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: Avian Reovirus P17 Suppresses Angiogenesis by Promoting DPP4 Secretion

doi: 10.3390/cells10020259

Figure Lengend Snippet: ARV p17-mediated release of Dipeptidyl Peptidase 4 (DPP4) expression. ( A ) Wound healing assay performed after HUVECs’ overnight stimulation with conditioned medium from Mock- or ARV p17-nucleofected HUVEC cells. Confluent cell monolayers were scratched using a 200 μL pipette tip and cell migration was recorded by light microscopy 10 h after wound scratch (original magnification, 4×). The wound width was measured, and the relative wound area was calculated as the ratio of the remaining area at the 10 h time point to the 0 h starting point. Pictures are representative of one out of two independent experiments with similar results. Values are the mean ± SD of one representative experiment out of two with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001. ( B ) Tube formation assay performed with HUVECs co-cultivated for 48 h with Mock- or ARV p17-nucleofected HUVEC cells. The pictures were taken 6 h after cell seeding (original magnification, 4×). Closed rings were counted as a parameter for quantification of tube formation. Pictures are representative of one out of two independent experiments with similar results. Values are the mean ± SD of one representative experiment out of two with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001. ( C ) Angiogenesis array performed with the supernatants of Mock- or ARV p17-nucleofected cells recovered 24 h post-nucleofection. Relative pixel intensity was calculated using ImageJ software and expressed as the mean of duplicate dots. Values are representative of one experiment out of two with similar results. ( D ) Analysis of DPP4 gene expression performed using quantitative real-time PCR in Mock- and ARV p17-nucleofected cells. Analysis of real-time PCR data were performed with the 2 -DDCt method using relative quantitation study software. Quantification of DPP4 mRNA was normalized according to the internal β-actin control. Values represent the mean ± SD of one representative experiment out of two with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001. ( E ) soluble (s) DPP4 levels in supernatants of HUVECs stimulated with GST-ARV p17 for 48 h were quantified with human sDPP4 ELISA. Supernatant from GST stimulated cells was used as a negative control. Bar graph displays the concentration of DPP4 determined by absorbance quantification. Values are the mean ± SD of one representative experiment out of three independent experiments with similar results, performed in triplicates. Statistical analysis was performed by Student’s t test. *** p < 0.001.

Article Snippet: The release of DPP4 in the concentrated conditioned medium was evaluated using a human CD26 ELISA Kit (R&D systems) according to manufacturer’s instructions.

Techniques: Expressing, Wound Healing Assay, Transferring, Migration, Light Microscopy, Tube Formation Assay, Software, Gene Expression, Real-time Polymerase Chain Reaction, Quantitation Assay, Control, Enzyme-linked Immunosorbent Assay, Negative Control, Concentration Assay