Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: ELISA analysis of secreted biomarkers. SB525334, nintedanib, and sorafenib suppressed secretion of hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium after 48 h of incubation. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 8–10 liver slices for each condition. **P < 0.01 and ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Ligation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: The small molecule αV integrins inhibitor CWHM12 attenuated fibrotic phenotype in fibrotic precision-cut liver tissue slices (PCLSs) after 48 h of incubation. A: CWHM12 suppressed expression of a panel of fibrotic genes. B: CWHM12 decreased secretion of procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 6–8 liver slices for each condition. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Incubation, Expressing, Binding Assay, Ligation

Journal: International Journal of Molecular Sciences

Article Title: Protective Effect of Paeoniae Radix Alba Carbonisata on Hepatic Amyloidosis by Regulating Calcium Homeostasis

doi: 10.3390/ijms27062582

Figure Lengend Snippet: PRAC-E modulates calcium homeostasis via the cGMP/PKG/ATP2A1 signaling axis, thereby alleviating inflammation and amyloid deposition. ( A , B ) The dose-dependent ( A ) and time-dependent ( B ) effects of LYSO-6 on NCTC1469 cells. ( C ) Screening of the effective concentration of PRAC-E on LYSO-6-stimulated cells. ( D – F ) The levels of IL-6, TNF-α, and IL-8 in the cell culture supernatant. ( G – I ) The levels of SAA, SAP ( G ), and APO-E ( H ) in NCTC1469 cells. ( J ) CR staining of NCTC1469 cells. ( K ) The dose-dependent effects of BAPTA-AM on NCTC1469 cells. ( L , M ) Representative Fluo-4 AM fluorescence staining and semi-quantification of NCTC1469 cells. ( N ) Ca 2+ -ATPase activity in NCTC1469 cells. ( O ) Intracellular cGMP levels in NCTC1469 cells were measured by ELISA. ( P – R ) Western blotting and semi-quantitative analysis of PDE5A, PKG, and ATP2A1 in NCTC1469 cells. Data are presented as the mean ± SD ( n = 6). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. solvent control group; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. LYSO-6; ns, no significance.

Article Snippet: Rabbit antibodies against β-actin (Cat# 20536-1-AP), PDE5A (Cat# 22624-1-AP), and ATP2A1 (Cat# 22361-1-AP) were obtained from Proteintech Group, Inc. (Wuhan, China).

Techniques: Concentration Assay, Cell Culture, Staining, Fluorescence, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Solvent, Control

Journal: International Journal of Molecular Sciences

Article Title: Protective Effect of Paeoniae Radix Alba Carbonisata on Hepatic Amyloidosis by Regulating Calcium Homeostasis

doi: 10.3390/ijms27062582

Figure Lengend Snippet: Characterization of components in PRAC-E and analysis of core components. ( A , B ) Total ion flow diagram of PRAC-E under negative ( A ) and positive ( B ) ion mode. Number 1 to 42: The active ingredients are detailed in . ( C ) The types of compounds in the PRAC-E. ( D ) The TCM–component–target network. ( E ) Six core component structure diagrams. ( F ) Molecular docking binding energy. ( G ) Benzoyl paeoniflorin-ATP2A1. ( H ) Catechin-ATP2A1. ( I ) Gallate-ATP2A1. ( J ) Methylgallate-ATP2A1. ( K ) Paeoniflorin-ATP2A1. ( L ) Albiflorin-ATP2A1. Note: In G–L, the green font indicates non-ligand residue names, and the black font indicates hydrophobic residue names.

Article Snippet: Rabbit antibodies against β-actin (Cat# 20536-1-AP), PDE5A (Cat# 22624-1-AP), and ATP2A1 (Cat# 22361-1-AP) were obtained from Proteintech Group, Inc. (Wuhan, China).

Techniques: Binding Assay, Residue

Journal: bioRxiv

Article Title: Keratin 17- and PKCα-dependent transient amplification of neutrophil influx after repeated stress to the skin

doi: 10.1101/2023.10.11.561954

Figure Lengend Snippet: A) Schematic of neutrophil transwell migration assay. B) Migration of human primary neutrophils towards conditioned medium (CM) from A431 keratinocyte cultures. Individual symbols depict data using neutrophils from different donors (n=4). Data are shown as mean ± SEM. One-way ANOVA. C-D) ELISA measurements of selected chemokine levels in A431 CM (pg/ml). N=3 measurements for CXCL1 (2 technical replicates each), n=2 measurements for CXCL11 (4 technical replicates each). Data are shown as mean ± SEM. One-way ANOVA. E) Migration of human primary neutrophils towards A431 CM in the presence of CXCR2 antagonist and/or CXCR3 antagonist. Individual symbols depict data using neutrophils from different donors (n=4-6). Data are shown as mean ± SEM. Two-way ANOVA. F) ELISA measurements of TNFα levels in A431 CM (pg/ml). n=2 measurements (4 technical replicates each). Data are shown as mean ± SEM. One-way ANOVA. Acet, Acetone.

Article Snippet: These inhibitors together with 1μg/ml of TNFα neutralizing antibody Infliximab (Novus Biologicals #NBP2-52655) do not alter neutrophil migration in response to fMLF, a potent neutrophil chemoattractant , suggesting that neutrophils can respond normally to other chemotactic cues in the presence of the inhibitors ( Suppl.

Techniques: Transwell Migration Assay, Migration, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Viral Induction of the Zinc Finger Antiviral Protein Is IRF3-dependent but NF-κB-independent

doi: 10.1074/jbc.m109.054486

Figure Lengend Snippet: FIGURE 1. IRF3-dependent induction of ZAP and OASL mRNAs by virus or dsRNA in cells deficient in both type I and III IFN signaling. A, immunoblot analysis of ISG56 in Hec1B cells mock treated, infected with 100 HAU/ml of SeV, or treated with 1000 units/ml of IFN for 16 h. The asterisk denotes a nonspecific band. B, immunoblot analysis of ISG15 in Hec1B cells stably expression the control vector (HecNeo) or DN IRF3 (HecF3DN), 293FT, and Huh7 cells treated with 10 ng/ml of recombinant human IL-29 (lanes 1–4) or 500 units/ml of recombinant human IFN (lanes 5–8) for 18 h. C, HecNeo and HecF3DN cells were mock infected or infected with SeV and subsequently immunoblotted for IRF3, SeV, and actin. In the IRF3 panel, FL denotes full- length IRF3, whereas N denotes DN IRF3. D, IFN production in culture super- natants of HecNeo and HecF3DN cells that were mock infected or infected with SeV for 24 h, determined by VSV plaque reduction assay. The dashed line indicates the detection limit of the assay (3 units/ml). E, Northern blot anal- ysis of ZAP, ISG56, and OASL mRNAs in HecNeo (lanes 1–7) and HecF3DN (lanes 8–11) cells growing in 100-mm dishes following the indicated treat- ments: IL-29 (10 ng/ml); IFN (1000 units/ml); poly I:C transfection (T-pIC, 6 g); poly(IC) added to culture medium (M-pIC, 50 g/ml); SeV (300 HAU); and transfection of a vector expressing IRF3/5D (6 g). All of the treatments were done for 8 h except IRF3/5D, which was transfected into cells for 20 h before cell lysis and RNA extraction.

Article Snippet: The following monoclonal or polyclonal antibodies were used: anti-actin and anti-FLAG monoclonal antibodies (Sigma); anti-HA monoclonal antibody (Invivogen); anti-ISG56 polyclonal antibody (33); antiISG15 polyclonal antibody (Rockland); anti-IRF3 polyclonal antibody (a gift from Michael David); anti-Sendai virus polyclonal antibody (a gift from Ilkka Julkunen); anti-phospho specific STAT1 and STAT2 (Cell Signaling Technology); and peroxidase-conjugated secondary anti-mouse and anti-rabbit polyclonal antibodies (Southern Biotech).

Techniques: Virus, Western Blot, Infection, Stable Transfection, Expressing, Control, Plasmid Preparation, Recombinant, Northern Blot, Transfection, Lysis, RNA Extraction

Journal: Journal of Biological Chemistry

Article Title: Viral Induction of the Zinc Finger Antiviral Protein Is IRF3-dependent but NF-κB-independent

doi: 10.1074/jbc.m109.054486

Figure Lengend Snippet: FIGURE 2. IRF3 deficiency abrogates viral induction of ZAP. A, left panel, HeLa and HeLa cells stably expressing BVDV Npro (HeLaNpro) were mock infected or infected with 100 HAU/ml of SeV for 16 h. The cell lysates were immunoblotted for Npro (using anti-HA monoclonal antibody), IRF3, actin, andISG56.Rightpanel,HeLaandHeLaNprocellsweremocktreatedortreated with 100 nM of epoxomicin for 12 h prior to immunoblot analysis of Npro, IRF3,andactin.B,Q-PCRanalysisofZAP,IFN-,andOAS1mRNAlevelsinHeLa and HeLaNpro cells mock treated or stimulated with 500 units/ml IFN, 100 HAU/mlofSeVortransfectedwith2gofinvitrotranscribedHCVRNAfor8h. mRNA abundance was normalized to cellular 28 S ribosomal RNA. Fold changes were calculated by dividing normalized mRNA abundance following various treatments by that of the mock treated HeLa cells.

Article Snippet: The following monoclonal or polyclonal antibodies were used: anti-actin and anti-FLAG monoclonal antibodies (Sigma); anti-HA monoclonal antibody (Invivogen); anti-ISG56 polyclonal antibody (33); antiISG15 polyclonal antibody (Rockland); anti-IRF3 polyclonal antibody (a gift from Michael David); anti-Sendai virus polyclonal antibody (a gift from Ilkka Julkunen); anti-phospho specific STAT1 and STAT2 (Cell Signaling Technology); and peroxidase-conjugated secondary anti-mouse and anti-rabbit polyclonal antibodies (Southern Biotech).

Techniques: Stable Transfection, Expressing, Infection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Viral Induction of the Zinc Finger Antiviral Protein Is IRF3-dependent but NF-κB-independent

doi: 10.1074/jbc.m109.054486

Figure Lengend Snippet: FIGURE 4. Activation of hZAP and hOASL promoters by induction of the TLR3 and RIG-I/MDA5 signaling pathways. A and B, promoter activities of hZAP(2881) and pGL3basic in HeLa and Hec1B (A) and PH5CH8 cells (B) following indicated treatments. M-pIC, poly(IC) added to culture medium. C, activities of hZAP(2881) and hOASL promoters in Hec1B cells transiently expressing an control vector or individual signaling components in the RIG- I/MDA5 or TLR3 pathways. N-RIG and N-MDA5 denote the constitutively active CARD domain of RIG-I and MDA5, respectively. D, activation of hOASL promoter in Hec1B cells by ectopic expression of various forms of IRFs or the constitutively active RelA. IRF7 is a constitutive IRF7 mutant which has a internal deletion (deletion of amino acids 238–408). E, activation of wild type (WT) or the indicated mutant hOASL promoters or human OAS2 p69 pro- moter in Hec1B cells by IRF3/5D. mISREp and mISREd denote mutation in the proximal (291 to 271) and distal (335 to 315) ISRE, respectively (see supplemental Fig. S2A for further details). F, activation of various hZAP pro- moters by IRF3/5D in Hec1B cells. The wild type hZAP(2881) promoter was also tested for its activation by other indicated IRFs or RelA.

Article Snippet: The following monoclonal or polyclonal antibodies were used: anti-actin and anti-FLAG monoclonal antibodies (Sigma); anti-HA monoclonal antibody (Invivogen); anti-ISG56 polyclonal antibody (33); antiISG15 polyclonal antibody (Rockland); anti-IRF3 polyclonal antibody (a gift from Michael David); anti-Sendai virus polyclonal antibody (a gift from Ilkka Julkunen); anti-phospho specific STAT1 and STAT2 (Cell Signaling Technology); and peroxidase-conjugated secondary anti-mouse and anti-rabbit polyclonal antibodies (Southern Biotech).

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Control, Plasmid Preparation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Viral Induction of the Zinc Finger Antiviral Protein Is IRF3-dependent but NF-κB-independent

doi: 10.1074/jbc.m109.054486

Figure Lengend Snippet: FIGURE 7. Differential binding of IRF3 and STAT transcription factors to the hZAP promoter following virus infection or IFN stimulation. A, immu- noblot analysis of ISG56 expression and phosphorylation status of STAT1 and STAT2 in HeLa cells following stimulation of IFN (500 units/ml) for 1 and 8 h or infection with SeV (100 HAU/ml) for 16 h. Actin blot was shown as a loading control. B, ChIP analyses of IRF3 and STAT binding to the ISRE1–2, ISRE3–5, and STAT V sites within hZAP promoter in HeLa cells mock treated, stimulated with IFN (400 units/ml) for 1 h, or infected with SeV (200 HAU/ml) for 8 h. The ChIP-enriched DNA levels analyzed by Q-PCR were normalized to input DNA, followed by subtraction of nonspecific binding determined with control IgG.

Article Snippet: The following monoclonal or polyclonal antibodies were used: anti-actin and anti-FLAG monoclonal antibodies (Sigma); anti-HA monoclonal antibody (Invivogen); anti-ISG56 polyclonal antibody (33); antiISG15 polyclonal antibody (Rockland); anti-IRF3 polyclonal antibody (a gift from Michael David); anti-Sendai virus polyclonal antibody (a gift from Ilkka Julkunen); anti-phospho specific STAT1 and STAT2 (Cell Signaling Technology); and peroxidase-conjugated secondary anti-mouse and anti-rabbit polyclonal antibodies (Southern Biotech).

Techniques: Binding Assay, Virus, Infection, Expressing, Phospho-proteomics, Control

Journal: Heliyon

Article Title: Ellagic acid improves the symptoms of early-onset Alzheimer's disease: Behavioral and physiological correlates

doi: 10.1016/j.heliyon.2024.e37372

Figure Lengend Snippet: A) i. Total protein estimation for cerebral peptide levels; ii. Total protein quantification for Ca 2 ⁺ estimation procedure (n = 6). B) Effects of EA treatments on i. hippocampus, ii. PFC, iii. AMD, and iv. serum Aβ levels (n = 6). C) Effects of AB and EA treatments on calcium levels in i. hippocampus, ii. PFC, and iii. AMD (n = 6). D) Effects of AB and EA treatments on brain calpain levels in i. hippocampus, ii. PFC, and iii. AMD (n = 6). E) Effects of AB and EA on the gene expression profile of GSK-3β and CDK5. i) SH: a) Hippocampus, b) PFC; AB: c) Hippocampus, d) PFC; EA70: e) Hippocampus, f) PFC; EA140: g) Hippocampus, h) PFC. mRNA expression of GSK-3β: ii. hippocampus; PFC and CDK5: iii. Hippocampus; PFC, after AB and EA treatments. The relative intensity for each group was calculated relative to the expression profile of the SH group (n = 3). F) Effects of AB and EA treatments on cerebral i. hippocampus, ii. PFC, iii. AMD, and iv. serum p-Tau levels (n = 6). All data are represented as Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical analysis was conducted by one-way ANOVA followed by Dunnett's multiple comparison test.

Article Snippet: ELISA kits for Aβ, p-Tau, CREB, and calpain-1 as well as Ca 2+ assay kits were purchased from Elabscience, USA.

Techniques: Gene Expression, Expressing, Comparison

Journal: Cell Reports Methods

Article Title: Optogenetic induction of hibernation-like state with modified human Opsin4 in mice

doi: 10.1016/j.crmeth.2022.100336

Figure Lengend Snippet:

Article Snippet: Plasmid: pcDNA3-Nano-lantern(Ca2+)_600 , Saito et al. (2012) , Addgene #51982.

Techniques: Virus, Recombinant, Transfection, Modification, Cloning, Synthesized, Titration, Plasmid Preparation, Software