Journal: ACS chemical neuroscience

Article Title: Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

doi: 10.1021/acschemneuro.7b00219

Figure Lengend Snippet: NPFF EC50 in functional calcium mobilization assays. (A) NPFF response in stable NPFF1-RD-HGA16 cells (●) and parental RD-HGA16 CHO cells (○). (B) NPFF response in stable NPFF2-RD-HGA16 cells (■) and parental RD-HGA16 CHO cells (○). Representative data from one experiment is shown and each data point is the mean ± SD of duplicate determinations.

Article Snippet: Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from PerkinElmer and used with the Lance Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates.

Techniques: Functional Assay

Journal: ACS chemical neuroscience

Article Title: Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

doi: 10.1021/acschemneuro.7b00219

Figure Lengend Snippet: Antagonist activity of compound 16 in NPFF1 (A) and NPFF2 (B) calcium mobilization functional Ke assays. (A) Concentration-response curves of NPFF alone (○) and NPFF + 5 μM final 16 (□) in stable NPFF1-RD-HGA16 cells. (B) Concentration-response curves of NPFF alone (○) and NPFF + 10 μM final 16 (□) in stable NPFF2-RD-HGA16 cells. The right shift of the NPFF curve in the presence of test compound was used to calculate Ke values as described in the Methods. Representative data from one experiment are shown and each data point is mean ± SD of duplicate determinations.

Article Snippet: Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from PerkinElmer and used with the Lance Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates.

Techniques: Activity Assay, Functional Assay, Concentration Assay

Journal: ACS chemical neuroscience

Article Title: Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

doi: 10.1021/acschemneuro.7b00219

Figure Lengend Snippet: Antagonist activity of compounds 16 and 33 in NPFF1 (A) and FF2 (B) cAMP functional Ke assays. (A) Concentration-response curves of NPFF alone (○), NPFF + 4 μM final 16 (□), and NPFF + 2 μM final 33 (◊) in stable NPFF1-CHO cells. (B) Concentration-response curves of NPFF alone (○), NPFF + 10 μM final 16 (□), and NPFF + 10 μM final 33 (◊) in stable NPFF2-CHO cells. The right shift of the NPFF curve in the presence of test compound was used to calculcate Ke values as described in the Methods. Each data point is mean ± SEM of at least N=3 conducted in duplicate.

Article Snippet: Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from PerkinElmer and used with the Lance Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates.

Techniques: Activity Assay, Functional Assay, Concentration Assay

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: Cell reports

Article Title: Chronic stress physically spares but functionally impairs innate-like invariant T cells

doi: 10.1016/j.celrep.2021.108979

Figure Lengend Snippet:

Article Snippet: Mouse: B16-F10-Red-FLuc (B16-FLuc) melanoma cells , PerkinElmer , Cat # BW124734.

Techniques: Control, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, In Situ, Selection, cDNA Synthesis, 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: 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

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 1. Identification and charac- terization of GPCR135 ligand activity in rat brain extracts. A, GPCR135 li- gand activity in different rat tissues. Ex- tracted rat tissues at the dilutions shown were used as ligands in the GTPS bind- ing assay using human GPCR135 ex- pressing cell membrane. B, molecular weight characterization of GPCR135 li- gand from rat brain extract. Crude rat brain ethanol/HCl extract was run through a HPLC gel filtration column. Fractions were collected and assayed for GPCR135 ligand activity in GTPS bind- ing assays using human GPCR135 ex- pressing cell membranes. In a parallel ex- periment, peptides and nucleotides with known molecular weights were run using the same conditions to serve as the molec- ular mass standards.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Activity Assay, Membrane, Molecular Weight, Filtration

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 2. Purification and characterization of porcine GPCR135 ligand. After extraction, fractionation, two rounds of ion exchange, and two rounds of reverse phase chromatographies, homogeneous peptide was obtained. A, the final RP-HPLC. Fractions 39–40 showed ligand activity for GPCR135 as demonstrated in GTPS binding assays. B, GTPS binding assays for GPCR135 ligand activity in the RP-HPLC fractions. C, the porcine GPCR135 ligand in fraction 39-40 was subjected to amino acid sequence analysis. The resulting amino acid sequences are aligned to human, mouse, and rat relaxin-3. All cysteine residues are highlighted in bold letters. The three amino acids in porcine GPCR135 ligand that differ from human relaxin-3 are underlined.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Purification, Extraction, Fractionation, Activity Assay, Binding Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 3. Expression, purification, and characterization of recombinant human relaxin-3. A, schematic diagram of the predicted amino acid sequence for the human relaxin-3 expression construct, relaxin-3RR. Arrows indicate an enterokinase cleavage site (DDDDK) and two furin cleavage sites (RWRR and RGRR). The amino acid R with the star (*) indicates the mutation made from the natural sequence of RGSR to RGRR, an artificial furin site in the construct. The numbers indicate the corresponding positions of the amino acid in the natural human relaxin-3 pre-propeptide. B, RP-HPLC analysis of purified recombinant human relaxin-3. The purified human relaxin-3 was run in a C-18 RP-HPLC analytical column in 0.1% trifluoroacetic acid with an acetonitrile (ACN) gradient. C, fractions from B were assayed for GPCR135 ligand activity in GTPS binding assays using GPCR135 expressing cell membranes.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Expressing, Purification, Recombinant, Sequencing, Construct, Mutagenesis, Activity Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 4. Characterization of GPCR135 using 125I-relaxin-3 as the radioligand. A, saturation isotherm binding for 125I-relaxin-3 and GPCR135. GPCR135 expressing cell membranes were used in binding assays with different concentrations of 125I-relaxin-3 as the radioligand. Nonspecific binding was determined using the same conditions in the presence of 5 M unlabeled relaxin-3. B, competition binding analysis of GPCR135. GPCR135 expressing cell membranes were used to perform radioligand binding assays with 100 pM 125I-relaxin-3 as the radioligand. Different unlabeled peptides at various concentrations were used as competitors.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Binding Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 5. Functional characterization of GPCR135 using relaxin-3 and related peptides as ligands. A, relaxin-3 stimulates 35S-GTPS binding in GPCR135 expressing cells. Different peptides were added at various concentrations to the human GPCR135 expressing cell membranes to stimulate GTPS incorporation. The specific 35S-GTPS incorporation was obtained by subtracting counts without ligand from the counts with ligand. B, inhibition of forskolin-stimulated cAMP production by relaxin-3. Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences). C, dose response of relaxin-3 inhibition of cAMP production in GPCR135 expressing cells. Chinese hamster ovary cells stably expressing GPCR135 were stimulated with different concentrations of peptides, including relaxin-3, at various concentrations. Forskolin was then added to all samples at a final concentration of 5 M. cAMP from the stimulated cells was extracted and measured using cAMP flash plates. D, relaxin-3 stimulates Ca2 mobilization in HEK293 cells co-expressing GPCR135 and Gqi5. HEK293 cells, either mock transfected (293), transfected with Gqi5 (293/Gqi5), human GPCR135 (GPCR135), or co-transfected with human GPCR135 and Gqi5 (GPCR135/Gqi5), were used for Ca2 mobilization assays. Relaxin-3 stimulated intracellular Ca2 mobilization was monitored by FLIPR.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Functional Assay, Binding Assay, Expressing, Inhibition, Stable Transfection, Control, Concentration Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 6. A, RT-PCR detection of GPCR135 and relaxin-3 mRNA expression profiles in different human tissues. The PCR products were run in 2% agarose gels, stained with ethidium bromide, and visualized under UV irradiation. In parallel, PCRs amplifying human -actin cDNA served as the controls. B–E, coronal brain sections showing expression of GPCR135 and relaxin-3 mRNA by in situ hybridization. B, autoradiogram shows that GPCR135 mRNA distribution is distinct in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). C, brightfield photomicrograph of the paraventricular nucleus showing expression of GPCR135 mRNA. Strong hybridization signal is seen as dark grains. Magnification 20. D, relaxin-3 mRNA distribution in the central gray and nucleus incertus. E, brightfield photomicrograph of central gray (CG) and nucleus incertus (NI) showing expression of relaxin-3 mRNA. Magnification 2. B and D in the figure are pseudocolor images from autoradiograms processed on the Fuji Film Bio-Imaging Analyzer System. Colors represent relative levels of hybridization densities with the rank order of red yellow green blue black.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Irradiation, In Situ Hybridization, Hybridization, Imaging

Journal:

Article Title: Expression of the nuclear transport protein importin ?-1 and its association with the neurokinin 3 receptor in the rat hypothalamus following acute hyperosmotic challenge

doi: 10.1016/j.neuroscience.2010.08.015

Figure Lengend Snippet: Western blots from CLU209 cells and rat PVN (A) showing the labeling of the neurokinin 3 receptor (NK3R, sheep anti-NK3R in CLU209, K7 anti-NK3R in rat) and importin β-1 (Imp β-1, KPNB1 labeling for both samples). RT-PCR from the CLU209 and rat PVN (B) also demonstrate that mRNA for NK3R and imp β-1 are expressed in these samples along with vasopressin (VP). The center marker shows the 300 bp, 150 bp and 50 bp bands respectively.

Article Snippet: 2.1.2 Cell Culture CLU209 cells were purchased from Cedarlane (Ontario, Canada).

Techniques: Western Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Marker

Journal:

Article Title: Expression of the nuclear transport protein importin ?-1 and its association with the neurokinin 3 receptor in the rat hypothalamus following acute hyperosmotic challenge

doi: 10.1016/j.neuroscience.2010.08.015

Figure Lengend Snippet: Quantification of the labeling density of nuclear neurokinin 3 receptor (NK3R) in the CLU209 cells following importin β-1 immuno-neutralization. Lamin B was used as an internal loading control. The decrease in the nuclear presence of the NK3R was dependent on the time of incubation with the importin β-1 antibody.

Article Snippet: 2.1.2 Cell Culture CLU209 cells were purchased from Cedarlane (Ontario, Canada).

Techniques: Labeling, Neutralization, Control, Incubation

Journal:

Article Title: Expression of the nuclear transport protein importin ?-1 and its association with the neurokinin 3 receptor in the rat hypothalamus following acute hyperosmotic challenge

doi: 10.1016/j.neuroscience.2010.08.015

Figure Lengend Snippet: Western blots from CLU209 cell homogenate following incubation with the anti importin β-1 antibody. These data show that there was no decrease in the expression levels of neurokinin 3 receptor (NK3R, sheep anti-NK3R antibody) or lamin B following incubation with the importin β-1 antibody. These data also show that there was no activation of caspases-3 or increase in cell death due to incubation with the antibody.

Article Snippet: 2.1.2 Cell Culture CLU209 cells were purchased from Cedarlane (Ontario, Canada).

Techniques: Western Blot, Incubation, Expressing, Activation Assay