1 Supplemental Information Development of an in Vitro Human

Home , ACADS, HADHA

Supplemental Information

Development of an In Vitro Human Liver System for Interrogating Non-Alcoholic

Steatohepatitis

Ryan Feaver1, Banumathi K. Cole1, Mark J. Lawson1, Stephen A. Hoang1,

Svetlana Marukian1, Robert A. Figler1, Arun J. Sanyal2, Brian R. Wamhoff*1, Ajit

Dash1

1HemoShear Therapeutics LLC, Charlottesville, VA

2Virginia Commonwealth University School of Medicine Richmond, VA

*Corresponding Author:

Brian R. Wamhoff, 501 Locust Ave., Charlottesville, VA 22902

[email protected]

434-872-0196

1 Supplemental Figures and Tables:

log2 Fold Change Values

●

● ● ● ● ●

● ● 5 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●●●● ●● ● ● ● ● ●●● ● ● ● ● ●● ●●●● ●●●●●● ● ● ● ● ● ●● ● ● ● ● ●● ●●●●●●●●●●●●●●●● ● ● ● ● ● ●●● ●●●●●●●●●●●●●● ● ● ● ●●●●●●●●●●●●●●●●● ● ●● ● ● ●●● ●● ●●●●●●●● ●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ● ● ●● ●● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●● ● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●● ●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● 0 ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●● ●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ● ● ● NASH BGA0302 ● ● ●● ●●●● ●●●●●●●●●●●●●●●● ●●●●● ● ● ●●● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●● ● − ● ● ● ● ●● ● ●●●●●●●●●●●●●●●●●●●●●●●●● ●●●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●● ●● Healthy ● ●● ●●●●●● ●●●●●●●●●● ●● ● ● ● ● ● ●●●● ●●●●●●●●●● ●●●●●●● ●●●●●● ●●●●● ● ● ● ●●●●●●●●●●●●●●●●●● ●●●●●●● ●●● ● Lipotoxic ● ● ●●● ● ●●●●●●●●●●●●●● ● ●● ●● ●●●●●●●●●●●●● ●●●●●●●● ●● ●●● ● ● ●● ● ●●●●●●●● ●● ● ● ● ● ● ● ● ● ●● ●●● ●●●●●●●● ●● ● ● ●●●● ●●●●●● ●● ● ●●●●●●●●● LGLI ● ● ● ● ● ●● ●●● ●●●● ● ● ● ● ● ●●●●●●●●●●● ● ●● ●● ● ● ●● ●●● ● ●●● ● ● ● ● ●●● ● ● ●● ●●● ● ●●● ● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ●●●● ●● ●● ●●●● ● ●●●● ●● ● Experiment#1 ● ● ● ●● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ●● ● ● ● ● ●●●● ● ● ● ● ● ● ● ●● ● ● ● ● (Log2Fold Change) ● ● ● ● ●● ● ● ● ● ●● ● −5 ● ●● ● ● ● ● ● ● ●

●● ● ● Pearson correlation = 0.8890599

● ● ● at a p value <2.2e-16

−5 0 5 LGLI−NASH BGA0301 Healthy Lipotoxic (Log2 Fold Change) Experiment #2

Supplemental Figure 1. Correlation plot demonstrating the reproducibility of data from different experiments in the lipotoxic system. Hepatocyte transcriptomic data of all genes (each gene is an individual dot) from two different experiments (#1 and #2) performed 2 months apart with n=3 and n=4 donors, respectively, is represented in this correlation plot. Comparison is healthy vs. lipotoxic + 0.1ng/ml TNFα milieu for each experiment. The scatterplot shows that genes from both experiments exhibit a strong positive correlation in gene response, and thus high reproducibility.

2 Fatty Acid Biosynthesis

Oxaloacetate

PC TCA Cycle

Fatty Acid

Pyruvate Citrate ACSL3 ACSL1 ACLY ACAS2

Acetyl-CoA ACSL4 ACACB MLYCD ACSL5 ACACA ACSL6 Malonyl-CoA CPT1A

FASN

Palmitic Acid Acyl-CoA

Cytosol ELOVL6 Cytosol Steric Acid EC HEALTH SCD CPT1A Oleic Acid Acetyl-CoA

Beta Oxidation Long-Chain fatty acid 2 cell EC ACSL1 SMC ACSL3 * * * * PTGS2 MitochondrionMitochondrion 1 ACSL4 cell_type ACSL5 i ACSL6 p ACAS2 * * * * * EDN1 0 log2fold Fatty acyl CoA −1 DGAT1 DGAT2 Lipotoxic * * PTGS1 Triacylglyceride Synthesis pathway Healthy −2

* HSP90AB1 Supplemental Figure 2. Transcriptomic data from hepatocytes exposed to

the lipotoxic compared to the healthy milieu is overlaid onto a diagram * CALM1 representing the fatty acid biosynthesis pathway. A red-filled box indicates

* * * * HSP90B1 the gene is upregulated while a blue-filled box indicates the gene is downregulated; the intensity of the color corresponds to greater log2fold-

* CAV1 changes. Boxes with a green perimeter indicate the gene expression log2fold-

change is significant (FDR<10%).

* * * * NOS3

* * * * KLF2 3 * * * * KLF4

* * * * ARG2

CCA−STATIC CCA−STATIC ICS−CCA ICS−CCA CCA−STATIC CCA−STATIC ICS−CCA ICS−CCA

http://tinyurl.com/ph6nka9

Cholesterol Biosynthesis

Lipotoxic Fatty Acid Degradation Healthy

Acetyl-CoA

HMGCS1

Cholesterol HMG-CoA

HMGCR DHCR7

7-Dehydrocholesterol Mevalonic acid

SC5DL EC HEALTH MVK Lathosterol Mevalonic acid-5P NSDHL PMVK 2 SC4MOL cell CYP51A1 Mevalonic acid 5-pyrophosphate EC SMC IDI1 MVD Lanosterin * * * * PTGS2 1 Dimethylallylpyrophosphatecell_type isopentenyl pyrophosphate i LSS p 0 FDPS * * * * * EDN1 (S)-2,3-Epoxysqualene log2fold SQLE Geranyl-PP farnesyl pyrophosphate −1 Squalene * * PTGS1 FDPS −2 FDFT1

* HSP90AB1 Supplemental Figure 3. Transcriptomic data from hepatocytes exposed to

the lipotoxic compared to the healthy milieu is overlaid onto a diagram * CALM1 representing the cholesterol biosynthesis pathway. A red-filled box indicates

* * * * HSP90B1 the gene is upregulated while a blue-filled box indicates the gene is downregulated; the intensity of the color corresponds to greater log2fold-

* CAV1 changes. Boxes with a green perimeter indicate the gene expression log2fold-

change is significant (FDR<10%).

* * * * NOS3

* * * * KLF2

* * * * KLF4 4

* * * * ARG2

CCA−STATIC CCA−STATIC ICS−CCA ICS−CCA CCA−STATIC CCA−STATIC ICS−CCA ICS−CCA

http://tinyurl.com/ph6nka9 Glycolysis/Gluconeogenesis

Glucose SLC2A1 Glycolysis SLC2A2 SLC2A3 Gluconeogenesis SLC2A4 SLC2A5

Glucose

HK1 HK2 G6PC HK3

GCK Glucose-6P Pentose Phosphate Pathway

GPI Glycogen metabolism

Fructose 6P PFKM FBP1 PFKL FBP2 PFKP

Fructose-1,6BP

ALDOA ALDOB ALDOC

Glyceraldehyde 3P Dihydroxyacetone-P Triglyceride synthesis

GAPDHS TPI1 GAPDH

1,3BP-Glycerate

PGK1 PGK2 Cytosol

3P-Glycerate Mitochondrion PGAM2

PGAM1

2P-Glycerate EC HEALTH ENO1 Aspartate Aspartate ENO3 ENO2 P-enolpyruvate GOT1 GOT2

PKM2 2 Oxaloacetate Oxaloacetate cellPKM2 PKLR EC PCK1 MDH1 SMC PC MDH2 * * * * PTGS2 Malate Malate Pyruvate 1 LDHAL6B LDHAcell_type MPC1 LDHB Pyruvate i MPC2 LDHC p Lactate 0 PDHA1 TCA Cycle * * * * * EDN1 PDHA2

log2fold PDHB DLAT DLD

−1 PDHX

Lipotoxic Acetyl-CoA * * PTGS1 Healthy −2

* HSP90AB1 Supplemental Figure 4. Transcriptomic data from hepatocytes exposed to the lipotoxic compared to the healthy milieu is overlaid onto a diagram * CALM1 representing the glycolysis/gluconeogenesis pathway. A red-filled box indicates the gene is upregulated while a blue-filled box indicates the gene is * * * * HSP90B1 downregulated; the intensity of the color corresponds to greater log2fold- changes. Boxes with a green perimeter indicate the gene expression log2fold- change is significant (FDR<10%). * CAV1

5 * * * * NOS3

* * * * KLF2

* * * * KLF4

* * * * ARG2

CCA−STATIC CCA−STATIC ICS−CCA ICS−CCA CCA−STATIC CCA−STATIC ICS−CCA ICS−CCA

http://tinyurl.com/ph6nka9

6 6 4 4 8 Gene8 ResponseGene HeatmapGene Response Response Heatmap HeatmapGeneGene Response Response Heatmap HeatmapGeneGene Response Response Heatmap HeatmapGene Response HeatmapGene Response HeatmapGene Response Heatmap 2 2 6 6 3 3 2 2 4 4 Count Count Count Count Count Count 0 0 0 0 Count Count Count Count 0 0 0 0 0 0

−2 −2−1 −01 10 2 1 2 −2 −2 −−1 0 1 0 2 1 2 −2 −2−1−1 0 10 2 1 2 −2 −1 0 1 2 −2−2 −1−10 1 0 2 1 2 −2 −1 0 1 2 ValueValue ValueValue ValueValue Value ValueValue Value

comparisoncomparison comparison comparison comparison comparisoncomparison Lipotoxic Lipotoxic Lipotoxic Lipotoxic Lipotoxic cellcell cell cell cell cellcell Healthy Healthy Healthy Healthy Healthy ...... FADDFADD CYP1A1 . . CXCL6 . . . . . NUMA1NUMA1 ...... HADHA CCR6 PYCARDPYCARD . . . . CAT . . CCL16 PTK2PTK2 . . TXNRD2 ...... BIDBID CXCL1 ...... NOX4 . . . HADHB . . TLR4 . P2RX7P2RX7 CASP7CASP7 . . CXCL12 . . . . . SPTAN1SPTAN1 . MGST1 ...... CXCL8 . RIPK1RIPK1 . . XDH ACADS . . TLR2 . TXNIPTXNIP . . TNFRSF10BTNFRSF10B ...... NQO1. . . . CCL20 . FASFAS . . . TLR1 . CASP6CASP6 NFKB1 EC HEALTH ACADM . . STAT1 . CASP1CASP1 . SATB1SATB1 . . . . . GPX1 . . IL10RBIL10RB . TNFRSF1ATNFRSF1A . MAPK14 CXCL16 CASP2CASP2 ECHS1 . . IFNAR2IFNAR2 . . BCL2L1 NFIX . . BCL2L1 MADDMADD . . CCL15 TRADDTRADD SOD3 ECI1 . . STAT6 . . 2 CASP8CASP8 UGT1A6 IFNGR2IFNGR2 NFKB2NFKB2 cell . . APAF1APAF1 STAT5A . . MAPK10 DECR1 . . CSF1 EC CASP10CASP10 . . . . DIABLODIABLO GSR STAT2 APPAPP . . . . IL1R1IL1R1 SMC Apoptosis CRADDCRADD GCLC . . * * * * MUT STAT5B PTGS2 1 DFFADFFA SOD2 MIF . . NFKB1 . . . . . TNFRSF10ATNFRSF10A . Inflammation NFKB1 cell_type . . . . TOP1TOP1 SP1 ACADL IL15IL15 Inflammasome Genes (0.9) Genes (0.9) IFNAR1IFNAR1 Genes (0.73) Genes (0.86) Genes (0.86) . . Genes (0.73) . . . . TXNRD1 Genes (0.97) Genes (0.97) i CASP9CASP9 Genes (0.76) Genes (0.76) . . . TLR3 . TXNTXN CASP3CASP3 CYBA TNFRSF11B p . MAP3K1MAP3K1 . . . PCCA . . TXN2 IFNLR1IFNLR1 . 0 . CYCSCYCS . . . IL7IL7 . RELARELA . * . GAS2GAS2 *. * NFE2L2 * . . * . . . TNFSF13B EDN1 Oxidative Stress Response Oxidative Stress

MCEE log2fold . . . . . DFFBDFFB . . . SOD1 . . IL1RNIL1RN . . BAXBAX . . JUNB CXCR5 PANX1PANX1 . . KRT18KRT18 . . PCCB ...... FOS . . IFNGR1IFNGR1 −1 . BCL2L1BCL2L1 . . . Mitochondrial Fatty Acid Beta Oxidation TLR6 . . . . . VIMVIM . GPX3 . . . . . TLR5 . HSP90AB1HSP90AB1 . . . . . TNFSF10TNFSF10 . . . HMOX1 HADH . . STAT3 . . . . . APPAPP . . . . . AIMP1 . . . * . * MAOA . . PTGS1. . SUGT1 . GSNGSN ...... ACADVL . . CXCL2 SUGT1 . . . . LIMK1LIMK1 MT1X CXCL13 −2

Responses (NA) * Responses (NA) Responses (NA) Responses (NA) Responses (NA) ResponsesHSP90AB1 (NA) Responses (NA) Responses (NA) Responses (NA) Responses (NA)

20357 21077 * 20357 21077 CALM1

* * * * HSP90B1

* CAV1

Supplemental* * Figure* 5. Transcriptomic* data from hepatocytesNOS3 exposed to

the * lipotoxic* compared* * to the healthy milieu is presentedKLF2 for various biological pathways associated with cellular stress and inflammation. * * * * KLF4 Expression of each gene is represented as log2fold-change of lipotoxic vs. * * * * ARG2

healthyCCA−STATIC milieuCCA−STATIC (red=upregICS−CCA ICS−ulation,CCA CCA −STATIC blue=downregulation).CCA−STATIC ICS−CCA ICS− CCAn=6 experiments, 3

donors. http://tinyurl.com/ph6nka9

CULTURE: 0.01 CULTURE: <0.01 CULTURE: 0.53 MILIEU: <0.01 MILIEU: <0.01 MILIEU: <0.01 INTERACTION: <0.01 INTERACTION: 0.14 INTERACTION: 0.26 ** ** 2048 65536 ** 512 ** ** ** 32768 256 1024 16384 * 128 Mono-culture Multi-culture 512 ** 8192 64 4096 32 256 2048 16 IL6 (fg/ml) IL8 (pg/ml)

1024 CXCL10 (pg/ml) 8 128 512 4 64 256 2 Healthy Lipotoxic Healthy Lipotoxic + Healthy Lipotoxic + TNF TNF + TNF CULTURE: <0.01 CULTURE: 0.04 CULTURE:MONO MULTI <0.01NASH NASH MILIEU: <0.01 MILIEU: <0.01 MILIEU: 0.08 MULTI HEALTHY MONO INTERACTION:HEALTHY 0.08 INTERACTION: 0.76 INTERACTION:MONOMULTI NASH 0.14 NASH MONOMULTI NASH NASH MONOMULTI HEALTHY HEALTHY ** MONOMULTI HEALTHY HEALTHY ** p=0.10 ** ** 512 4096 p=0.13 64 * ** 256 ** 2048 32 ** 128 1024 16 64 512 8 32 256 MCP1(pg/ml) VEGF (pg/ml) YKL40 (pg/ml) 16 128 4

8 64 2 Healthy Lipotoxic + Healthy Lipotoxic Healthy Lipotoxic TNF + TNF + TNF MONOMULTI NASH NASH MONOMULTI HEALTHY HEALTHY MONOMULTI NASH NASH Supplemental Figure 6. The MONO presenceMULTI NASH NASH of non-parenchymal cells (NPCs) in MONOMULTI HEALTHY HEALTHY MONOMULTI HEALTHY HEALTHY the in vitro human liver system significantly impacts its inflammatory profile. Secreted analytes were measured in the media effluent from devices at day 10. The lipotoxic milieu for these experiments is composed of 65µM oleic acid and 0.1ng/ml TNFα. n=4 experiments, 1 donor. Triangles indicate samples were below lower limit of quantification. Tables above charts provide overall significance from 2-way ANOVA for cell culture and milieu treatment conditions and the interaction p-value. Multiple comparison were performed as indicated with *p<0.05, **p<0.01, 2-way ANOVA, Tukey.

Mono-culture ● Mono-culture Healthy Milieu ● Lipotoxic Milieu

● 50 ● ● ●

Mono-culture Cell Type ● 2_Mono_HEPMulti-culture ● 2_Tri_HEP 0 PC2 PC2 ● Milieu Healthy Milieu ● LGLI + DMSO ● NASH + DMSOLipotoxic Milieu ● ●

● ● −50

Multi-culture Multi-culture Healthy Milieu Lipotoxic Milieu

−50 0 50 100 PC1 PC1

Supplemental Figure 7. The presence of non-parenchymal cells (NPCs) in the in vitro human liver system significantly impacts the transcriptomic profile of hepatocytes. Transcriptomic data was acquired from hepatocytes exposed for 10 days to a lipotoxic milieu composed of 65µM oleic acid and

0.1ng/ml TNFα in the presence or absence of NPCs. n=3-6 experiments, 1 donor. Gene expression profile reveals that the hepatocytes from healthy and lipotoxic treatments separate along principle component 1, and these further separate along principle component 2 if co-cultured with NPCs.

8 OCA vs LGLI Effect on Lipid Species CE CER DAG DCER 2 2 2 2

1 ●● 1 1 1 ● ● ● ●●●● ● ● ● ● ●● ●● ● ● ●● ● ● ●● ●● ● ● 0 ● 0 ● ● 0 ● ● ● ● ● 0 ● ●●● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ● −1 −1 −1 ● −1 ●

−2 −2 −2 ● ● −2 ● −3 −3 −3 −3

−4 −2 0 2 −4 −2 0 2 −4 −2 0 2 −4 −2 0 2 FFA HCER LCER LPC 2 2 2 2

1 ● 1 1 1 ● ● ● ● ● 0 0 0 ● 0 ● ● ● ● ● ● ● ● ● −1 −1 ● ● ● −1 −1 −2 −2 −2 −2 −3 −3 −3 −3 RSI 1.0 (VEH) (OCA)

NASH VEH −4 −2 0 2 −4 −2 0 2 −4 −2 0 2 −4 −2 0 2 0.5 − 0.0

log2fold LPE PC PE PI 2 2 2 2 −0.5

● ● ●● ● ● −1.0 1 1 ● 1 ●●● 1 Lipotoxic ● ● ● Lipotoxic (log2 fold change) (log2 fold ●●● ● ●●●● ● ●●●●●●●● ●● ● ● ● ● ● ●●●●●● ● ● ●● ● ● ●●● ●●● ● ● ● ●●●●●●●● ●●●●●●●●● ● ● ● ● ● ●●●●●●●●●● ● ●● ● ● ● ● ● 0 ● 0 ● ●● 0 ●●●●● ● 0 ● ●● ●● ●●● ●● ● NASH OCA ● ●●● ●● ● ● ● ●● ● ● ●●●● ●● ● ● ● ●●● ● ● ● ● ● ● ● −1 −1 ● −1 ● −1 ● ● ● ● −2 −2 −2 −2 −3 −3 −3 −3

−4 −2 0 2 −4 −2 0 2 −4 −2 0 2 −4 −2 0 2 SM TAG 2 2 ● ● ● ● ●●● ● 1 1 ● ●● ● ●● ● ●● ●● ● ● ● ●●●●●● ● ●● ●● ● ●● ●● ●●●●● ●●● ●● ●●●● ●●● ● ●● ● ●●● ●●●●●● ●●● ●●●●●●●●●●●●●●●●●●● ● ●●● ●●● ●●●●●●●●● ● 0 ● 0 ●●●●●● ●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●● ●● ●●●●●●●●●●●●●●●●●●●●● ●●● ●●●●●●● ●●●●●●●●●●●● ● ●●●●●●● ● ●●●●●●●●●●●●●●●●●● ●● ● ●● ●●●●●●●●●●●●●●●●●●● −1 −1 ● ● ●● ●●●●●●●● ●●●●● ● ●● ● ●●●●●●●●● ● ● ●●●● ●● ●●●●●● ● ● ●● ● ●●●●●●●●● ● ● ● ● ● ●● ● ●●● ●● ●● ●● ● ● −2 −2 ● ● ● ● ● ● ● ● ● ●●●●● ● ●● ● −3 −3 ● ●● ● ● ●

−4 −2 0 2 −4 −2 0 2 LGLI VEHlog2fold − NASH VEH (log2Healthy fold change)(VEH) Lipotoxic (VEH)

Supplemental Figure 8. OCA treatment in the lipotoxic milieu promotes a change in lipid profile towards the healthy controls. Lipids from hepatocytes from device exposed to the healthy or lipotoxic milieu with 0.5µM OCA or vehicle control were measured by metabolomics. Scatterplot representation of differentially expressed lipids in these hepatocytes are shown as log2fold-change and colored by RSI. n=4 experiments, 2 donors. Each lipid species is represented in an individual plot: CE (cholesterol ester), CER (ceramide), DAG (diacylglycerol), DCER (dihydroceramide), FFA (free fatty acid), HCER (hexosylceramide), LCER (lactosylceramide), LPC (lysophosphatidylcholine), LPE (lysophosphatidylethanolamine), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PI (phosphatidylinositol), SM (sphingomyelin), TAG (triacylglycerol).

p=0.12

1.2

1.0 (Fold VEH)

0.8 Total Intracellular Cholesterol Cholesterol Intracellular Total Veh OCA ATOR

Supplemental Figure 9. Obeticholic acid (OCA) increases total intracellular cholesterol levels. Total intracellular cholesterol was measured from hepatocytes from the device exposed to vehicle control, 0.5µM OCA, or 300nM atorvastatin (ator) in the lipotoxic milieu for 10 days and represented as fold change relative to vehicle controls. Atorvastatin serves as a positive control as it inhibits HMG-CoA reductase, the rate-limiting enzyme of the cholesterol- producing mevalonate pathway. n=4 experiments, 4 donors. *p<0.05, student’s

2-tailed t-test.

In Vitro References / Figure/Table Model Reviews Steatosis / Lipogenesis Yes Fig 2 1-3 Hypertriglycermia Yes Table 2 2-5 Increased Cholesterol Yes Table 2 2-5 Increased Glucose Output Yes Fig 3A 2,3 6 Insulin Resistance Yes Fig 3 1-3,6,7 Cell Stress / Elevated ALT Yes Fig 4 1-3,7,8 Inflammatory Signaling Yes Fig 4 2,3,7,8 Increased Apoptosis Yes Fig 4 2,3,7-9 Histological Features (e.g. ballooning) NA 1-3 Early Fibrosis Yes Fig 5 2,3,7,10 Advanced Fibrosis Fig 5 2,3,10 Cirrhosis NA 2,3,10 Oncogenesis NA 2,3

Supplemental Table 1. Pathological features of NAFLD in the in vitro human liver lipotoxic system.

Donor Donor ID Age Gender BMI Vendor 1 HC5-30 21 Male 25 XenoTech 2 QHuf14024 55 Female 29 QPS 3 QHuf15028 58 Female 21 QPS 4 HC3-31 42 Female 31 XenoTech 5 QHu13035 39 Male 30 QPS

Supplemental Table 2. Hepatic donors used in this study with their sourcing details and demographic information.

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