Supplementary MaterialsMMC1. the response from the liver organ microenvironment. Autophagy is usually a mechanism involved in the regulation of this initial response and its manipulation can change the progression of liver damage. All procedures were performed in accordance with Spanish legislation and approved by the Animal Research Committee of the University of Barcelona and were conducted in accordance with the European Community guide-lines for the protection of animals used for experimental and other scientific purposes (EEC Directive 86/609). Generation of endothelial Previously described in endothelial cells. Induction of fibrosis in mice Carbon tetrachloride (CCl4) (Sigma) was used to induce moderate acute liver injury. Mice received 3 intraperitoneal (i.p.) injections of 10% CCl4 (diluted in olive oil) at a dose of 0.5 l/g body weight13 or vehicle (olive oil) every other day for 1 week.8 Animals were sacrificed 48 h after the last dose under ketamine/midazolam anesthesia. At least 5 animals per group were used in isolation experiments and 6 Anlotinib HCl to 12 animals per group in total tissue experiments making a total of 81 mice analyzed. All experiments were performed with Anlotinib HCl mice between 10 and 14 weeks of age. We analyzed the effect of liver injury in male and female mice and the effect was comparable in both genders, so all subsequent experiments were performed indistinctly in male and female mice. Induction of fibrosis in rats Hepatic injury was induced in 250C300 g male Sprague-Dawley (SD) rats (Charles River) by CCl4 (50% CCl4 diluted in olive oil at a dose Anlotinib HCl of 1 1 l/g of body weight) with 3 i.p. injections per week for 1, 4 or 6 weeks and compared with control rats injected with vehicle (olive oil).13 A minimum of 3 animals per group were used in isolation experiments accounting for a total of 36 rats. Animals were sacrificed 48 h after the last dose (CCl4) under ketamine/midazolam anesthesia. Cell lines and culture conditions Unless normally specified, chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Culture media and supplements for cell culture were from Gibco-Invitrogen (Carlsbad, CA, USA) and Anlotinib HCl plastic ware was from TPP (Trasadingen, Switzerland). Human umbilical vein endothelial cells (HUVEC, Lonza) were cultured on gelatin covering with M-199 (Gibco) medium supplemented with 20% FBS, 1% L-Glutamine, 1% penicillin/ streptomycin (PS) and 1% endothelial cellgrowth product (ECGS). Mouse LSECs (TSECs) were kindly provided by Dr. V Shah14 and cultured with endothelial cell medium (ECM, Scien-Cell) with 5% FBS, 1% PS and 1% ECGS. Rat and mouse LSECs as well as mouse hepatic stellate cells (HSCs) were isolated as previously explained.15,16 In brief, livers were perfused through the portal vein Anlotinib HCl and digested with a collagenase answer. After mincing the liver, cells were filtered and centrifuged at 50xG to remove hepatocytes. Non-parenchymal cells were then separated by differential centrifugation using a Percoll gradient. Kupffer cells were eliminated by plastic pre-culture for 30 min. LSEC were plated in collagen covering dishes in RPMI-1640 medium with 10% FBS, 1% L-Glutamine, 1% PS, 1% Fungi-zona (Reactiva) and 1% ECGS. HSC cells were cultured in DMEM/F12 with 20% FBS, 1% PS and 1% Fungizona. ROS induction studies HUVECs and TSECs were seeded in 6-well plates and produced for 24 h before treatment with hydrogen peroxide (0.25 and 120 M, respectively) and collected after 72 h. Western blotting Extracted IKK-alpha proteins were analyzed by western blot. Antibodies used were: SQSTM1/p62 (Cell Signaling, 1/1000), LC3B [MAP1LC3B] (Cell Signaling, 1/1000), ATG7 (Cell signaling, 1/1000), SMA [ACTA2] (Sigma, 1/1000), PDGFRB (Santa Cruz, 1/500),.