Supplementary material Gut SUPPLEMENTARY FIGURES LEGENDS Figure S1. (A) Gene Ontology enrichment analysis of biological processes in which selected TS/ONC identified by proteomic analysis are involved. Inter-connections between biological processes and genes involved are represented in B. Figure S2. mRNA expression of TSs/ONCs in constitutive hepatocytes-specific PTEN knockout mice. (A) qRT-PCR analyses of TS/ONC in liver tissues (4 controls and 5 LPTENKO mice) from 4-months-old control and constitutive hepatocyte-specific PTEN knockout (LPTENKO) mice. Cyclophilin-A was used to normalize qRT-PCR analyses. Data represent the mean+/-SD. (B) The mRNA level of TS/ONCO candidates identified in the proteomic analysis were investigated in a transcriptomic dataset from the Gene Expression Omnibus Database (GSE70681, LPTENKO 3-months old. For each candidate, Log2-Fold Change between Control and LPTENKO mice were calculated and represented in a heatmap. ***P<0.001, **P<0.01, and *P<0.05 (t-test). Figure S3. Hepatic steatosis and AKT phosphorylation in inducible hepatocytes-specific PTEN knockout mice (LIPTENKO). A. Hematoxylin/Eosin (H&E) staining of histological sections from 5-month old control and LIPTENKO mice treated with tamoxifen for 3 months. The protein level of PTEN, pAKTser473, pAKTthr308 and AKT were analyzed by Western Blot (B). Corresponding quantifications are reported in C. Data represent the mean +/- SD. *P<0.05, **P<0.01, ***P<0.001 compared with controls (t-test). Figure S4. Hepatic steatosis in ob/ob and db/db mice and analysis of TS/ONC mRNA expression in ob/ob mice. (A) Hematoxylin/Eosin (H&E) staining of histological sections from 2-month old Control, ob/ob and db/db (n=5-6 mice/group) mice. (B) A transcriptomic datasets from the GEO database was used to assess the relative mRNA levels of TS/ONC candidates in control vs ob/ob mice. For each candidate, Log2-Fold Change between Control and ob/ob mice were calculated and represented in a heatmap. *P<0.05, **P<0.01, ***P<0.001 compared with controls (t-test). Figure S5. S100a11, Anxa2 and Lgals1 mRNA expression in steatotic livers of aged mice. (A) Hematoxylin/Eosin (H&E) staining of histological sections from 3-months (n=4) and 1- year old mice (n=9). mRNA expression levels of S100a11, Anxa2 and Lgals1 were assessed by qRT-PCR. (B) Data represent the mean +/- SD. *P<0.05, **P<0.01, ***P<0.001 compared with controls (t-test). Sobolewski C, et al. Gut 2020;0:1–14. doi: 10.1136/gutjnl-2019-319019 Supplementary material Gut Figure S6. Anxa2 and Lgals1 mRNA expression in mice fed a high-fat-containing diet (HFD) and exercising. (A) Control mice were fed a HFD for 10 weeks and subjected or not to treadmill exercise for the last 4 weeks of the HFD (PMID: 30138163). (B) Relative mRNA levels of S100a11, Anxa2 and Lgals1 were investigated by qRT-PCR analyses. Data represent the mean+/- SD (Control: n=6: mice fed a HFD: n=3; mice +HFD/exercise: n=3). *P<0.05, **P<0.01, ***P<0.001 compared with controls (One-way ANOVA). Figure S7. Analysis of TS/ONC mRNA expression in different mice models of NAFLD. Transcriptomic datasets (see Table S3) from the GEO database were used to assess the relative mRNA levels (Fold Change) of TS/ONC candidates (S100a11, Anxa2, Lgals1, Mgll, Fasn, Acaca, Cd36 and Entpd5) between mice having hepatic steatosis (A, GSE57425, GSE38856, GSE53131 and GSE1432: mice fed a High-Fat Diet, HFD) or NASH (B, GSE63027: GNMTKO and MAT1AKO mice, GSE55747: CCL4-treated mice) and their related controls. Dashed lines indicate the 0.66 and 1.5 Fold Change limits. Data represent the mean +/- SD. *P<0.05, **P<0.01, ***P<0.001 compared with controls (t-test). Figure S8. Summary of TSs/ONCs mRNA alterations in human/mouse transcriptomic datasets: the table summarizes the data from Figure 2C-D and Figure S7). Figure S9. S100a11, Anxa2 and Lgals1 mRNA expression in mice mice fed a methionine/choline-deficient diet (MCD) or in Huh-7 cells treated with inflammatory cytokines. (A) Representative trichrome Masson staining of liver sections and mRNA expression of markers of inflammation/fibrosis in C57BL6/J mice were fed a standard (n=4) or MCD diet (n=6) for 2 weeks (B) Relative mRNA expression of S100a11, Anxa2 and Lgals1 in mice fed a control or MCD diet for 2 weeks. (C and D) Relative mRNA expression of S100A11 in Huh-7 or differentiated HepaRG cells treated with pro-inflammatory cytokines. Cyclophilin-A was used to normalize qRT-PCR analyses. Data represent the mean +/- SD (n=3). *P<0.05, **P<0.01, ***P<0.001 compared with controls (t-test). Figure S10. Expression of pro-inflammatory cytokines in hepatic tumoral tissues of LPTENKO mice. qRT-PCR analyses of S100a11, Anxa2 and Lgals1 in hepatic tumoral tissues from 15-months-old control (4 mice) and tumors from LPTENKO mice (10 LPTENKO mice). Cyclophilin-A was used to normalize qRT-PCR analyses. Data represent the mean+/-SD. ***P<0.001, **P<0.01, and *P<0.05 (t-test). Figure 11. S100A11 and ANXA2 expressions are upregulated in HCC from LPTENKO mice. (A) Anatomy of explanted livers with tumors in 1 year- and 15-months old LPTENKO Sobolewski C, et al. Gut 2020;0:1–14. doi: 10.1136/gutjnl-2019-319019 Supplementary material Gut mice and representative histologies (H&E staining) of hepatic tissues showing the presence of steatotic nodules, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) in the liver of 15-months old LPTENKO mice (B) Representative Western blot and protein/mRNA quantifications of S100A11/ANXA2 expression in control hepatic tissues and HCC tumors from 15-months old LPTENKO mice (n=5 controls and 8 liver tumors from 8 different LPTENKO mice). (C) The relative mRNA level of S100a11 (Fold Change) in liver tissues of controls and LPTENKO mice were compared at the age of 4 months (presence of steatosis), 8 months (presence of steatosis and inflammation), 12 months (presence of steatotic nodules, hepatocellular adenoma and hepatocellular carcinoma) and 15 months (presence of hepatocellular carcinoma and intrahepatic cholangiocarcinoma) (this study and Horie et al, JCI, 2004). Data represent the mean+/- SD (4-months: n=4: 8-months old: n=4; 12-months-old: n=6; 15-months-old: n=10). *P<0.05, **P<0.01, ***P<0.001 compared with controls (One-way ANOVA). Figure S12. S100A11, ANXA2 and LGALS1 expressions are upregulated in primary hepatocytes from LPTENKO mice. qRT-PCR analyses of S100a11, Anxa2 and Lgals1 in isolated primary hepatocytes from 4-months-old control and LPTENKO mice (4 controls and 4 LPTENKO mice). Cyclophilin-A was used to normalize qRT-PCR analyses. Data represent the mean+/-SD. ***P<0.001, **P<0.01, and *P<0.05 (t-test). Figure 13. Deregulated signaling pathways in HCC and hallmarks of cancer associated with candidate TSs/ONCs identified in this study. (A) Number of potential interactions of TSs/ONCs regulating specific pathway (enrichment analysis based on Figure 3D). (B) Classification of TSs/ONCs candidates within typical hallmarks of cancer. Figure S14. Liver tumors development in DEN-treated wild type mice. (A). Anatomy of 11 months old wild type mice injected at15 days of age with a single dose of DEN (25mg/kg) (left panel). The right panel shows representative histologies (H&E staining) of hepatic tissues showing the presence of HCC. (B) A transcriptomic dataset (GSE50431) from the GEO database was used to assess the relative mRNA levels (Fold Change) of S100a11, Anxa2 and Lgals1 in cancerous hepatocytes isolated from control or HCC from DEN-treated mice. Data represent the mean+/- SD. *P<0.05, **P<0.01, ***P<0.001 compared with controls (t-test). Figure S15. mRNA levels of S100 family members in liver tissues from LPTENKO mice. Relative mRNA expression of S100 protein family members in hepatic tissues of 3- and 15- months-old Control and LPTENKO mice. Data are derived from GEO datasets GSE70681 Sobolewski C, et al. Gut 2020;0:1–14. doi: 10.1136/gutjnl-2019-319019 Supplementary material Gut and are represented as LOG2 Fold Change between control and LPTENKO mice (n=5 for the 3-months old group and n=4 for the 15-months old group) in a heatmap. Figure S16. Expression of S100a11/Lgals1/Anxa2 and proliferation/apoptosis markers in hepatic tissues of 48h DEN-treated mice having or not in vivo silencing of S100A11. C57BL6/J mice were treated with DEN (100mg/kg) or NaCl 0.9%, sacrificed 48h after (A) and relative mRNA levels of S100a11, Lgals1 and Anxa2 were assessed by qRT-PCR (B). In panel (C), control C57BL6/J mice were transduced with control AAV8 (6 mice) or shS100a11-AAV8 (5 mice) for 10 days before i.p. injection of DEN (100mg/kg). 48h after DEN injection, mice were sacrificed and relative mRNA expression of key regulators of proliferation (Ccnd1, Mki67), DNA repair (Rad51, Mpg) and apoptosis (Tp53, Bax, Fas) were analyzed by qRT-PCR. Cyclophilin-A was used as a housekeeping gene for the PCR analyses. Data represent means+/-SD. ***P<0.001, **P<0.01, and *P<0.05 (t-test). Figure S17. S100A11 and ANXA2 are upregulated in human and mouse hepatic cancer cells. (A). mRNA expression of S100A11 and ANXA2 in human primary hepatocytes (HPH) and human hepatic cultured cancer cells. Cyclophilin-A was used as a housekeeping gene for the PCR analyses. Representative Western blots (B) and related quantifications (C) of S100A11 and Anxa2 protein/mRNA expression in mouse primary hepatocytes (MPH) and mouse hepatic cultured cancer cells (i.e, AML12 and Hepa1-6).