Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Inhibition of HCVcc entry into Huh7.5 cells by anti-HAVCR1 antibody. Huh7.5 cells were treated with decreasing concentrations of the following antibodies following 2-fold dilutions from 4 μg/ml to 0.0625 μg/ml: anti-HAVCR1 MAb (1D12) (A), anti-CD81 MAb (JS-81) (B), and isotype control (C). Cells were treated with antibodies 1 h prior to infection with J6/JFH1. After 72 h, HCVcc infection was evaluated by automatic counting of stained foci and the percentage of inhibition calculated by comparing focus counts in treated and mock-treated cells. (D) Huh7.5 cells were treated with increasing concentrations of anti-CD81 and anti-HAVCR1 MAbs individually or in combination. The percentage of inhibition and dose effect plot was created using CalcuSyn 2.0 software. Testing for all assays was performed in duplicate. Error bars represent standard errors of the means. Data are representative of three independent experiments.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Inhibition, Control, Infection, Staining, Software

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Comparison of inhibitory activities and cell membrane localization of CD81 and HAVCR1. JFH1-Nanoluc was added to cells at 4°C for 1 h before moving the cells to 37°C to allow entry and infection to proceed. Antibody against CD81 (JS-81) (A), HAVCR1 (1D12) (B), or mouse isotype control (C) was added hourly to cells before and after HCVcc binding. The amount of antibody used was the concentration that was previously found to result in 50% inhibition of infection or the highest equivalent concentration in the case of the isotype control. The levels of luciferase expression at different time points were measured, and the percentages of inhibition were calculated by comparing the signals in mock-treated and infected cells at each time point. Error bars represent standard errors of the means. (D) Confocal analysis of Huh7.5 cells surface stained with 4′,6-diamidino-2-phenylindole (DAPI), anti-CD81 MAb (JS-81), and anti-HAVCR1 MAb (HAVCR1-1 MAb).

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Comparison, Membrane, Infection, Control, Binding Assay, Concentration Assay, Inhibition, Luciferase, Expressing, Staining

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Dose-dependent inhibition of HCVcc entry with soluble HAVCR1 receptors, phosphatidyl serine, or annexin V. HCVcc was incubated 1 h prior to infection of Huh7.5 cells with increasing amounts of soluble HAVCR1 receptor containing the IgV- and mucinlike domains (HAVCR1-Fc) (A), soluble monkey HAVCR1 (mkHAVCR1-Fc) or mouse Havcr1 ortholog (mHavcr1-Fc) (B), HAVCR1-Fc or soluble HAVCR1 containing the IgV-like domain [HAVCR1(IgV)-Fc] (C), HAVCR1-Fc or the same receptor with an N94A mutation in the phosphatidylserine binding domain (HAVCR1-N94A-Fc) (D), phosphatidylserine (PS) or phosphatidylcholine (PC) liposomes (E), or annexin V (F). Testing for all assays was performed in duplicate. Error bars represent standard errors of the means. Data are representative of three independent experiments. All experiments were performed with HCVcc J6/JFH1 except for that shown in panel A, which used HCVcc chimeric viruses 1a/2a, 1b/2a, 2a/2a (J6/JFH1), and 3a/2a.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Inhibition, Incubation, Infection, Mutagenesis, Binding Assay, Liposomes

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Reduced HAVCR1 expression on Huh7.5 cells results in reduced levels of HCVcc and HCVpp entry. Huh7.5 HAVCR1-1 knockdown (KD) cells were generated by transfection of parent Huh7.5 cells with a plasmid expressing HAVCR1 shRNA. (A) Percentages of parent and KD cells positive for HAVCR1, CD81, and SRB1 surface expression. Bars represent the mean values from 5 independent experiments for HAVCR1 and CD81 and 4 independent experiments for SRB1. (B) Mean fluorescence signals of HAVCR1, CD81, and SRB1 surface expression on parent and KD cells. Bars represent the mean values from 5 independent experiments for HAVCR1 and CD81 and 4 independent experiments for SRB1. (C) Western blot detection of claudin and occludin after cell surface biotinylation of Huh7.5 and HAVCR1 KD cells. Whole-cell lysate (WCL) is cell lysate prior to pulldown treatment. Nonbiotinylated control represents cells treated exactly as for the biotinylated cells but without the addition of EZ-Link sulfo-NHS-LCLC-biotin for cell surface biotinylation. Anti-claudin antibody was used for detection and shows the specificity of the NeutrAvidin beads for precipitation. (D) Luciferase activities at 24, 48, and 72 h postinfection in lysates of parent and KD cells infected with JFH1-Nanoluc. Bars represent the mean relative light units (RLU) calculated from 4 independent experiments using 9 replicates per experiment. (E) Titers of virus recovered from the supernatants of parent and KD cells at 72 h postinfection with JFH1-Nanoluc. Bars represent the mean titers from 4 independent experiments. (F) Luciferase activities in parent and KD cells 48 h after infection with HCVpp. (G) Luciferase activities in parent and KD cells 48 h after infection with VSV-G. Bars represent the mean values from 4 independent experiments. Error bars in all graphs represent standard errors of the means. Statistical analyses were performed using the Mann-Whitney test.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Expressing, Knockdown, Generated, Transfection, Plasmid Preparation, shRNA, Fluorescence, Western Blot, Control, Luciferase, Infection, Virus, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Complete silencing of HAVCR1 expression in Huh7.5 cells significantly reduces HCVcc infection. Surface expression levels of receptors on Huh7.5 cells, Huh7-A-I cells, and different clones of Huh7-A-I HAVCR1 knockout (KO) cells were assessed by FACS using MAbs specific to HAVCR1 (REA383) (A), CD81 (JS-81) (B), and SRB1 (m1B9) (C). Data for positive cells are shown in dark gray, and data for mouse PE-isotype control are shown in light gray. Data are representative of three independent experiments. (D) Western blot detection of claudin and occludin after cell surface biotinylation of Huh7-A-I and clone 12 KO cells. Whole-cell lysate (WCL) is cell lysate prior to pulldown treatment. Nonbiotinylated control represents cells treated exactly as for the biotinylated cells but without the addition of EZ-Link sulfo-NHS-LCLC-biotin for cell surface biotinylation. Anticlaudin antibody was used for detection and shows the specificity of the NeutrAvidin beads for precipitation. Antiactin antibody was used for detection of actin, a cytoplasmic protein that is not expressed on the cell surface, demonstrating biotinylation of surface proteins only. (E) Luciferase activities in lysates from Huh7-A-I HAVCR1 KO clones and parent cell line at 72 h postinfection with JFH1-Nanoluc. Data were obtained from 3 independent experiments, and each experiment was carried out using 12 replicates for each cell clone. Each symbol represents an individual well from the 3 independent experiments. Horizontal bars represent the mean luciferase activities. P values were calculated using the mean values from each of the 3 independent experiments. (F) Time course of luciferase expression relative to the expression at 3 h posttransfection in Huh7-A-I HAVCR1 KO clones 2 and 12 and parent cells following transfection with JFH1-Nanoluc RNA transcribed in vitro. Data are the mean values from 6 replicates at each time point. Error bars represent standard errors of the means. Data are representative of two independent experiments. RLU, relative light units.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Expressing, Infection, Clone Assay, Knock-Out, Control, Western Blot, Luciferase, Transfection, In Vitro

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Expression of HAVCR1 in KO clone 12 enhances HCV infection. HAVCR1 KO clone 12 cells were transfected with plasmids expressing HAVCR1, mutated forms of HAVCR1 (E88A, N94A, Y330A, Y336A, and del324–331 mutations), or vector. (A) Expression of HAVCR1 on the surface of clone 12 cells transfected with vector or plasmid expressing HAVCR1 48 h posttransfection was assessed using anti-HAVCR1-1 MAb. (B) Apoptotic binding activities of clone 12 cells transfected with vector or plasmid expressing HAVCR1. (C) Luciferase expression in cell lysates of clone 12 cells transfected with HAVCR1 expression plasmid and infected with JFH1-Nanoluc. Data at 24 h postinfection are shown. Four independent experiments were performed; each time point included 3 replicates. (D) Expression of E88A or N94A mutant on the surface of transfected clone 12 cells 48 h posttransfection assessed using anti-HAVCR1-1 MAb. (E) Apoptotic binding of clone 12 cells transfected with vector or plasmids expressing E88A and N94A mutant forms of HAVCR1. (F) Luciferase expression in cell lysates of clone 12 cells transfected with plasmids expressing E88A or N94A mutants and infected with JFH1-Nanoluc. Data are from 4 independent experiments, each including 3 replicates, at 24 h postinfection. (G) Expression of Y330A, Y336A, or del324–331 mutant on the surface of transfected clone 12 cells 48 h posttransfection was assessed using anti-HAVCR1-1 MAb. (H) Apoptotic binding of clone 12 cells transfected with vector or plasmids expressing Y330A, Y336A, and del324–331 mutant forms of HAVCR1. (I) Luciferase expression in cell lysates of clone 12 cells transfected with Y330A, Y336A, or del324–331 mutant plasmids and infected with JFH1-Nanoluc. Data are from 4 independent experiments, each including 3 replicates, at 24 h postinfection. Horizontal bars represent the mean values. Asterisks represent significance values obtained using the Mann-Whitney test to compare the mean values for vector-transfected cells with the mean values for each of the other transfectants. NS, not significant; **, P < 0.001; ***, P < 0.0001. For FACS histograms, HAVCR1 expression on transfected cells is shown as filled dark-gray areas, and data for untransfected cells stained with anti-HAVCR1-1 MAb are shown as filled light-gray areas. RLU, relative light units; MFI, mean fluorescence intensity.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Expressing, Infection, Transfection, Plasmid Preparation, Binding Assay, Luciferase, Mutagenesis, MANN-WHITNEY, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: Schematic representation of the HAVCR1, mHavcr1, and chimeric proteins used in transient-transfection experiments. (A) Receptor domains and mutations in the human and mouse proteins. (B) Sequence alignment of HAVCR1, mouse Havcr1 (mHavcr1), and chimeric protein. Yellow highlighting denotes residues conserved between the molecules. The different receptor regions are defined by colored underlining as follows: red, IgV domain; blue, mucin domain; green, transmembrane domain; black, cytoplasmic domain. Mutated or deleted residues in HAVCR1 are shown in black boxes.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Transfection, Sequencing

Journal: Journal of Virology

Article Title: Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

doi: 10.1128/JVI.01742-17

Figure Lengend Snippet: The IgV domain of human HAVCR1 is responsible for facilitating HCV entry. (A) Expression of HAVCR1 or mHavcr1 on the surface of transfected clone 12 cells 48 h posttransfection. Receptor expression is shown as filled dark-gray areas, and data for stained, untransfected cells are shown as filled light-gray areas. (B) Apoptotic binding of clone 12 cells transfected with vector or plasmids expressing HAVCR1, mHavcr1, or chimeric human/mouse receptor. MFI, mean fluorescence intensity. (C) Luciferase expression in cell lysates of clone 12 cells transfected with expression plasmids and infected with JFH1-Nanoluc. RLU, relative light units. Data shown are from 5 experiments with transient transfection of the control vector and HAVCR1 and 3 independent experiments for mHavcr1, including 3 replicates each, at 24 h postinfection. Asterisks represent significance values obtained using the Mann-Whitney test to compare the data for vector-transfected cells with the data for each of the other transfectants or the parent cell line. NS, not significant; **, P < 0.001; ***, P < 0.0001.

Article Snippet: To characterize receptor expression on the surface of Huh7.5 cells, we performed staining with the following antibodies: anti-CD81 mouse MAb (JS-81; BD Pharmingen, San Jose, CA); anti-SRB1 antibody (m1B9; BioLegend, San Diego, CA), anti-HAVCR1 phycoerythrin (PE)-labeled mouse MAb (REA384; Miltenyi Biotec, Inc., Auburn, CA), or purified HAVCR1-1 MAb, an antibody directed against the IgV domain of HAVCR1 that blocks binding of phospholipids.

Techniques: Expressing, Transfection, Staining, Binding Assay, Plasmid Preparation, Fluorescence, Luciferase, Infection, Control, MANN-WHITNEY

Journal: Autophagy

Article Title: SMAD3 promotes autophagy dysregulation by triggering lysosome depletion in tubular epithelial cells in diabetic nephropathy

doi: 10.1080/15548627.2020.1824694

Figure Lengend Snippet: Inhibition of SMAD3 improves autophagic flux and protects HK-2 cells from injury after treatment with AGE-BSA. (A) Immunofluorescence staining of LC3 and SQSTM1 in HK-2 cells with or without SMAD3 gene knockdown after exposure to 30 μg/ml AGE-BSA or 30 μg/ml Co-BSA for 24 h. (B and C) Quantification of LC3 and SQSTM1 puncta. (D–G) Western blot analysis of phospho-SMAD3, LC3, and SQSTM1 expression in AGE-BSA or Co-BSA-treated HK-2 cells. (H) Fluorescent microscopic analysis of autophagic flux in RFP-GFP-LC3 plasmid-transfected HK-2 cells. After transient transfection with RFP-GFP-LC3 plasmids, HK-2 cells were pretreated with 10 µM SIS3 or 10 µM dimethyl sulfoxide (DMSO) for 1 h and exposed to AGE-BSA or Co-BSA. The yellow puncta indicate autophagosomes (arrowheads), and red puncta indicate autolysosomes (arrows). (I) Quantitative data for autophagosomes or autolysosomes in each cell. (J–L) Western blot analysis of HAVCR1 and FN1 expression in AGE-BSA or Co-BSA-treated HK-2 cells. (M) Immunofluorescence staining of FN1 in HK-2 cells. (N and O) ELISA of HAVCR1 and FN1 in the HK-2 cell culture supernatants. Bars represent means ± SEM for at least 3 independent experiments. *P< 0.05, **P< 0.01, and ***P< 0.001. DAPI was used to stain nuclei. Scale bar: 10 μm

Article Snippet: ELISA for FN1 and HAVCR1 The quantity of FN1 and HAVCR1 in cultured cell supernatants was measured by human fibronectin ELISA kit (R&D Systems, DFBN10) and human HAVCR1 ELISA kit (R&D Systems, DKM100) respectively, according to the manufacturer’s instructions.

Techniques: Inhibition, Immunofluorescence, Staining, Knockdown, Western Blot, Expressing, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Autophagy

Article Title: SMAD3 promotes autophagy dysregulation by triggering lysosome depletion in tubular epithelial cells in diabetic nephropathy

doi: 10.1080/15548627.2020.1824694

Figure Lengend Snippet: Smad3 gene knockout alleviates autophagic blockage and protects TECs. (A–C) Immunofluorescence staining of LC3 and SQSTM1 in TECs from db/m mice and db/db mice with or without smad3 gene knockout. DAPI was used to stain nuclei. Scale bar: 10 µm. (D–F) Western blot analysis of LC3 and SQSTM1 expression in the renal tissues of mice. (G) Fluorescent microscopic analysis of HAVCR1 and FN1 in renal tissues of mice. Scale bar: 10 μm. (H–J) Western blot analysis of FN1 and HAVCR1 expression in the renal tissues of mice. Each bar represents the mean ± SEM for groups of five to eight mice. Smad3+/+ db/m (n= 8), smad3−/- db/m (n= 5), Smad3+/+ db/db (n= 8), Smad3± db/db (n= 8), and smad3−/- db/db (n= 8).*P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: ELISA for FN1 and HAVCR1 The quantity of FN1 and HAVCR1 in cultured cell supernatants was measured by human fibronectin ELISA kit (R&D Systems, DFBN10) and human HAVCR1 ELISA kit (R&D Systems, DKM100) respectively, according to the manufacturer’s instructions.

Techniques: Gene Knockout, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Autophagy

Article Title: SMAD3 promotes autophagy dysregulation by triggering lysosome depletion in tubular epithelial cells in diabetic nephropathy

doi: 10.1080/15548627.2020.1824694

Figure Lengend Snippet: TFEB knockdown abolishes the protective effects of SMAD3 inactivation on lysosome biogenesis and autophagic flux in HK-2 cells. (A) Double immunofluorescence staining of LAMP1 and RAB7 to detect primary and/or secondary lysosomes. HK-2 cells with or without TFEB siRNA knockdown, were pretreated with SIS3 10 μM for 1 h and exposed to 30 μg/ml AGE-BSA for indicated time periods. LAMP1 and RAB7 double-positive dots represent secondary lysosomes (arrowheads), while single LAMP1-positive dots indicate primary lysosomes (arrows). Scale bar: 10 μm. (B and C) Number of primary lysosomes or percentage of primary and secondly lysosomes per cell. (D) Western blot analysis of LC3, SQSTM1, and HAVCR1 levels in HK-2 cells. Briefly, HK-2 cells with or without TFEB siRNA knockdown were pretreated with 10 μM SIS3 or DMSO for 1 h and exposed to 30 μg/ml AGE-BSA or Co-BSA for 24 h. (E–G) Densitometry results; the ratio of LC3-II, SQSTM1, or HAVCR1 to ACTB is expressed as a fold change compared with the level in controls. Data represent the means ± SEM of at least 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: ELISA for FN1 and HAVCR1 The quantity of FN1 and HAVCR1 in cultured cell supernatants was measured by human fibronectin ELISA kit (R&D Systems, DFBN10) and human HAVCR1 ELISA kit (R&D Systems, DKM100) respectively, according to the manufacturer’s instructions.

Techniques: Knockdown, Double Immunofluorescence Staining, Western Blot

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: HAVCR1 was upregulated in GAC tissues and cell lines, and high HAVCR1 expression was related with poor prognosis. (a) HAVCR1 expression was upregulated in GAC tissues ( n = 375) compared with normal gastric tissues ( n = 32). (b) Kaplan-Meier's survival analysis showed that the high regulation of HAVCR1 had a close relationship with the poor outcome of GAC patients, p = 0.012. (c) QRT-PCR reveals the expression levels of HAVCR1 in GAC cells (MKN-45 and AGS) and in a normal gastric cell line (GES-1). (d) Western blot analysis was performed to detect the expression of HAVCR1. (e) The gray value of protein bands was quantified. ∗∗ p < 0.01 versus GES-1 cells.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: Correlation between HAVCR1 expression and clinicopathological characteristics of GAC patients.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: Expressing

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: Univariate and multivariate analysis of clinic pathologic factors for overall survival in GAC patients.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: Expressing

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: HAVCR1 expression was significantly decreased in MKN-45 and AGS cells using siRNA strategy. (a) QRT-PCR was used to assess the HAVCR1 mRNA expression in MKN-45 cells after si-HAVCR1#1 and si-HAVCR1#2 transfection, respectively, ∗∗ p < 0.01 versus the si-con group. (b and c) The HAVCR1 protein level was evaluated using MKN-45 cells that were transfected with si-HAVCR1#1 or si-HAVCR1#2. The gray value of the protein bands was quantified. ∗∗ p < 0.01 versus the si-con group. (d) QRT-PCR analysis showed the silencing efficiency of si-HAVCR1#1 and si-HAVCR1#2 in AGS cells, ∗∗ p < 0.01 versus the si-con group. (e and f) The expression of HAVCR1 in AGS cells was detected by western blot and quantified, ∗∗ p < 0.01 versus the si-con group.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: Downregulation of HAVCR1 impaired the proliferation and colony formation of GAC cells. (a and b) A CCK-8 assay was used to measure the proliferation of MKN-45 and AGS cells. (c and d) A colony formation assay was conducted in MKN-45 and AGS cells to further determine the impact of HAVCR1 deficiency on clonogenic capability. And the number of clones was counted. ∗∗ p < 0.01 versus the si-con group.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: CCK-8 Assay, Colony Assay, Clone Assay

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: Reduction of HAVCR1 attenuated the migration and invasion of GAC cells. A wound healing assay was implemented to assess the migration of (a) MKN-45 cells and (b) AGS cells. And the distance of the wound was also quantified. (c and d) The migration and invasion of GAC cells was further detected by transwell assays. ∗∗ p < 0.01 versus the si-con group.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: Migration, Wound Healing Assay

Journal: Gastroenterology Research and Practice

Article Title: HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

doi: 10.1155/2019/6746970

Figure Lengend Snippet: Activation of MEK/ERK signaling pathway was associated with HAVCR1 expression. (a) The MEK/ERK related protein levels were examined by western blot analysis in MKN-45 cells after si-HAVCR1#1 transfection. (b) The gray values of protein bands were quantified. ∗∗ p < 0.01 versus the si-con group. (c) The effects of HAVCR1 deficiency in MEK, p-MEK, ERK, and p-ERK expression levels in AGS cells were measured by western blot. (d) The expression levels of the above proteins were quantified and normalized to GAPDH. One specific siRNA (si-HAVCR1#1) was used as an experimental group because of its higher knockdown efficiency, and nonspecific siRNA was used as a negative control group (si-con). ∗∗ p < 0.01 versus the si-con group.

Article Snippet: Membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies (dilution 1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA) against MEK (Cat no. 9126), p-MEK (Cat no. 3958), ERK (Cat no. 4695), p-ERK (Cat no. 8544), GAPDH (Cat no. 5174), and HAVCR1 (Cat no. 14971).

Techniques: Activation Assay, Expressing, Western Blot, Transfection, Knockdown, Negative Control

Journal: Translational Cancer Research

Article Title: shRNA-based PD-1 suppression preserves memory phenotype and function of CD19-targeted CAR-T cell

doi: 10.21037/tcr-2025-938

Figure Lengend Snippet: PD-1 knockdown maintains memory phenotype and reduces exhaustion after repeated antigen stimulation. (A) Schematic of in vitro repeated antigen stimulation (“stress test”) model. CAR-T cells were challenged every 48 h with RAJI-PD-L1 cells at a 1:5 E:T ratio. (B) Representative flow plots of Annexin V/7-AAD staining after repeated antigen stimulation rounds. (C) Apoptosis rates of CAR-T cells after repeated antigen exposure (n=4 donors). (D) Representative CCR7 and CD45RA expression after repeated stimulation. (E) Summarized results of memory T cell subsets: TN + TSCM (CCR7 + CD45RA + ), TCM, TEM, and TEFF (n=4 donors) after repeated stimulation. (F) Expression of memory-related genes after repeated stimulation rounds (one representative donor from three is shown; n=3 independent wells). (G) Representative flow plot of the expression of exhaustion markers (e.g., TIM-3, LAG-3) on CAR-T cells after repeated stimulation. (H) Percentages of TIM-3 + and LAG-3 + CAR-T cells after repeated stimulation (n=4 donors). (I) Expression of exhaustion-related genes after repeated stimulation rounds (one representative donor from three is shown; n=3 independent wells). (J) Cytotoxic activity of CAR-T cells after 3 rounds of antigen stimulation (one representative donor from three is shown; n=4 independent wells). Data are presented as mean ± SD. Unpaired Student’s t -test (C,E,H), two-way ANOVA followed by Sidak’s multiple-comparison test (F,I,J) is used. *, P<0.05, **, P<0.01; ***, P<0.001; ****, P<0.0001. AAD, 7-aminoactinomycin D staining; ANOVA, analysis of variance; CAR-T, chimeric antigen receptor T cell; CD19, cluster of differentiation 19; E:T, effector-to-target; PD-1, programmed cell death protein 1; PD-L1, programmed death ligand 1; SD, standard deviation; TCM, central memory T cell; TEFF, effector T cell; TEM, effector memory T cell; TN, naïve T cells; TSCM, stem cell memory T cells.

Article Snippet: TaqMan ® Gene Expression Assays (20×, Life Technologies, Carlsbad, CA, USA) was used for the detection of the following human genes: PD-1 (Hs01550088_m1), CCR7 (Hs01013469_m1), SELL (Hs00174151_m1), TCF7 (Hs01556515_m1), HAVCR2 (Hs00958618_m1), LAG3 (Hs00958444_g1), CTLA4 (Hs00175480_m1), IL2 (Hs00174114_m1), IFNG (Hs00989291_m1), granzyme B (GZMB) (Hs00188051_m1), TNF (Hs00174128_m1), and the reference gene GAPDH (Hs99999905_m1).

Techniques: Knockdown, In Vitro, Staining, Expressing, Activity Assay, Comparison, Standard Deviation