Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: 4T1-Her2 cells were seeded in 96 well plates at increasing numbers and the luciferase level was determined after 2 hours. Both cell number and luciferase quantity were converted into percentage by dividing the individual figure with the maximal cell number seeded (100,000) or the luciferase reading from the well with the maximal cell number. The correlation coefficient (R) is indicated in the figure.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase

Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: Initially 10,000 4T1-Her2 cells were seeded. The supernatants were collected at the indicated times for measurement of luciferase.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase

Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: A . Cell-associated and supernatant luciferase activity during cTCR-splenocyte mediated cytolysis of 4T1-Her2 cells. cTCR-splenocytes were added to 4T1-Her2 cells at a ratio of 10∶1. Supernatants and cells were harvested at the indicated time for quantification of luciferase activity. B . 4T1-Her2 monolayer without cTCR-transduced splenocytes. C . Seventy-two h after cTCR-tranduced splenocytes were added to 4T1-Her2 monolayer.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: Mock-transduced ( A ) or cTCR-transduced ( B ) splenocytes were added to 4T1-Her2 cells at the indicated ratio. Cells were harvested 72 later for quantification of luciferase activity. C . Using the formula: % specific luc reduction = (% luc reduction from engrafted T-cell)/(% luc reduction from control T-cell)×100, the data from A and B were converted into percentage of specific luciferase release and plotted.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase, Activity Assay, Control

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: EGFR and ErbB2 concentrations, EGF-stimulated heterodimerization, and phosphorylation of EGFR and ErbB2 in various breast cancer cell lines. (A) MDA453, SKBR3, BT474, and BT20 cells were lysed with radioimmunoprecipitation assay buffer, and 20 μg of protein from each lysate were separated by 10% SDS-PAGE, transferred onto a nitrocellulose filter, and incubated with rabbit polyclonal anti-ErbB2 or anti-EGFR antibody. (B) MDA453, SKBR3, BT474, and BT20 cells were serum starved or stimulated with EGF (100 ng/ml) at 4°C for 60 min and then lysed with immunoprecipitation buffer. The total lysates were immunoprecipitated with monoclonal anti-ErbB2 antibody. Then, of the total protein from both the anti-ErbB2 immunoprecipitates and the nonprecipitated supernatants was separated by 10% SDS-PAGE, transferred onto a nitrocellulose filter, and incubated with a rabbit polyclonal anti-EGFR antibody or a mouse monoclonal anti-pTyr antibody. Bands were visualized on x-ray film by using an HRP-conjugated secondary antibody and chemiluminescence reagents.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Radio Immunoprecipitation, SDS Page, Incubation, Immunoprecipitation

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Determination by immunofluorescence analysis of ErbB2 and EGFR endocytosis after EGF stimulation. MDA453, SKBR3, BT474, and BT20 cells on glass coverslips were serum starved or stimulated with EGF (100 ng/ml) at 4°C for 60 min followed by further incubation at 37°C for 30 min. The cells were fixed with methanol and stained with mouse monoclonal anti-ErbB2 or anti-EGFR antibody followed with FITC-conjugated secondary antibody, as described in MATERIALS AND METHODS. Magnification, 200×.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Immunofluorescence, Incubation, Staining

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Relative ErbB2 and EGFR concentrations, the EGFR–ErbB2 dimerization level, and EGFR endocytosis level in various breast cancer cell lines

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques:

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Analysis by subcellular fractionation and immunoblotting of EGFR and ErbB2 endocytosis after EGF stimulation. MDA453, SKBR3, BT474, and BT20 cells were cultured in serum-free medium or incubated with EGF (100 ng/ml) at 4°C for 60 min, followed by further incubation with EGF at 37°C for 15, 30, and 60 min as indicated. The cells were fractionated into PM, EN, and Cyt fractions as described in MATERIALS AND METHODS. Proteins (10 μg) from each fraction were separated by SDS-PAGE, transferred onto a nitrocellulose filter, and incubated with rabbit polyclonal anti-ErbB2 or anti-EGFR antibody. Bands were visualized on x-ray film by using an HRP-conjugated secondary antibody and chemiluminescence reagents.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Fractionation, Western Blot, Cell Culture, Incubation, SDS Page

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Inhibition of EGFR endocytosis in BT20 cells after microinjection of the ErbB2 expression plasmid. (A) BT20 cells were microinjected with the ErbB2 expression plasmid pcDNA3.1(−)/ErbB2 (A–D) or the control plasmid pcDNA3.1(−)/Myc-His/LacZ (E–H) and incubated at 37°C for 24 h. After treatment with EGF (100 ng/ml; (A–C and E–G) or TR-EGF (100 ng/ml; D and H), the cells were fixed with methanol and stained by immunofluorescence. ErbB2 and LacZ expression-positive cells were identified with a mouse anti-ErbB2 (A, C, and D) or mouse anti-myc (B, G, and H) antibody followed by FITC-conjugated anti-mouse IgG. EGFR endocytosis was assayed with sheep polyclonal anti-EGFR antibody followed by a TRITC-conjugated anti-sheep antibody (B, C, F, and G). The endocytosis of TR-EGF was directly examined under fluorescent microscope (D and H). Magnification, 200×. (B) Quantification of inhibition of EGFR and TR-EGF endocytosis after microinjection of ErbB2 expression plasmid. Data are means ± SE of three independent experiments.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Inhibition, Expressing, Plasmid Preparation, Incubation, Staining, Immunofluorescence, Microscopy

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Analysis by subcellular fractionation and immunoblotting of EGFR and ErbB2 endocytosis after EGF stimulation in the transiently transfected 293T cells. (A) Western blot analysis of EGFR and ErbB2 expression in 293T cells transiently transfected with pcDNA3.1(−)/EGFR and/or pcDNA3.1(−)/ErbB2. (B) Determination of EGF-induced EGFR and ErbB2 endocytosis in 293T cells transfected with pcDNA3.1(−)/EGFR and/or pcDNA3.1(−)/ErbB2. 293T cells were cultured in serum-free medium or stimulated with EGF at 4°C for 60 min followed by further incubation in serum-free medium for 15 or 30 min. The cells were subcellular fractionated and immunoblotted for EGFR and/or ErbB2 as described in MATERIALS AND METHODS.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Fractionation, Western Blot, Transfection, Expressing, Cell Culture, Incubation

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Determination by immunofluorescence analysis of EGF-induced EGFR and ErbB2 endocytosis in the transiently transfected 293T cells. 293T were transiently transfected with pcDNA3.1(−)/ErbB2 (A and B), pcDNA3.1(−)/EGFR (C and D), or both (E–H). After the transfection, the cells were cultured in serum-free medium (A, C, E, and G) or incubated with EGF (100 ng/ml) at 4°C for 60 min followed by further incubation for 30 min at 37°C (B, D, F, and H). The cells were then single immunofluorescent stained for ErbB2 (A and B) and EGFR (C and D) or double immunofluorescent stained for both ErbB2 (E and F) and EGFR (G and H), as described in MATERIALS AND METHODS. Magnification, 200×.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Immunofluorescence, Transfection, Cell Culture, Incubation, Staining

Journal:

Article Title: Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ErbB2 Heterodimers in Response to EGF Stimulation

doi:

Figure Lengend Snippet: Determination by immunofluorescence analysis of EGF-induced endocytosis of an ErbB2/EGFR chimera. BT20 cells were microinjected with the chimeric ErbB2/EGFR expression plasmid pcDNA3.1(−)/ErbB2/EGFR/Myc/His and incubated at 37°C for 24 h. The cells were either untreated (A and B) or treated with EGF (100 ng/ml) at 37°C for 30 min (C and D). The cells were then fixed with methanol and stained by immunofluorescence. The endocytosis of the chimeric ErbB2/EGFR was assayed by mouse anti-myc antibody (B, G, and H) followed by FITC-conjugated anti-mouse IgG (A and C). The EGFR endocytosis was assayed with sheep polyclonal anti-EGFR antibody followed by a TRITC-conjugated anti-sheep antibody (B and D). Magnification, 200×.

Article Snippet: The supernatants, containing 1 mg of total proteins, were then incubated with 1 μg of mouse anti-ErbB2 antibody (immunoglobulin G1 [IgG1]) 9G6 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h with gentle mixing by inverting.

Techniques: Immunofluorescence, Expressing, Plasmid Preparation, Incubation, Staining

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: FASN phosphorylation by HRG in SKBR3 cells . (A) SKBR3 cells were incubated in serum-free medium overnight and then treated with 50 ng/mL heregulin (HRG) for the indicated times. FASN was immunoprecipitated and immunoblotted by the use of a monoclonal phosphotyrosine (PT66) and anti-FASN antibodies. (B) Immunoprecipitated FASN substrate was incubated with HER2 kinase and radiolabeled with (γ-P 32 )-ATP for 30 minutes at 30ºC, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by autoradiography. Recombinant HER2 kinase was used as a positive control. Western blotting for FASN and HER2 was performed to confirm that equal amounts of FASN or HER2 were used in each kinase assay.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Recombinant, Positive Control, Western Blot, Kinase Assay

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Colocalization of FASN and HER2 in breast cancer cells . Treatment of SKBR3 cells with HRG caused HER2 (red) and FASN (green) to colocalize at the cell membrane (yellow on merged images).

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Association of FASN and HER2 in breast cancer cells . SKBR3 (A) and BT474 (B) cells were treated for one hour with: 1) no agent (control); 2) 50 ng/mL HRG; 3) 1 μM lapatinib or 4) 50 ng/mL HRG plus 1 μM lapatinib. The immunoprecipitated FASN complexes were subjected to Western blotting for FASN, HER2 and PT66. (C) Whole-cell lysates from SKBR3 (left panel) and BT474 cells (right panel) were subjected to Western blotting for pHER2 (Y1248), pHER3 (Y1289), pAkt (S473), pErk1/2, HER2, HER3, Akt, Erk1/2, FASN and β-actin.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: FASN phosphorylation in breast cancer cells treated with C75 . SKBR3 (A) and BT474 (B) cells were pretreated with 10 μM C75 for five hours and then treated for one additional hour with 1) no agent (control); 2) 50 ng/mL HRG; 3) 10 μM C75; or 4) 50 ng/mL HRG plus 10 μM C75. The immunoprecipitated FASN complexes were assessed for FASN, HER2 and PT66 by Western blotting. (C) Whole-cell lysates from SKBR3 and BT474 cells were subjected to Western blotting for pHER2 (Y1248), pHER3 (Y1289), pAkt (S473), pErk1/2, HER2, HER3, Akt, Erk1/2, FASN and β-actin.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: HRG-induced invasion in HER2-positive BT474 breast cancer cells treated with lapatinib or siRNA targeting FASN . (A) BT474 cells were treated with 50 ng/mL HRG or 50 ng/mL HRG plus 0.2 μM lapatinib for 36 hours. The invaded BT474 cells were stained with crystal violet and counted under a microscope. Representative microscopic images are shown. The changes in cell invasion compared with HRG-induced cell invasion are shown in a bar graph. All experiments were done three times. A simple t -test was used to assess differences in the number of invaded cells between any two experimental conditions. *, statistically significant compared with cells treated with HRG; P < 0.05 was considered statistically significant. (B) BT474 cells transfected with scrambled siRNA or siRNA targeting FASN (si-FASN) were treated with 50 ng/mL HRG for 36 hours. The efficiency of si-FASN knockdown was confirmed by Western blotting for FASN and β-actin (lower left). The changes in cell invasion compared with the invaded scrambled siRNA-transfected untreated cells are shown as a percentage in the bar graph (lower right). All experiments were done three times. A simple t -test was used to assess differences in the number of invaded cells between any two experimental conditions. *, statistically significant compared with scrambled si-RNA-transfected cells treated with HRG; P < 0.05 was considered statistically significant.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Staining, Microscopy, Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Cell invasion and activity of MMP9 in HER2-positive BT474 breast cancer cells with suppressed FASN . (A) BT474 cells were treated with 50 ng/mL HRG or 50 ng/mL HRG plus 10 μM C75 for 36 hours. Cells shown in the microscopic image represent invasive cells. The changes in cell invasion compared with HRG-induced cell invasion are shown as a percentage in the bar graph. All experiments were done three times. A simple t -test was used to assess differences in the number of invaded cells between any two experimental conditions. *, statistically significant compared with cells treated with HRG; P < 0.05 was considered statistically significant. (B) BT474 cells were treated as described in Figures 7A, B and 8A, and the medium was harvested for zymogram assay as described in Materials and methods. MMP-9 activity is shown as the digested clear band in Coomassie blue-stained gel containing 0.2% gelatin.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Activity Assay, Staining

Journal: Breast Cancer Research : BCR

Article Title: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

doi: 10.1186/bcr2777

Figure Lengend Snippet: Regulation of cell invasion through functional interaction between FASN and HER2 in HER2-overexpressing breast cancer cells . When HRG binds to HER3, HER2 interacts with HER3 and induces tyrosine phosphorylation of HER3. The activated HER2 not only stimulates the PI3K/Akt and MAPK signaling pathways but also induces tyrosine phosphorylation of FASN and upregulation of FASN activity, leading to increased cell invasion. Suppression of FASN indirectly (for example, with lapatinib) or directly (for example, with C75 or si-FASN) inhibits invasion of HER2-overexpressing breast cancer cells.

Article Snippet: To measure kinase activity, we mixed the FASN substrate and HER2 kinase (endogenous HER2 or human recombinant HER2 (BPS Bioscience Inc., San Diego, CA, USA) as positive control) in kinase reaction buffer (10 mM MnCl 2 , 5 μC 32 P-γ- adenosine-5'-triphosphate (ATP), 20 mM Tris-Cl [pH, 7.5], 50 mM NaCl, 10 mM MgCl 2 , 1 mM NaF, 1 mM Na 3 VO 4 , 20 mM β-glycerophosphate, 1 mM dithiothreitol and 20 μM ATP) for 30 minutes at 30°C.

Techniques: Functional Assay, Activity Assay

Journal: Molecular Medicine

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: TWEAK regulated the Th17/Treg cell ratio in AC mice. ( A ) The effect of TWEAK knockdown on Th17 and Treg cell ratio in spleen of AC mice was observed by flow cytometry. ( B-C ) WB and IHC were employed to evaluate the expression levels of FoxP3 and RORγt in mice conjunctival tissue. ( D ) The effect of TWEAK knockdown on the levels of Th17 and Treg cytokines in mice spleen were evaluated by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Con + AAV-shNC; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. AC + AAV-shNC; ns, no significant difference

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Knockdown, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine

Article Title: TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice

doi: 10.1186/s10020-024-01004-5

Figure Lengend Snippet: Inhibition of Nrf2/HO-1 signaling pathway affected Th17/Treg cell ratio in AC mice with TWEAK knockdown. ( A ) The effect of Nrf2 inhibitor on Th17/Treg cell ratio in AC mice with TWEAK knockdown was assessed by flow cytometry. ( B-C ) WB and IHC assays were employed to evaluate the expression of FoxP3 and RORγt in conjunctival tissue of AC mice. ( D ) The levels of Th17 and Treg cytokines in mice spleen were detected by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. AC + AAV-shNC; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. AC + AAV-shTWEAK

Article Snippet: After deparaffinization and rehydration, tissue slides were treated with 100 μL endogenous peroxidase blockers for 10 min. Then, the slides were incubated with primary antibody against RORγt (14-6981-82, 1:200; ThermoFisher, Waltham, MA, USA), FoxP3 (BA2032-1, 1:200; Boster, Pleasanton, CA, USA), TWEAK (BM4635, 1:200; Boster), Fn14 (GTX85216; 1:200, Genetex, Alton, CA, USA), Nrf2 (PA5-27882; 1:200, ThermoFisher) and HO-1 (PA5-77833, 1:200; ThermoFisher) overnight at 4 °C.

Techniques: Inhibition, Knockdown, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay