Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Control, Plasmid Preparation, Solvent

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Immunoprecipitation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress oxidative stress–induced parthanatos. A , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 42 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001 ( versus CTX 1 mg/ml, rucaparib 0 μM cells). B , HT1080 and PARP-1 KO HT1080 were treated with CTX (1 mg/ml) for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus WT control cells), ### p < 0.001 ( versus WT CTX 1 mg/ml cells). C , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗ p < 0.01, ( versus control cells), # p < 0.05, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). D , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of Na 2 S 4 for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ## p < 0.01, ### p < 0.001, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 36 h or CHX (10 μg/ml) and TNF-α (25 μg/ml) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. F , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). G , HT1080 cells were treated with CTX (1 mg/ml) with indicated concentration of I3MT-3 for 48 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 48 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). I , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. J , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01 ( versus CTX 1 mg/ml, I3MT-3 0 μM cells). K , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.051, ∗∗∗ < 0.001 ( versus CTX 1 mg/ml, PAG 0 mM cells). L , HT1080 cells were treated with CTX (1 mg/ml) with the indicated concentration of PAG for 24 h. Dead cells were labeled with PI for 15 min and analyzed by FACS. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM cells). M , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 36 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. N , nuclear AIF expressions were quantified using Image Lab software from Bio-Rad. Graphs depict the mean ± SEM of three independent experiments. Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05 ( versus CTX 1 mg/ml, PAG 0 mM cells). All data are representative of at least three independent experiments. AIF, apoptosis-inducing factor; CHX, cycloheximide; CTX, cefotaxime; FACS, fluorescence-activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; PI, propidium iodide; PMS, phenazine methosulfate; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: MTS Assay, Control, Concentration Assay, Labeling, Western Blot, Software, Fluorescence, FACS

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS suppress the ALIS formation. A , HT1080 and p62 KO HT1080 cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. B , HT1080 cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. C , HT1080 cells were treated with CTX (1 mg/ml) and/or NAC (2 mM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. D , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. Scale bar represents 10 μm. G , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, Na 2 S 4 0 μM cells). H , HT1080 cells were treated with CTX (1 mg/ml) and/or I3MT-3 (10 μM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. I , HT1080 cells were treated with CTX (1 mg/ml) and/or PAG (5 mM) for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were treated with the indicated reagents for 24 h, then performed immunofluorescence staining with the indicated antibody, and DAPI nuclear staining. CTX (1 mg/ml). I3MT-3 (10 μM). PAG (5 mM). Scale bar represents 10 μm. K , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus CTX 1 mg/ml, I3MT-3 0 μM PAG 0 mM cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; PAG, DL-propargylglycine; NAC, N-acetylcysteine; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Western Blot, Immunofluorescence, Staining, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS disaggregate the ALIS by inducing HSP70. A , HT1080 cells were treated with the indicated reagents for 24 h and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. CTX (1 mg/ml). PAG (5 mM). I3MT-3 (20 μM). Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), ### p < 0.001, ( versus CTX 1 mg/ml, PAG 0 mM, I3MT-3 0 μM cells). B , HT1080 cells were treated with CTX (1 mg/ml) and/or Na 2 S 4 (100 μM) for 24 h and then incubated with 10 μM DCFH-DA. Quantification of ROS was calculated by detecting the fluorescence intensity of DCFH-DA. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗∗ p < 0.001, ( versus control cells), N.S. p > 0.05 ( versus CTX 1 mg/ml Na 2 S 4 0 μM cells). C , HT1080 cells were treated with the indicated reagents for 24 h, and then the detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. Bafilomycin A1 (5 nM). CTX (1 mg/ml). Na 2 S 4 (100 μM). D , HT1080 cells were treated with CTX (1 mg/ml) for 20 h and then treated with MG132 (10 μM) and/or Na 2 S 4 (100 μM) for 4 h. The detergent-soluble and detergent-insoluble fractions and whole cell lysate were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the indicated concentration of Na 2 S 4 for 24 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. G , HT1080 cells were treated with CTX (1 mg/ml) and Na 2 S 4 (100 μM) for 18 h, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with CTX (1 mg/ml) and/or rucaparib (1 μM) for 24 h, and then whole cell lysate were subjected to immunoblotting with the indicated antibodies. I , HT1080 and PARP-1 KO cells were treated with Na 2 S 4 (100 μM) for indicated period, and then whole cell lysates were subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, and then the detergent-soluble and detergent-insoluble fractions were subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with siRNA for negative control or HSP70 (HSP70 #1 or HSP70 #2). After 24 h, the cells were treated with CTX (1 mg/ml) for 24 h, then performed immunofluorescence staining with the indicated antibody, and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar represents 10 μm. L , the number of p62 and ubiquitin-colocalized puncta were quantified using Image J. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗ p < 0.05, ∗∗ p < 0.01, ( versus siRNA Ctr, CTX 1 mg/ml cells). All data are representative of at least three independent experiments. ALIS, aggresome-like induced structure; CTX, cefotaxime; HSP, heat shock protein; PAG, DL-propargylglycine; PARP-1, poly (ADP-ribose) polymerase-1; ROS, reactive oxygen species; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Incubation, Fluorescence, Control, Western Blot, Concentration Assay, Transfection, Negative Control, Immunofluorescence, Staining, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos

doi: 10.1016/j.jbc.2023.104710

Figure Lengend Snippet: RSS activate HSF1 by promoting its dissociation from HSP90. A , HT1080 cells were treated with the Na 2 S 4 (100 μM) for indicated period, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s t test; ∗∗∗ p < 0.001, ( versus control cells). B , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. C , HT1080 cells were treated with the Na 2 S 4 (100 μM) for the indicated period. Cell lysates were subjected to immunoblotting with the indicated antibodies. D , HT1080 and PARP-1 KO cells were treated with the Na 2 S 4 (100 μM) for 6 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. E , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F , HT1080 cells were treated with the Na 2 S 4 (100 μM) and KRIBB11 (10 μM) for 12 h, and then the mRNA levels were measured by quantitative real-time PCR. Data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Tukey–Kramer test; ∗∗ p < 0.01, ( versus control cells), ## p < 0.01 ( versus Na 2 S 4 100 μM, KRIBB11 0 μM cells). G , HT1080 cells were transfected with FLAG-HSP90 and/or Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. H , HT1080 cells were treated with indicated concentration of Na 2 S 4 for 4 h, then immunoprecipitated protein G-Sepharose beads with the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. I , HSP90 was immunoprecipitated using anti-HSP90 antibody with protein G beads. Beads were washed four times with PBS and then treated with Na 2 S 4 (100, 1000 μM) for 1 h. After reaction, beads were washed four times with PBS and subjected to immunoblotting with the indicated antibodies. J , HT1080 cells were transfected with FLAG-HSP90 (WT/C412A/C564A/C521A) and Myc-HSF1 plasmid for 24 h and treated with Na 2 S 4 (100 μM) for 4 h, then immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. K , HT1080 cells were transfected with FLAG-Empty or FLAG-HSP90 (WT/C521A) plasmid for 24 h and treated with Na 2 S 4 (100 μM) for indicated periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. All data are representative of at least three independent experiments. HSF, heat shock factor; HSP, heat shock protein; PARP-1, poly (ADP-ribose) polymerase-1; RSS, reactive sulfur species.

Article Snippet: HT1080 cells were transfected with 10 nM nontargeting siRNA pool (Dharmacon) as control or HSP70 siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen), according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Concentration Assay

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: CX3CR1 and HIF-1α expression by OvCa tissues: Ovarian tissues from normal and various cancer stages [well-differentiated (Stage I), moderately differentiated (Stage II), and poorly differentiated (Stage III)] were stained with anti-CX3CR1 and anti-HIF-1α antibodies. Magenta (AP) color shows CX3CR1, and Brown (DAB) color shows HIF-1α staining. An Aperio ScanScope CS system with a 40X objective captured digital images of each tissue. Representative cases are immuno-intensities of CX3CR1 and HIF-1α using image analysis Aperio ImageScope v.6.25 software

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Expressing, Staining, Software

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Expression of CX3CR1 and CX3CL1 by OvCa cells. OVCAR-3, SW 626, and TOV-112D cells were stained with FITC-conjugated or anti-CX3CR1 and PE-conjugated isotype or anti-CX3CL1 antibodies, and the expressions were quantified by flow cytometry. The gray and white histograms represent isotypes and CX3CR1/CX3CL1 fluorescence intensity, respectively

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Western blot expression of hypoxia regulatory markers in OvCa cells. Immunoblot detection of OVCAR-3, SW 626, and TOV-112D cells exposed to 3, 6, 9, or 12 h hypoxia respectively. Immunoblotting was accomplished with primary antibodies against HIF-1α, CX3CR1, EMT marker (Snail), and MMP-9. As an internal standard for equal loading, anti-GAPDH antibody was used to probe blots

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Western Blot, Expressing, Marker

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: mRNA analysis of hypoxia-induced signaling molecules in OvCa cells. Ovarian tumor cells (OVCAR-3, SW 626, and TOV-112D) were exposed to hypoxia for 3, 6, 9, or 12 h. Quantitative RT-PCR results for expressions of CX3CR1, HIF-1α, MMP9, Snail, Twist, N- and P-cadherin are shown. The data were normalized to the levels of the housekeeping gene (18 S), and the analyses were accomplished in triplicate. Data were presented as fold change in expression (± standard error); the asterisks indicate significant differences as determined by Student’s t -test (* P < 0.05; ** P < 0.01)

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Migration and invasion induced by the CX3CR1/CX3CL1 interaction in hypoxic OvCa cells. OVCAR-3, SW 626, and TOV-112D cells were cultured in the presence of CoCl 2 (150 μM) (to mimic hypoxia), and with or without CX3CL1 (chemoattractant) and KC7F2 (inhibitor) in the medium. Over 6 days, migration and invasion were assessed. ( a ) Morphology and ( b ) quantitative analysis of surface area, representing the invading cells projecting out of the spheroid into the medium when using CX3CL1. For cells under hypoxia, there was minimal growth, or they remained as aggregates when treated with or without the inhibitor, KC7F2

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Migration, Cell Culture