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: Cancer Immunology, Immunotherapy

Article Title: Regulatory T cells promote glioma cell stemness through TGF-β–NF-κB–IL6–STAT3 signaling

doi: 10.1007/s00262-021-02872-0

Figure Lengend Snippet: Regulatory T cells (Tregs) secrete TGF-β which promotes GSC expansion. a A multiple-cytokine kit was used to detect cytokines in the supernatants from the U251/Tregs co-culture and from the U251 and Tregs cultures alone. b The TGF-β protein level, validated using ELISA. c TGF-β expression in cells from the co-cultured system and from the U251 and Tregs cultures alone, based on reverse-transcription PCR (RT-PCR). d Immunofluorescence localization analysis of Foxp3 (red) and TGF-β (green) in glioma tissues; colocalization of Foxp3 and TGF-β is indicated in yellow. Scale bars: 200 μm. e CD133 + proportions in U251 cells co-cultured with Tregs in the presence of TGF-β-neutralizing antibody (1 μg/ml) and of control cells. f The number of spheres generated from U251 cells co-cultured with Tregs, with or without the TGF-β-neutralizing antibody (1 μg/ml). The number of spheres per field was averaged from five randomly selected fields. g Expression of cancer stemness genes CD133 , SOX2 , NESTIN , and MUSASHI1 in U251 cells co-cultured with Tregs in the presence of TGF-β-neutralizing antibody (1 μg/ml) and of control cells, determined using RT-PCR. h Sphere numbers in U251 cells transfected with shTGFBR2 and control cells with or without Tregs. The number of spheres per field was averaged from five random fields. i CD133 + proportions in shTGFBR2-U251 cells and control cells with or without Tregs, analyzed using flow cytometry. j Expression of CD133 , SOX2 , NESTIN , and MUSASHI1 in shTGFBR2-U251 cells and control cells with or without Tregs, evaluated using RT-PCR analysis. Results are based on three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: ELISAs were processed using the Human IL6 ELISA Kit (R&D Systems) and Human TGF-β1 ELISA Kit (BioLegend), according to the manufacturer’s instructions.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Control, Generated, Transfection, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy

Article Title: Regulatory T cells promote glioma cell stemness through TGF-β–NF-κB–IL6–STAT3 signaling

doi: 10.1007/s00262-021-02872-0

Figure Lengend Snippet: IL6-IL6R signaling mediates GSC properties induced by TGF-β. a Cytokine levels with or without TGF-β treatment (10 ng/ml), evaluated using a multiple-cytokine kit. b IL6 protein expression levels, validated using ELISA. c U251 cells were infected with siRNA to inhibit the expression of IL6. IL6 expression was examined by reverse-transcription PCR (RT-PCR). Expression of CD133 , SOX2 , NESTIN , and MUSASHI1 in d U251 cells transfected with either si-scramble or siIL6, following TGF-β (10 ng/ml) treatment, and in e U251 cells treated with tocilizumab (5 μg/ml) following TGF-β treatment (10 ng/ml), evaluated using RT-PCR. f IL6 expression, examined by western blotting analysis, in U251 cells transfected with lentiviral sh-IL6. g Sphere-formation capacity of U251 cells transfected with either sh-scramble or sh-IL6 following TGF-β treatment (10 ng/ml), assessed by sphere-formation assay. h CD133 , SOX2 , and NESTIN expression in U251 cells transfected with either sh-scramble or shIL6 following TGF-β treatment (10 ng/ml), assessed using western blotting analysis. Results are based on three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: ELISAs were processed using the Human IL6 ELISA Kit (R&D Systems) and Human TGF-β1 ELISA Kit (BioLegend), according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Infection, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Tube Formation Assay

Journal: Cancer Immunology, Immunotherapy

Article Title: Regulatory T cells promote glioma cell stemness through TGF-β–NF-κB–IL6–STAT3 signaling

doi: 10.1007/s00262-021-02872-0

Figure Lengend Snippet: The STAT3 signaling pathway mediates IL6-induced GSC properties. a Expression of phospho-p65 under TGF-β treatment (10 ng/ml) at different times, assessed using western blotting analysis. b U251 cells were pretreated with NF-κB inhibitor (EVP4593) for 30 min, followed by TGF-β treatment (10 ng/ml) for 30 min and 24 h. The expression of phospho-p65 and IL6 was examined using western blotting analysis. c–f : U251 cells were pretreated with Stattic (10 umol/L, 30 min), followed by IL6 treatment (10 ng/ml, 24 h). c Cells were harvested to determine the proportion of CD133 cells. d Sphere numbers, determined using sphere-formation assay. The number of spheres per field was averaged from five random fields. e Expression of CD133 , SOX2 , NESTIN , and MUSASHI , analyzed using reverse-transcription PCR. f Expression of phospho-STAT3, STAT3, CD133, SOX2, and NESTIN, assessed using western blotting analysis. Results are based on three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. NC normal control

Article Snippet: ELISAs were processed using the Human IL6 ELISA Kit (R&D Systems) and Human TGF-β1 ELISA Kit (BioLegend), according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Tube Formation Assay, Reverse Transcription, Control