Fig 2: SCD1-overexpressing fibroblasts promote lung cancer growth. (A and B) The BODIPY staining and CD36 expression of tumor cells (LLC cells) after co-cultured with or without NIH 3T3-SCD1-MOCK cells and NIH 3T3-SCD1-OVER cells (N = 5). (C) The distribution and LD contents of tumor cells (LLC-GFP cells) that were co-cultured with SCD1-MOCK and SCD1-OVER fibroblasts (NIH 3T3) by lipid-tox red staining (Scale bar: 50 µm, N = 3). (D) The LLC cells with or without NIH 3T3-SCD1-MOCK cells or NIH 3T3-SCD1-OVER cells were subcutaneously co-injected into the C57 mice (N = 8 mice/group) and the tumor volumes were measured every two days. (E) The representative tumor images and tumor weights are presented. (F) Representative immunofluorescence images of PCNA (red) in the frozen sections from the tumor tissues (Scale bar: 50 µm). Data are shown as the Mean±SD; NS: p ≥ 0.05, *p < 0.05, **p < 0.01.

Fig 3: Activation of PIK3CA downstream effectors and the lipogenic phenotype in induced tumors. (A) Representative immunohistochemical analyses of preneoplastic clusters in one animal (upper row) and a tumor in one animal (lower row) generated by hydrodynamic tail vein injection of PIK3CA H1047R into WT mice. FASN and SCD1 represent master regulators of lipogenesis, while pAKT and pERK comprise downstream effectors of activated PIK3CA. Magnification inset highlighting focal cholangiocellular differentiation with the corresponding loss of CPS1 and positive staining for CK7 in one animal. Scale bars: 50 µm (except for magnification inset: 100 µm). (B) Representative immunohistochemical analyses of a preneoplastic lesion (upper row) and a tumor (lower row) generated by hydrodynamic tail vein injection of PIK3CA E545K into WT mice. Images from two different mice. Tumor magnification inset illustrates spindle cell morphology with combined weak expression of CPS1 and CK7. T, tumor. Scale bars: first row and second row large panel 100 µm, magnification inset 50 µm. (C) Western blot analysis shows effective upregulation of PIK3CA, HA-tagged PIK3CA, FASN, SCD1, and pro-inflammatory COX2 with increasing intensity in liver tissue containing preneoplastic lesions (n = 5 mice) and tumors (n = 5 mice) induced by PIK3CA E545K injections as compared to empty vehicle (EV) injections (n = 4 mice). (D) Western blot of PIK3CA downstream effectors pAKT, p-SGK3, pERK1/2, and p-STAT3 in liver lysates with preneoplastic lesions and tumors induced by PIK3CA E545K injections and liver lysates from EV injections with corresponding total protein levels of AKT, SGK3, ERK1/2, and STAT3. Loading control: ß-actin. Molecular weights of observed bands are marked on the right. (E) Phosphorylated protein/total protein ratios calculated from adjusted band intensities of the above western blot displayed as Tukey method box-and-whisker plots. (p)AKT, (phosphorylated) AKT; (p-)SGK3, (phosphorylated) SGK3; (p-)STAT3, (phosphorylated) STAT3.

Fig 4: Hepatic lipid metabolism in 0.1MCD‐fed mice treated with vehicle or with Aramchol. (A) Schematic representation of hepatic lipid metabolism. Hepatic FAs originate from serum and through de novo lipogenesis. FAs can either be oxidized in the mitochondria or esterified to form TGs. The rate‐limiting step in mitochondrial β‐oxidation is CPT1A. The accumulation of FAs in the cytoplasm increases their oxidation and generates ROS, which induce GSH depletion and produce oxFA, such as oxLA, which can lead to fibrosis and cell death. (B) Relative fold change (log2) in the hepatic content of the main metabolites involved in lipid metabolism in mice fed a 0.1MCD diet and treated with vehicle or with Aramchol compared to mice fed a normal diet. A 0.1MCD diet led to reduced levels of glucose, serum FAs, and MUFAs and to a reduction in the FA(16:1/16:0) ratio. In contrast, feeding the mice a 0.1MCD diet led to increased levels of FAs, DGs, TGs, CEs, oxFA, and oxLA. (C) Relative fold change (log2) in the content of proteins involved in liver lipid metabolism in mice fed a 0.1MCD diet and treated with vehicle or with Aramchol compared to mice fed a normal diet. Feeding the mice a 0.1MCD diet resulted in reduced levels of ACLY, ACC1, ACC2, FAS, and SCD1. The 0.1MCD mice also showed increased levels of CD36, AGPAT3, CPT1A, HADHA, HADHB, UCP2, and CYP4A14. *P < 0.05. Abbreviations: ACLY, adenosine triphosphate citrate lyase; AGPAT3, 1‐acylglycerol‐3‐phosphate O‐acyltransferase 3; CYP4A14, cytochrome P450, family 4, subfamily a, polypeptide 14; FAS, fatty acid synthase; HADHA, HADHB, 3‐hydroxyacyl‐CoA dehydrogenase A and B; LA, linoleic acid; oxFA, oxidized FA; ND, normal diet; UCP2, uncoupling protein 2. Data were represented as mean ± SEM.

Fig 5: Suppression of hepatocarcinogenesis following HSF1 inactivation is accompanied by downregulation of activated AKT and its downstream effectors in the mouse liver(A, B) Representative Western blot analysis of livers from control mice (injected with empty vector), AKT mice (injected with myr-AKT1) and AKT/HSF1dn mice (co-injected with myr-AKT1 and HSF1dn). Inactivation of HSF1 in AKT/HSF1dn mice resulted in downregulation of phosphorylated/activated AKT. Levels of some AKT upstream (PIK3CA) and downstream effectors (p-RPS6, p-NDRG1, FASN, ACLY, ACAC, SCD1, PPARG, ALDOA, LDHA/C, PKM2) were downregulated in AKT/HSF1dn mice when compared with AKT mice. As expected, livers from AKT/HSF1dn mice were the only ones to express the transfected HSF1dn construct (V5-tagged). Of note, the levels of some of the proteins tested were even lower in AKT/HSF1dn mice than in control mice. Ten livers from each group of mice were used for the analysis, and representative images are shown. β-Actin was used as a loading control. (C–F) Fatty acid biosynthesis, cholesterol and triglyceride content, and lactate dehydrogenase activity were strongly induced in the livers of AKT mice, and equally lower in control and AKT/HSF1dn livers. Tukey–Kramer test: P < 0.0001 a, vs control (mice injected with empty vector); b, vs AKT mice. Abbreviations: FA, fatty acid; CE, cholesterol; TG, triglycerides; LDH, lactate dehydrogenase.