Journal: Frontiers in Physiology

Article Title: Impact of Bilateral Sympathetic Stellate Ganglionectomy on TGF-β1 Signaling Pathway in Rats With Chronic Volume Overload

doi: 10.3389/fphys.2020.00375

Figure Lengend Snippet: Expressions of norepinephrine (NE) and acetylcholine (ACh) in the plasma, right ventricle (RV), and left ventricle (LV) by ELISA. (A) The expression of NE was significantly increased in the abdominal aorta-cava fistula (AV) group and decreased in the bilateral sympathetic stellate ganglionectomy after abdominal aorta-cava fistula (AD) group in the plasma, RV, and LV. (B) The expression of ACh was significantly decreased in the AV group and increased in the AD group in the RV (* P < 0.05, ** P < 0.01) (three replicate experiments were performed).

Article Snippet: Acetylcholine (ACh) concentrations of the plasma, RV, and LV were determined with the ELISA kit (BioVision, E4454-100, Milpitas, CA, United States) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: Moreover, phosphorylation levels of calcium/calmodulin-dependent protein kinase II isoforms CAMK2A and CAMK2D at Thr286 (p-CAMK2A, p-CAMK2D) were quantified using the Colorimetric Cell-Based ELISA Kit (CAMK2A/CAMK2D (Phospho-Thr286), Boster Bio, #EKC2366).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Reproductive Medicine

Article Title: The role of nGPx4 in resisting DEHP-induced DNA damage and reducing caspase‐independent cell death in male germ cells

doi: 10.5653/cerm.2024.07521

Figure Lengend Snippet: Effects of nuclear glutathione peroxidase 4 (nGPx4) overexpression on DNA damage and apoptosis-related protein expression in germ cell line (GC-1) spg cells after mono-2-ethylhexyl phthalate (MEHP) exposure. Quantitative analysis of mRNA expression levels of (A) phosphorylated H2A histone variant (γ-H2A.X), (B) Caspase-3, (C) Bcl 2-associated X (Bax), and (D) B-cell lymphoma 2 (Bcl-2), demonstrating MEHP’s impact on these genes. (E) Western blot analysis of apoptosis-related proteins. (F) Quantitative immunofluorescence analysis of the DNA damage marker γ-H2A.X. Image analysis was performed using ImageJ, and the data are presented as mean±standard deviation. Scale bar: 20 µm. ns, not significant; NC, negative control; DMSO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-2-phenylindole. a) p <0.05; b) p <0.01; c) p <0.001; d) p <0.0001 compared with the control group.

Article Snippet: GC-1 cells and mouse testicular tissues were digested and lysed in radioimmunoprecipitation assay (RIPA) buffer (AR0105; Boster) containing protease inhibitors (AR1182; Servicebio) for 30 minutes to extract proteins, including AIF, γ-H2A.X, caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2). nGPx4 and truncated AIF (tAIF) were isolated using a cytoplasmic and nuclear separation kit (AR0106; Boster).

Techniques: Over Expression, Expressing, Variant Assay, Western Blot, Immunofluorescence, Marker, Standard Deviation, Negative Control, Control

Journal: Clinical and Experimental Reproductive Medicine

Article Title: The role of nGPx4 in resisting DEHP-induced DNA damage and reducing caspase‐independent cell death in male germ cells

doi: 10.5653/cerm.2024.07521

Figure Lengend Snippet: Impact of nuclear glutathione peroxidase 4 (nGPx4) overexpression on truncated apoptosis-inducing factor (tAIF)/phosphorylated H2A histone variant (γ-H2A.X) axis activation and apoptosis in germ cell line (GC-1) spg cells following mono-2-ethylhexyl phthalate (MEHP) exposure. (A) Western blot analysis of AIF and γ-H2A.X protein expression. (B) Quantitative immunofluorescence analysis of the DNA damage marker AIF. (C) Expression of nGPx4. (D) Translocation of AIF from the cytoplasm to the nucleus. (E) Immunoprecipitation analysis from MEHP-treated and untreated groups using anti-γ-H2A.X/anti-immunoglobulin G (IgG) antibodies. (F) Quantitative mRNA expression analysis of nGPx4, AIF, and γ-H2A.X. (G, H) Flow cytometry analysis of apoptotic GC-1 cells following MEHP exposure. Data were analyzed using ImageJ and are expressed as mean±standard deviation. Scale bar: 20 µm. OE, overexpress; NC, negative control; IP, immunoprecipitation; IB, immunoblotting; DMSO, dimethyl sulfoxide; DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant; 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin-conjugated. a) p <0.01; b) p <0.001; c) p <0.0001 compared with the control group.

Article Snippet: GC-1 cells and mouse testicular tissues were digested and lysed in radioimmunoprecipitation assay (RIPA) buffer (AR0105; Boster) containing protease inhibitors (AR1182; Servicebio) for 30 minutes to extract proteins, including AIF, γ-H2A.X, caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2). nGPx4 and truncated AIF (tAIF) were isolated using a cytoplasmic and nuclear separation kit (AR0106; Boster).

Techniques: Over Expression, Variant Assay, Activation Assay, Western Blot, Expressing, Immunofluorescence, Marker, Translocation Assay, Immunoprecipitation, Flow Cytometry, Standard Deviation, Negative Control, Control

Journal: Clinical and Experimental Reproductive Medicine

Article Title: The role of nGPx4 in resisting DEHP-induced DNA damage and reducing caspase‐independent cell death in male germ cells

doi: 10.5653/cerm.2024.07521

Figure Lengend Snippet: Flow cytometry analysis of acridine orange (AO) and chromomycin A3 (CMA3) data. (A) Western blot analysis of phosphorylated H2A histone variant (γ-H2A.X). (B, C) Immunofluorescence staining of γ-H2A.X in testis sections. (D) Quantitative mRNA expression analysis of γ-H2A.X. (E, F) AO orange fluorescence intensity. (G, H) CMA3 binding positivity rates. (I) Sperm chromatin status assessed by sperm chromatin structure analysis, showing DNA fragmentation index (DFI) and high DNA staining (HDS) percentages. Data are expressed as mean±standard deviation. Scale bar: 50 µm. ns, not significant; WT, wild-type; DEHP, di(2-ethyl-hexyl) phthalate; nGPx4, nuclear glutathione peroxidase 4; NC, negative control; DAPI, 4′,6-diamidino-2-phenylindole; PerCP-A, phycoerythrincy5.5; PE-A, phycoerythrin; FITC, fluorescein isothiocyanate. a) p <0.05; b) p <0.01; c) p <0.001; d) p <0.0001 compared with the control group.

Article Snippet: GC-1 cells and mouse testicular tissues were digested and lysed in radioimmunoprecipitation assay (RIPA) buffer (AR0105; Boster) containing protease inhibitors (AR1182; Servicebio) for 30 minutes to extract proteins, including AIF, γ-H2A.X, caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2). nGPx4 and truncated AIF (tAIF) were isolated using a cytoplasmic and nuclear separation kit (AR0106; Boster).

Techniques: Flow Cytometry, Western Blot, Variant Assay, Immunofluorescence, Staining, Expressing, Fluorescence, Binding Assay, Standard Deviation, Negative Control, Control

Journal: Clinical and Experimental Reproductive Medicine

Article Title: The role of nGPx4 in resisting DEHP-induced DNA damage and reducing caspase‐independent cell death in male germ cells

doi: 10.5653/cerm.2024.07521

Figure Lengend Snippet: Di (2-ethyl-hexyl) phthalate (DEHP) exposure induces caspase-independent cell death in male mouse germ cells via the formation of the apoptosis-inducing factor (AIF)/phosphorylated H2A histone variant (γ-H2A.X) complex. After mice ingest DEHP, it is metabolized into mono-2-ethylhexyl phthalate (MEHP), which activates Bcl 2-associated X (Bax) to trigger the release of AIF from the mitochondria. The released truncated AIF (tAIF) translocates to the nucleus and forms a DNA degradation complex with γ-H2A.X/cyclophilin A (CypA), leading to DNA fragmentation. In severe cases, this process results in caspase-independent cell death. In contrast, nuclear glutathione peroxidase 4 (nGPx4) overexpression induces chromatin condensation and effectively downregulates γ-H2A.X expression, thereby mitigating caspase-independent cell death. Collectively, these mechanisms protect male mouse germ cells.

Article Snippet: GC-1 cells and mouse testicular tissues were digested and lysed in radioimmunoprecipitation assay (RIPA) buffer (AR0105; Boster) containing protease inhibitors (AR1182; Servicebio) for 30 minutes to extract proteins, including AIF, γ-H2A.X, caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2). nGPx4 and truncated AIF (tAIF) were isolated using a cytoplasmic and nuclear separation kit (AR0106; Boster).

Techniques: Variant Assay, Over Expression, Expressing

Journal: Cells

Article Title: Dual CXCR4 and E-Selectin Inhibitor, GMI-1359, Shows Anti-Bone Metastatic Effects and Synergizes with Docetaxel in Prostate Cancer Cell Intraosseous Growth

doi: 10.3390/cells9010032

Figure Lengend Snippet: In vivo experiments: comparison of the effects assessed for GMI-1359, GMI-1271 and CTCE-9908 administered at the subcutaneous 22rv1 xenograft model. ( A ) Diagram of treatments: tumour bearing animals were randomized approximately 10 days or when tumors reached 80–100 mm 3 of volume in four experimental groups as follows Group 1: mice receiving intraperitoneal (i.p.) injections of 100 µL PBS (vehicle); Group 2: mice receiving CTCE-9908 (25 mg/kg, i.p); Group 3: mice receiving GMI-1359 twice a day ip at 40 m for consecutive 14 days; Group 4: mice receiving GMI-1271 40 mg/Kg BID for consecutive 10 days; ( B ) 22rv1 tumour weights assessed after 35 days from cell injection and 28 from start of treatments. ( C ) Time To Progression analysis (median values and 95% CI); ( D ) Kaplan Meyer representation for the analyses of effects of GMI-1359, CTCE-9908 and GMI-1271; ( E ) hazard ratio values and relative significance for the different comparisons; ( F ) dot blots for CXCR4 performed on tissue extracts obtained from eight tumors/group. Two hundred µg of extracted proteins were spotted in a nitrocellulose paper. ( G ) CXCR4 densitometric units analyzed by Image J software as described in the Material and Methods section (H) ELISA determination for HECA-542 in the plasma of animal of control or treated withGMI-1359, CTCE-9908 and GMI-1271. Statistics for all comparisons are in the text and . * p < 0.05 versus respective controls (vehicle).

Article Snippet: Phospho-CXCR4 (Ser339) Colorimetric Cell-Based ELISA Kit (OKAG01771) was purchased from Aviva Systems Biology, Corp (San Diego, CA, USA).

Techniques: In Vivo, Injection, Software, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Durable multitransgene expression in vivo using systemic, nonviral DNA delivery

doi: 10.1126/sciadv.aax0217

Figure Lengend Snippet: ( A ) Serum hG-CSF levels are shown from groups of mice (three mice per group) treated with liposomes followed by either an EF1/hG-CSF or an EF1/Luc–EF1/hG-CSF expression vector or LRS (control) at indicated times. Differences between EF1/hG-CSF vector and EF1/Luc–EF1/hG-CSF vector are significant ( P < 0.05) by t test at days 1, 22, and 29. hG-CSF levels in sera from untreated control mice, as well as from mock-treated control mice (mice receiving HEDGES-mAb cDNAs), were undetectable in this hG-CSF ELISA. ( B ) Neutrophil counts are displayed from groups of mice treated as in (A). Differences are significant ( P < 0.01) at days 8, 15, and 22. ( C ) Serum hG-CSF levels from groups of mice (five per group) injected with the following DNA/liposome combinations: mCMVenh:hCMVpro/hG-CSF and MLV, mCMVenh:EF1pro/hG-CSF and SUV, and hCMVenh:hCMVpro/hG-CSF and MLV. Differences between MLV and SUV groups subsequently injected with EF1/hG-CSF are significant at days 154, 161, 182, and 197. ( D ) Hematoxylin and eosin–stained spleen (top) and bone marrow (bottom) tissue sections harvested from control mice or mice injected 582 days earlier with SUV liposomes and EF1/hG-CSF. Scale bars, 25 μm. ( E ) hG-CSF serum levels from groups of mice ( n = 3) injected 24 hours earlier with EF1/hG-CSF plasmid DNA or PCR-generated (linear, closed-end linear, or circularized) EF1/hG-CSF DNA. ( F ) hG-CSF serum levels at indicated time points from groups of mice ( n = 3) injected with EF1/hG-CSF either as plasmid DNA or as PCR-generated circularized DNA. A third group, initially injected with circularized PCR DNA underwent reinjection 35 days after the initial injection. Reinjected PCR and plasmid groups are significantly different at day 106. (A, B, C, E and F) Graphs represent mean ± SEM. One representative result from two to five independent experiments is shown. Statistical significance was tested by the two-tailed Student’s t test or ANOVA where appropriate.

Article Snippet: Serum rituximab levels were analyzed using the rituximab ELISA kit (IG-AB106; Eagle Biosciences).

Techniques: Liposomes, Expressing, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Injection, Staining, Generated, Two Tailed Test

Journal: Science Advances

Article Title: Durable multitransgene expression in vivo using systemic, nonviral DNA delivery

doi: 10.1126/sciadv.aax0217

Figure Lengend Snippet: ( A ) Alanine transaminase (ALT) and aspartate transaminase (AST) levels at 24 and 48 hours from mice injected with LRS (control) or 1000 nmol of DOTAP liposomes, followed by 100 or 120 μg of an EF1pro/hG-CSF DNA vector. Means ± SEM are displayed. ** P < 0.01 compared to control by ANOVA. ns, not significant. No mortality was observed in HEDGES-injected mice receiving up to a 120-μg dose of DNA. ( B ) hG-CSF and ( C ) rituximab serum levels 24 hours after HEDGES injection. DOTAP only received 1120 nmol DOTAP liposomes; DOTAP, neutral lipid, and Dex received 1120 nmol DOTAP liposomes incorporating 2.5 mol % Dex-Palm, 1000 nmol DMPC liposomes incorporating 5 mol % Dex-Palm, and intraperitoneal water-soluble dexamethasone (40 mg/kg) 2 hours before intravenous injections. Mice receiving liposomes also received 88 μg of DNA vector encoding hG-CSF or rituximab. ( D ) Serum ALT levels from groups of mice in (B) and (C) 24 hours after injection. * P < 0.05 and ** P < 0.01 by the two-tailed unpaired Student’s t test. One representative result from two to three independent experiments is shown.

Article Snippet: Serum rituximab levels were analyzed using the rituximab ELISA kit (IG-AB106; Eagle Biosciences).

Techniques: Injection, Control, Liposomes, Plasmid Preparation, Two Tailed Test

Journal: Science Advances

Article Title: Durable multitransgene expression in vivo using systemic, nonviral DNA delivery

doi: 10.1126/sciadv.aax0217

Figure Lengend Snippet: On day 0, groups of CD-1 mice ( n = 3) were given dexamethasone intraperitoneally 2 hours before intravenous coinjection of 1120 nmol DOTAP + Dex and 1000 nmol DMPC + Dex liposomes and 2 minutes later intravenously, and then 88 μg of mAb heavy- and light-chain cDNA vector intravenously. Control mice received LRS. ( A ) Mean ± SEM rituximab serum levels at indicated time points were measured by ELISA. mAb levels in sera from untreated control mice, as well as from mock-treated control mice (mice receiving HEDGES encoding unrelated mAb cDNAs), were undetectable in the rituximab, 5J8 mAb, and mepolizumab ELISAs, respectively. ( B ) Mean ± SEM viability of CD20+ Raji lymphoma target cells is shown following incubation with human plasma and sera from anti-CD20 or control HEDGES-treated mice isolated at indicated time points. Target Raji cells incubated with recombinant rituximab (10 mg/ml) is also presented for comparison. *** P < 0.001 and * P < 0.05 by ANOVA method. ( C ) Mean ± SEM anti–IL-5/mepolizumab serum levels are shown over time by ELISA. 5J8 anti-H1 IAV mAb serum levels were measured by ELISA ( D ) and tested in parallel for capacity to neutralize Cal09 H1N1 IAV in a microneutralization assay (MNT) at indicated time points ( E ). Serum from HEDGES-rituximab–treated mice (control) served as controls at all time points in ELISA and MNT. ( F ) Mepolizumab and hG-CSF serum levels over time following intravenous HEDGES injection of an 8.8-kb triple-cassette cDNA vector encoding hG-CSF and mepolizumab heavy- and light-chain cDNAs. Graphs represent mean ± SEM. One representative result from two to three independent experiments is shown.

Article Snippet: Serum rituximab levels were analyzed using the rituximab ELISA kit (IG-AB106; Eagle Biosciences).

Techniques: Liposomes, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Incubation, Clinical Proteomics, Isolation, Recombinant, Comparison, Microneutralization Assay, Injection

Journal: medRxiv

Article Title: Elevated levels of IL-6 in IgA nephropathy patients are induced by an epigenetically-driven mechanism triggered by viral and bacterial RNA

doi: 10.1101/2022.03.05.22271944

Figure Lengend Snippet: (A) ELISA assay showed significant increased levels of IL-6 in IgAN patients with VTRNA2-1/PKR/CREB activated pathway respect to healthy group. (B) An IL-6 increase was found in T-IgAN patients with VTRNA2-1/PKR/CREB activated pathway compared to TP group, even if not statistically significant. Both the levels of pPKR (C) and pCREB (D) significantly correlated with IL-6 levels in IgAN patients (r=0.97, p= 0.0006 and r=0.89, p=0.0064, respectively). Data are representative of 8 independent experiments (means ±SEM; * p-value < 0.05).

Article Snippet: Colorimetric Cell-Based ELISA assays were used to detect the levels of CREB/pCREB (CREB (Phospho-Ser142) Aviva Systems Biology, USA), and PKR/pPKR (PKR (Phospho-Thr258) Aviva Systems Biology, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular oral microbiology

Article Title: Tannerella forsythia produced methylglyoxal causes advanced glycation endproducts (AGEs) accumulation to trigger cytokine secretion in human monocytes

doi: 10.1111/omi.12224

Figure Lengend Snippet: MGO in the spent media from the wild-type (Tf43037) and the mutant TFM-1726 strains was estimated by ELISA (a) and HPLC (b); sterile medium (TFB) was used as control. For HPLC, MGO was measured as the 6, 7-dimethoxy-2-methylquinoxaline derivative and Hexanedione (HDO) used as an internal standard was measured as 6, 7-dimethoxy-2-methyl-3-pentylquinoxaline derivative. *P< 0.05 ***P< 0.001.

Article Snippet: MGO in spent media from the T. forsythia 43037 and TFM-1726 strains was quantified by an ELISA kit (MGO Competitive EIA Kit, LifeSpan Biosciences) as per manufacturer’s instructions and by a high-performance liquid chromatography (HPLC) based quantitative assay described by McLellan et al. 22 with minor modifications.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Sterility, Control