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1 Supplemental Information Development of an in Vitro Human 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)
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