considered co-localization. At least 104 single cells were analyzed as described in Supplemental Experimental Procedures. 11

Shown above the distribution graph is a series of representative photos from a single cell within the >1 similarity range.

(C) qRT-PCR analysis of representative SREBP2, SREBP1 and YAP target gene expression in HepG2 cells grown in normal medium and transfected with siControl, siLATS1 or siLATS2 siRNA oligos. Values were normalized to HPRT. Error bars =

SD.

(D) qRT-PCR analysis of knockdown efficiency in HepG2 cells in the experiment shown in Fig. 1E.

(G) Quantification of Oil Red O (ORO) staining as depicted in Figure 1F. % ORO stained relates to the percentage of stained area within HepG2 cell clusters. At least ten fields were quantified for each condition.

Figure S3. Liver-specific deletion of Lats2 activates SREBP2 in vivo

(A) Lats2 liver conditional knockout scheme. Representative genotyping PCR using primers amplifying a portion of the cre recombinase gene (cre) and an internal positive control (marked by asterisk), and primers flanking the loxP sites adjacent to exon 5 (A+C), as depicted in the lower panel. A+C produce DNA fragments of approximately 1.3Kb and 350bp from the WT and exon 5-deleted alleles, respectively. Cre-specific primers amplify a fragment of approximately 140bp. In

Lats2-CKO animals, exon 5 is deleted only in liver tissue, but not in tail DNA. “M” denotes size marker wherein stronger bands represent 500bp and 1Kb.

(B) Western blot analysis of SREBP1 in liver lysates from three WT and three Lats2-

CKO 17 week old mice. N/P SREBP1 was calculated by averaging the N-SREBP1/P-

SREBP1 ratio for each genotype and normalizing to WT. 12

0.05) in livers from two WT and three Lats2-CKO 17 week old mice, derived from expression array analysis. Each row corresponds to an individual mouse.

(D) qRT-PCR analysis of Lats2 gene expression performed on RNA extracted from

WT or Lats2-CKO primary hepatocytes or from whole livers. Error bars = SD.

(E) Serum cholesterol (T-Cho) and triglyceride (TG) levels of WT and Lats2-CKO 17 week old mice.

(F) Normalized hepatic expression of SREBP target genes and YAP target genes in mice overexpressing a Yap transgene (tgYap) relative to control samples, as published previously (Yimlamai et al. 2014).

Figure S4. Hepatic deletion of Lats2 results in fatty liver

(A) Total body weight of WT and Lats2-CKO mice at the indicated times after birth.

(B) Heatmap portraying histological scores of H&E stains of liver sections from WT and Lats2-CKO mice under normal diet (ND), high cholesterol diet (HCD) and recovery conditions. At least 20 fields from at least 8 mice from each group were analyzed for each pathological feature. Colors indicate severity of observed pathological phenotype. Asterisks denote statistical significance of difference compared to same parameter of WT controls in same experimental setting.

(C) Quantification of Oil Red O (ORO) staining as depicted in Figure 4D. % ORO stained relates to the percentage of stained area within entire field. At least ten fields were quantified for each genotype.

(D) Quantification of Filipin (F) staining as depicted in Figure 4D. % Filipin stained relates to the percentage of stained area within entire field. At least ten fields were quantified for each genotype. 13

Figure S5. Hepatic deletion of Lats2 exacerbates diet-induced cholesterol accumulation but reduces fibrosis

(A) Quantification of Oil Red O (ORO) staining as depicted in Figure 5D. % ORO stained relates to the percentage of stained area within entire field. At least ten fields were quantified for each genotype.

(B) Quantification of free cholesterol (Filipin) staining as depicted in Figure 5D. %

Filipin stained relates to the percentage of stained area within entire field. At least ten fields were quantified for each genotype.

(D) Total body weight of WT and Lats2-CKO mice fed HCD for the indicated times, starting at age = 8 weeks.

(E) Quantification of fibrosis (Sirius Red, SR) staining as depicted in Figure 5F. %

SR stained relates to the percentage of stained area within entire field. At least ten fields were quantified for each genotype.

(F) Quantification of senescence (β-gal) staining as depicted in Figure 5F. % β-gal stained relates to the percentage of stained area within entire field. At least ten fields were quantified for each genotype.

Figure S6. High cholesterol diet induces different responses in WT and Lats2-

CKO livers

(A) Heatmap of genes most differentially expressed in HCD (fold change > 2; p-value

< 0.05) in livers from WT and Lats2-CKO fed either ND or HCD. Each row represents an individual mouse. Red bars below the heatmap mark genes upregulated in ND-fed Lats2-CKO mice, similar to WT and Lats2-CKO mice on HCD. 14

(B) GSEA representation of differentially expressed genes (FC > 2, p-value < 0.05) between WT mice fed normal diet (WT-ND) or high cholesterol diet (WT-HCD), compared to a signature of genes upregulated in obesity

(NADLER_OBESITY_UP)(Nadler et al. 2000).

(C) Pathway enrichment of genes whose expression is most differentially affected by high cholesterol diet in WT livers compared to Lats2-CKO livers (fold change >1.5; p-value < 0.05). Bar height represents –log10 of the corrected p-value.

(D) Quantification of activated macrophages (Mac2) staining as depicted in Figure 6A as defined by size threshold and as a percentage of the total Mac2-positive staining in each micron2. At least ten fields were quantified for each genotype.

(E) Quantification of apoptosis (TUNEL) staining as depicted in Figure 6A.

TUNEL+/µ2 relates to the number of stained nuclei in each micron2. At least ten fields were quantified for each genotype.

Figure S7. Analysis of SREBP2 and p53 expression in mouse livers and human liver-derived cells

(A) Western blot analysis of SREBP2 in liver lysates from three WT and three Lats2-

CKO mice fed ND or HCD; GAPDH = loading control. Note: Lanes 1-6 (ND) are the same as shown in the Western presented in Figure 2A.

(B) Western blot analysis of p53 in whole cell lysates of HepG2 cells grown in normal medium (NM) or cholesterol-enriched medium (NM+acLDL) and transfected with either empty vector DNA, MYC-LATS2 expression plasmid or siControl or siLATS2 siRNA oligos. GAPDH = loading control.

HepG2 cells grown in normal medium and transfected with siControl or siLATS2 15 siRNA oligos. When indicated, cells were treated with 10µM Nutlin-3 10 hours before harvesting. GAPDH = loading control.

Figure S8. Comparative gene expression in human fatty liver disease

(A) Graphic depiction of similarity between the global transcriptome of liver samples from WT and Lats2-CKO mice fed high cholesterol diet and human livers of NAFLD patients categorized according to fibrotic score (GEO accession GSE49541; (Moylan et al. 2014)). Fibrotic score 0/1 was defined as mild fibrosis (n=40), while fibrotic score 3/4 was defined as severe fibrosis (n=32).

(B) LATS2 expression (log2) in livers of NAFLD patients, extracted from dataset

GSE48452 (Ahrens et al. 2013); control n=14; NASH, Lats2-high expression (n=9);

NASH, Lats2-low expression (n=9). Bonferroni-corrected p-values for differential

LATS2 expression were calculated by t-test. Whisker denotes the most extreme data points within interquartile range x1.5. In each box plot, top and bottom regions represent second and third quartiles, respectively.

Table S1. LATS2 Interacting Metabolism-Related Proteins Entry Gene Name Entry Name Protein Name LATS2/ vector P15121 AKR1B1 ALDR_HUMAN Aldose reductase 3.26 P54886 ALDH18A1 P5CS_HUMAN Delta-1-pyrroline-5-carboxylate 5.91 synthase P00966 ASS1 ASSY_HUMAN Argininosuccinate synthase 5.40 P13804 ETFA ETFA_HUMAN Electron transfer flavoprotein 4.06 subunit alpha, mitochondrial Q49A26 GLYR1 GLYR1_HUMAN Putative oxidoreductase GLYR1 3.38 P11177 PDHB ODPB_HUMAN Pyruvate dehydrogenase E1 3.10 component subunit beta, mitochondrial P15259 PGAM2 PGAM2_HUMAM Phosphoglycerate mutase 2 5.10 P08607 VIM VIME_HUMAN Vimentin 5.04 Q12772 SREBF2 SRBP2_HUMAN Sterol regulatory element- 6.23 binding protein2

List of proteins with metabolic function pulled-down from HepG2 cell lysates by GFP-LATS2

(see Supplemental Experimental Procedures). Putative LATS2 interacting proteins were filtered

to possess at least 3 different identifying peptides and by at least threefold area ratio of

LATS2/vector in each of two biological replicates. “LATS2/vector” value represents an average

area ratio of the two experiments. SREBP2 (gene SREBF2) is indicated in bold font. 17

Table S2. LATS2-regulated SREBP Signature Gene Symbol RefSeq no. Gene name AHCY NM_001161766, NM_000687 adenosylhomocysteinase

SREBF1 NM_001005291, NM_004176 sterol regulatory element binding transcription factor 1

SQLE NM_003129 squalene epoxidase

GSS NM_000178 glutathione synthetase

HMGCS1 NM_002130, NM_001098272 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1

SREBF2 NM_004599 sterol regulatory element binding transcription factor 2

PPAT NM_002703 phosphoribosyl pyrophosphate aminotransferase

FDPS NM_002004, NM_001135821, farnesyl diphosphate synthase (farnesyl pyrophosphate NM_001135822 synthetase, dimethylallyltranstransferase, geranyltranstransferase)

LDLR NM_000527 low density lipoprotein receptor

STEAP4 NM_024636 STEAP family member 4

ACACA NM_198839, NM_198837, acetyl-Coenzyme A carboxylase alpha NM_198838, NM_198836, NM_198834

HMGCR NM_000859, NM_001130996 3-hydroxy-3-methylglutaryl-Coenzyme A reductase

MVK NM_001114185, NM_000431 mevalonate kinase

ACAT2 NM_005891 acetyl-Coenzyme A acetyltransferase

FDFT1 NM_004462 farnesyl-diphosphate farnesyltransferase 1

LIPG NM_006033 lipase, endothelial

ABCG8 NM_022437 ATP-binding cassette, sub-family G (WHITE), member 8

ABCG5 NM_022436 ATP-binding cassette, sub-family G (WHITE), member 5

LPIN2 NM_014646 lipin 2

IDI1 NM_004508 isopentenyl-diphosphate delta isomerase 1

AGPAT2 NM_001012727, NM_006412 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic acid acyltransferase, beta)

LPIN1 NM_145693 lipin 1

ACOT1 NM_001037161 acyl-CoA thioesterase 1

DHCR7 NM_001163817, NM_001360, 7-dehydrocholesterol reductase NM_001163817, NM_001360 18

Human SREBP target gene signature culled from Horton et al, 2013 (Horton et al. 2003) and filtered

for differential expression in Lats2-CKO livers compared to WT livers (FC>1.5, p-value<0.05).

Mouse-human gene conversion was performed using the DAVID Bioinformatic Database

(http://david.abcc.ncifcrf.gov/conversion.jsp).

Table S3. siGENOME Target Sequence RNAi target sequences used in this study. siRNA name RefSeq no. Sequence LATS2 NM_014572 (1) GUUCGGACCUUAUCAGAAA (2) GAAAGAGUCUAAUUACAAC (3) GAUCGGUGCCUUUGGAGAA (4) GAACGAUGCCAGCGAAGGU LATS1 M-004632-00-0005 (1) GAACCAAACUCUCAAACAA (2) GCAAGUCACUCUGCUAAUU (3) GAAAUCAAGUCGCUCAUGU (4) GAUAAAGACACUAGGAAUA YAP1 M-012200-00-0005 (1) GCACCUAUCACUCUCGAGA (2) GAACAUAGAAGGAGAGGAG (3) CCACCAAGCUAGAUAAAGA (4) GGUCAGAGAUACUUCUUAA siCONTROL D-001210-05-50 (1) UGGUUUACAUGUCGACUAA

Table S4. Experimental Setup Chart summarizing numbers of mice used in each experimental setup.

Diet Genotype 9 weeks 18 weeks Recovery WT 28 17 6 Normal diet Lats2-CKO 26 14 6 WT 23 13 4 High cholesterol diet Lats2-CKO 27 19 10

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