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demonstrated that silencing of HCs TAZ in murine models of nonalcoholic steatohepatitis (NASH) prevented or reversed hepatic inflammation, HCs death, and fibrosis 36

Posted on October 3, 2021 by president2010

demonstrated that silencing of HCs TAZ in murine models of nonalcoholic steatohepatitis (NASH) prevented or reversed hepatic inflammation, HCs death, and fibrosis 36. signaling. Conclusions: Our work has Sarafloxacin HCl identified a hepatocyte-specific lnc-HSER that regulates liver fibrosis, providing a proof that this molecule is a novel biomarker for damaged HCs and a potential target for anti-fibrotic therapy. in vitroand through inducing the EMT and the apoptosis of HCs. In addition, knockdown of lnc-Hser promoted HSCs activation through the signals derived from damaged HCs. we have also revealed that lnc-Hser inhibited HCs apoptosis via the C5AR1-Hippo-YAP pathway and suppressed HCs EMT through the Notch signaling. All these data suggest that lnc-HSER is a novel biomarker for damaged HCs and a potential target for anti-fibrotic therapy. Materials and methods Cell SLC3A2 culture and antibodies The non-tumorigenic mouse hepatocyte cell line AML12 was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Camarillo, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA), 1 insulin-transferrin-sodium selenite media supplement (ITS; Sigma-Aldrich), dexamethasone (40 ng/ml), penicillin (100 U/ml) and streptomycin (100 g/ml). The human hepatocyte cell line L02 and HEK293T were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 g/ml). All cells were cultured at 37C in an atmosphere containing 5% CO2. For co-culture experiment, lnc-Hser-silenced and lnc-Hser-overexpressed AML12 cells and the controls were washed with PBS after 24 – 48h of lentivirus infection. The cells were subsequently incubated in DMEM supplemented with 10% FBS for 48h and the supernatants were centrifuged at 1100 rpm for 5 min and mixed with DMEM containing 10% FBS at 1:1 ratio for preparing conditioned medium (CM). Cells were treated with the C5AR1 inhibitor PMX 205 (Med Chem Express, USA) or -secretase inhibitor RO4929097 (Med Chem Express, USA) for 24 hours at concentrations of 5 M. The antibodies were -SMA (rabbit polyclonal, Abcam, ab5694), Collagen1 (rabbit polyclonal, Abcam, ab34710; Millipore, #234167), TGF (rabbit polyclonal, Abcam, ab66043), MMP2 (rabbit monoclonal, Abcam, ab92536), TIMP1 (mouse monoclonal, Santa Cruz,sc-21734), Notch2 (rabbit monoclonal, Cell Signaling Technology, #5732), Notch3 (rabbit polyclonal, Abcam, ab23426), Hes1 (rabbit polyclonal, Abcam, ab71559), phospho-YAP (Ser-127) (rabbit monoclonal, Cell Signaling Technology, #13619), total YAP/TAZ (rabbit Sarafloxacin HCl monoclonal, Cell Signaling Technology, #8418), phospho-MST1/2 (rabbit monoclonal, Cell Signaling Technology, #49332), total MST1 (rabbit monoclonal, Cell Signaling Technology, #3682), phospho-LATS (Ser-909) (rabbit polyclonal, Cell Signaling Technology, #9157), total LATS1 (rabbit monoclonal, Cell Signaling Technology, #3477), C5AR1 (rabbit monoclonal, Proteintech, #21316-1-AP), Ki67 (rabbit monoclonal, Abcam, ab16667), Cleaved Caspase3 (rabbit polyclonal, Cell Signaling Technology, #9661), Caspase3 (rabbit monoclonal, Cell Signaling Technology, #9662), BAX (rabbit polyclonal, Abcam, ab32503), N-Cadherin (rabbit monoclonal, Cell Signaling Technology, #13116; Mouse monoclonal, Abcam, ab98952), E-cadherin (rabbit monoclonal, Cell Signaling Technology, #3195), -Catenin (rabbit monoclonal, Cell Signaling Technology, #8480), Vimentin (rabbit monoclonal, Cell Signaling Technology, #5741), Snail (rabbit monoclonal, Cell Sarafloxacin HCl Signaling Technology, #3879), rabbit IgG (Millipore, PP64B), goat anti rabbit IgG (Invitrogen, Alexa Fluor Sarafloxacin HCl 488/594), goat anti mouse IgG (Invitrogen, Alexa Fluor 594). Construction of plasmids gRNA design was based on CRISPR design (http://crispr.mit.edu/) or CHOPCHOP (https://chopchop.rc.fas.harvard.edu/) and cloned into lenti-CRISPRv2. Oligos encoding shRNA specific for lnc-Hser and the negative control shRNA were ligated into pSUPER.retro.puro, and the fragment containing the H1 promoter and hairpin sequences was subcloned into Sarafloxacin HCl the lentiviral shuttle pCCL.PPT.hPGK.GFP.Wpre (lnc-Hser-shRNA and Negative Control (NC)). The full-length lnc-Hser cDNA was sequentially amplified by PCR and ligated into the lentiviral shuttle pCCL.PPT.hPGK.IRES.eGFP/pre to generate the over-expression plasmid (LV-lnc-Hser and the empty plasmid as the LV-Control). These plasmids were used to produce lentivirus in HEK-293T cells with the packaging plasmids pMD2.BSBG, pMDLg/pRRE and pRSV-REV. Infectious lentiviruses were harvested at 36 h and 60 h after transfection and filtered through 0.45 m PVDF filters. Recombinant lentiviruses used were concentrated 100-fold by ultracentrifugation (2 h at 120,000 g). The virus-containing pellet was dissolved in PBS and injected in mice within 48 h. The primer sets used are shown in Table S1. Animals study Animal protocols were approved by Tianjin Medical University Animal Care and Use Committee. The methods were carried out in accordance with the approved guidelines. All Balb/c mice aged at 8 weeks were obtained from Institute of Laboratory Animal Sciences, CAMS & PUMC (Beijing, China). For bile duct ligation (BDL) -induced mouse liver fibrosis model, mice were treated with sham operation or BDL operation for 3 to 21 days and.

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