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. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: Hepatology. 2015 Dec 11;63(3):731–744. doi: 10.1002/hep.28252

Figure 2. CXCL10 is highly expressed in lipotoxic EVs in an MLK3-dependent manner.

Figure 2

(A) C-X-C ligand 10 (CXCL10) protein levels in extracellular vesicles (EVs) derived from wild type (WT) & mixed lineage kinase (Mlk3)−/− primary mouse hepatocytes (PMH) treated with either vehicle, or 20 μM lysophosphatidylcholine (LPC), and from Huh7 cells treated with either vehicle, or 20 μM LPC with or without 1 μM of one of the MLK3 inhibitors URMC099 (URMC) and CLFB1134 (CLFB), were assessed by western blot. Tumor susceptibility gene 101 (TSG101), cluster of differentiation (CD) 63, and ALG-2-interacting protein (Alix), were employed as EV marker and Cytochrome P450 2E1 (CYP2E1) as a marker of EVs of hepatocytes origin. CXCL10, Phospho-p38, Phospho-signal transducer and activator of transcription (PSTAT) 1 & STAT1 protein levels were also assessed by western blot on whole cell lysate from PMH and Huh7 treated with vehicle, LPC alone, or LPC in the presence of either 1 μM of one of the MLK3 inhibitors URMC, and CLFB or 100 μM of the STAT1 inhibitor Fludarabine (flud). CXCL10, P-STAT1, and STAT1 protein levels were also assessed on whole cell lysate from PMH isolated from chow, and high fat, fructose, and cholesterol diet (FFC)-fed mice, actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading control. (B) Immunogold reactivity for CXCL10 on EVs derived from WT & Mlk3−/− PMH was assessed by immunogold electron microscopy. (C) Total RNA was extracted from Huh7 cells treated with either vehicle, or 20 μM LPC with or without 1 μM of one of the MLK3 inhibitors URMC099, and CLFB1134, and mRNA CXCL10 expression was evaluated by real-time qPCR. CXCL10 content was assessed (D) in the whole conditioned media of LPC-treated Huh7 cells, the supernatants obtained after ultracentrifugation and the EVs by enzyme linked immunosorbent assay (ELISA), (E) and also in the serum of FFC-fed mice, the supernatants obtained after ultracentrifugation and the EVs by ELISA. Bar columns represent mean ± S.E.M. NS (non-significant) p> .05, *** P < .001.