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  • 92
    ATCC gc 2 cells
    HIF-1α deficiency decreases apoptosis and the expression levels of apoptosis-associated proteins in <t>GC-2</t> cells. HIF-1α-deficient GC-2 cells and control cells were subjected to hypoxic conditions for 48 h. (A) Cell apoptosis was detected by flow cytometry analysis. (B) Reverse transcription-quantitative polymerase chain reaction analysis detected miR-210 expression in GC-2 cells. (C) HIF-1α and apoptosis-associated proteins were evaluated by western blot analysis. **P
    Gc 2 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    ATCC gc 2 crl 2196 cells
    The phosphorylation level of PLK1 and inhibition assay of the PLKs in <t>GC2</t> cells. A , The expression and pT210 levels of PLK1 in testes and mixed tissues of different developmental stages were analyzed by Western blot, with β actin as a loading control. Mixed tissues is the pooling of equal amount of brain, brown fat, heart, liver, lung, kidney, pancreas, and spleen from 3-week or 8-week mice. The distribution patterns of GC2 cells in different phases of the cell cycle were compared after treatment with BI2536 and/or OA for 2 h B , 4 h C , and 6 h D .
    Gc 2 Crl 2196 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher gc 2 cells
    Differentially methylated genes verified with methylation-specific PCR and real-time PCR in 50 Hz ELF-EMF exposure. (a) Representative MSP results of the three genes ( Fut11 , Olfr969A , and Tagln ) methylation in <t>GC-2</t> cells at magnetic field intensity of 1.0 mT. M: methylated primers; U: unmethylated primers. (b) Validation of mRNA expression of the three genes ( Fut11 , Olfr969A , and Tagln ) by real-time PCR. (c) Representative MSP results of the three genes ( Fut11 , Olfr969B , and Lrrc9 ) methylation in GC-2 cells at magnetic intensity of 3.0 mT. (d) Validation of mRNA expression of the three genes ( Fut11 , Olfr969B , and Lrrc9 ) by real-time PCR.
    Gc 2 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Selleck Chemicals gc 2 cells
    DBP induces PTEN promoter demethylation and global decline in DNA methylation. a The schematic diagram and sequence of representative CpG Island of the PTEN promoter region. b Representative bisulfite-sequencing PCR results for the CpG island in the PTEN promoter region in GC-1 and <t>GC-2</t> cells. Each row represents the result of a clone sequence, each column represents a CpG site of the CpG island (10 clones, 53 CpG sites). The solid spots represent methylated CpG sites, the hollow spots represent unmethylated CpG sites. c – e Percentage of methylation for the CpG island above in GC-1 and GC-2 cells in the treatment of DBP, 5-Aza-CdR, or CpG Methyltransferase. All measurements are shown as the means ± SD from three independent experiments, ** p
    Gc 2 Cells, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Becton Dickinson gc2 cells
    The phosphorylation level of PLK1 and inhibition assay of the PLKs in <t>GC2</t> cells. A , The expression and pT210 levels of PLK1 in testes and mixed tissues of different developmental stages were analyzed by Western blot, with β actin as a loading control. Mixed tissues is the pooling of equal amount of brain, brown fat, heart, liver, lung, kidney, pancreas, and spleen from 3-week or 8-week mice. The distribution patterns of GC2 cells in different phases of the cell cycle were compared after treatment with BI2536 and/or OA for 2 h B , 4 h C , and 6 h D .
    Gc2 Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HIF-1α deficiency decreases apoptosis and the expression levels of apoptosis-associated proteins in GC-2 cells. HIF-1α-deficient GC-2 cells and control cells were subjected to hypoxic conditions for 48 h. (A) Cell apoptosis was detected by flow cytometry analysis. (B) Reverse transcription-quantitative polymerase chain reaction analysis detected miR-210 expression in GC-2 cells. (C) HIF-1α and apoptosis-associated proteins were evaluated by western blot analysis. **P

    Journal: Molecular Medicine Reports

    Article Title: Hypoxia-induced miR-210 contributes to apoptosis of mouse spermatocyte GC-2 cells by targeting Kruppel-like factor 7

    doi: 10.3892/mmr.2018.9644

    Figure Lengend Snippet: HIF-1α deficiency decreases apoptosis and the expression levels of apoptosis-associated proteins in GC-2 cells. HIF-1α-deficient GC-2 cells and control cells were subjected to hypoxic conditions for 48 h. (A) Cell apoptosis was detected by flow cytometry analysis. (B) Reverse transcription-quantitative polymerase chain reaction analysis detected miR-210 expression in GC-2 cells. (C) HIF-1α and apoptosis-associated proteins were evaluated by western blot analysis. **P

    Article Snippet: GC-2 cells (a mouse pachytene spermatocyte-derived immortalized cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin).

    Techniques: Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot

    miR-210 promotes apoptosis of GC-2 cell by targeting KLF7. GC-2 cells were transfected with pcDNA3.1, miR-210 mimics, KLF7 mimics and miR-210 mimics + KLF7 mimics following hypoxic for 48 h. (A) Analysis of apoptotic cells. (B) Analysis of miR-210 expression. (C) Analysis of apoptosis-associated protein expression. *P

    Journal: Molecular Medicine Reports

    Article Title: Hypoxia-induced miR-210 contributes to apoptosis of mouse spermatocyte GC-2 cells by targeting Kruppel-like factor 7

    doi: 10.3892/mmr.2018.9644

    Figure Lengend Snippet: miR-210 promotes apoptosis of GC-2 cell by targeting KLF7. GC-2 cells were transfected with pcDNA3.1, miR-210 mimics, KLF7 mimics and miR-210 mimics + KLF7 mimics following hypoxic for 48 h. (A) Analysis of apoptotic cells. (B) Analysis of miR-210 expression. (C) Analysis of apoptosis-associated protein expression. *P

    Article Snippet: GC-2 cells (a mouse pachytene spermatocyte-derived immortalized cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin).

    Techniques: Transfection, Expressing

    Hypoxia induces apoptosis of GC-2 cells at different time points. (A) TUNEL staining results of GC-2 cells. Scale bar, 20 µm. (B) Representative graphs of flow cytometry analysis. (C) Apoptosis rate of GC-2 cells was evaluated by TUNEL staining. (D) Apoptotic GC-2 cells were measured by flow cytometry. (E) Reverse transcription-quantitative polymerase chain reaction analysis of miR-210 expression in mouse GC-2 cells subjected to hypoxia for 12, 24, 48 and 72 h. (F) Western blot analysis for HIF-1α, caspase-3, Bax and Bcl-2 protein expression in mouse GC-2 cells subjected to hypoxia for 12, 24, 48 and 72 h. *P

    Journal: Molecular Medicine Reports

    Article Title: Hypoxia-induced miR-210 contributes to apoptosis of mouse spermatocyte GC-2 cells by targeting Kruppel-like factor 7

    doi: 10.3892/mmr.2018.9644

    Figure Lengend Snippet: Hypoxia induces apoptosis of GC-2 cells at different time points. (A) TUNEL staining results of GC-2 cells. Scale bar, 20 µm. (B) Representative graphs of flow cytometry analysis. (C) Apoptosis rate of GC-2 cells was evaluated by TUNEL staining. (D) Apoptotic GC-2 cells were measured by flow cytometry. (E) Reverse transcription-quantitative polymerase chain reaction analysis of miR-210 expression in mouse GC-2 cells subjected to hypoxia for 12, 24, 48 and 72 h. (F) Western blot analysis for HIF-1α, caspase-3, Bax and Bcl-2 protein expression in mouse GC-2 cells subjected to hypoxia for 12, 24, 48 and 72 h. *P

    Article Snippet: GC-2 cells (a mouse pachytene spermatocyte-derived immortalized cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin).

    Techniques: TUNEL Assay, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    Overexpression of miR-210 induces apoptosis in GC-2 cells. (A) Measurement of transfection efficiency. (B) Analysis of the rate of apoptotic cells transfected with miR-210 mimics, mimics NC, miR-210 inhibitor and inhibitor NC following hypoxic culture. (C) Analysis of apoptosis-associated protein expression in GC-2 cells transfected with miR-210 mimics, mimics NC, miR-210 inhibitor and inhibitor NC. **P

    Journal: Molecular Medicine Reports

    Article Title: Hypoxia-induced miR-210 contributes to apoptosis of mouse spermatocyte GC-2 cells by targeting Kruppel-like factor 7

    doi: 10.3892/mmr.2018.9644

    Figure Lengend Snippet: Overexpression of miR-210 induces apoptosis in GC-2 cells. (A) Measurement of transfection efficiency. (B) Analysis of the rate of apoptotic cells transfected with miR-210 mimics, mimics NC, miR-210 inhibitor and inhibitor NC following hypoxic culture. (C) Analysis of apoptosis-associated protein expression in GC-2 cells transfected with miR-210 mimics, mimics NC, miR-210 inhibitor and inhibitor NC. **P

    Article Snippet: GC-2 cells (a mouse pachytene spermatocyte-derived immortalized cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin).

    Techniques: Over Expression, Transfection, Expressing

    The role of hsa-miR-196a-5p in cell proliferation, apoptosis and cycle cycle in vitro . ( A ) The role of hsa-miR-196a-5p in cell proliferation. An MTT cell viability assay was performed at 24 h after the transfection of GC-2 cells with equal concentrations of hsa-miR-196a-5p mimics and hsa-miR-196a-5p inhibitor. ( B ) The role of hsa-miR-196a-5p in cell apoptosis. ( C ) The role of hsa-miR-196a-5p in cell cycle. For comparison, the expression levels of hsa-miR-196a-5p mimics or hsa-miR-196a-5p inhibitor transfected cells were compared with their respective negative controls (* P

    Journal: Scientific Reports

    Article Title: Common SNP in hsa-miR-196a-2 increases hsa-miR-196a-5p expression and predisposes to idiopathic male infertility in Chinese Han population

    doi: 10.1038/srep19825

    Figure Lengend Snippet: The role of hsa-miR-196a-5p in cell proliferation, apoptosis and cycle cycle in vitro . ( A ) The role of hsa-miR-196a-5p in cell proliferation. An MTT cell viability assay was performed at 24 h after the transfection of GC-2 cells with equal concentrations of hsa-miR-196a-5p mimics and hsa-miR-196a-5p inhibitor. ( B ) The role of hsa-miR-196a-5p in cell apoptosis. ( C ) The role of hsa-miR-196a-5p in cell cycle. For comparison, the expression levels of hsa-miR-196a-5p mimics or hsa-miR-196a-5p inhibitor transfected cells were compared with their respective negative controls (* P

    Article Snippet: Cell culture and transfections GC-2 cells were obtained from American Type Culture Collection (ATCC, Manassas VA, USA) maintained in high glucose DMEM with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin under a humid atmosphere including 5% CO2 at 37 °C.

    Techniques: In Vitro, MTT Assay, Viability Assay, Transfection, Expressing

    Effects of BPA on the downstream relative genes expression ( A , B ) GC-2 cells were treated as described above, Total RNA was extracted for qPCR. c-Fos a nd Cyclin D1 mRNA expression levels were evaluated following normalization to β-actin level. The values represent the mean ± SD of the data from three independent experiments. ** P

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: Effects of BPA on the downstream relative genes expression ( A , B ) GC-2 cells were treated as described above, Total RNA was extracted for qPCR. c-Fos a nd Cyclin D1 mRNA expression levels were evaluated following normalization to β-actin level. The values represent the mean ± SD of the data from three independent experiments. ** P

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    ( A ) Comet assay. DNA-damaged induced in different ways as described above in GC-2 cells. ( B ) The histogram of relative index in Comet assay. ( C ) Flow cytometry. Apoptosis induced in different ways as described above in GC-2 cells.

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: ( A ) Comet assay. DNA-damaged induced in different ways as described above in GC-2 cells. ( B ) The histogram of relative index in Comet assay. ( C ) Flow cytometry. Apoptosis induced in different ways as described above in GC-2 cells.

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Single Cell Gel Electrophoresis, Flow Cytometry, Cytometry

    Dose-dependent inhibition of GC-2 cells growth induced by BPA GC-2 cells planted in 96-well plate were treated with 1 nM-1 μM BPA for 96 h, and control cells were treated with ethanol. Cell growth relative to that of the control was plotted against the concentrations of BPA using sigmoid curve fitting and IC 50 was determined. Here, BPA induced a dose-dependent inhibition of growth in CC-2 cells and 0.1 μM was closer to IC 50 .

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: Dose-dependent inhibition of GC-2 cells growth induced by BPA GC-2 cells planted in 96-well plate were treated with 1 nM-1 μM BPA for 96 h, and control cells were treated with ethanol. Cell growth relative to that of the control was plotted against the concentrations of BPA using sigmoid curve fitting and IC 50 was determined. Here, BPA induced a dose-dependent inhibition of growth in CC-2 cells and 0.1 μM was closer to IC 50 .

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Inhibition

    Effects of BPA on Erk1/2 activation in GC-2 cells ( A , B ) BPA-induced activation of Erk1/2. Cells were treated for 30 min with the indicated concentrations of BPA, and the phosphorylation of Erk1/2 were examined by western blot. ( C , D ) Cells were treated for the indicated times with 100 nM of BPA. For the treatment with an estrogen antagonist, cells were pretreated with 10 μM ICI for 30 min and then treated with 100 nM BPA for 5 or 15 min. Western blot analyses of the amounts of phospho-Erk1/2 (p-Erk1/2) and total Erk1/2 (T-Erk1/2) were performed on 50 μg of total proteins extracted from GC-2 cells untreated (basal) or treated as indicated. Blots are representative of three independent experiments with similar results. ** P

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: Effects of BPA on Erk1/2 activation in GC-2 cells ( A , B ) BPA-induced activation of Erk1/2. Cells were treated for 30 min with the indicated concentrations of BPA, and the phosphorylation of Erk1/2 were examined by western blot. ( C , D ) Cells were treated for the indicated times with 100 nM of BPA. For the treatment with an estrogen antagonist, cells were pretreated with 10 μM ICI for 30 min and then treated with 100 nM BPA for 5 or 15 min. Western blot analyses of the amounts of phospho-Erk1/2 (p-Erk1/2) and total Erk1/2 (T-Erk1/2) were performed on 50 μg of total proteins extracted from GC-2 cells untreated (basal) or treated as indicated. Blots are representative of three independent experiments with similar results. ** P

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Activation Assay, Western Blot

    The expression of activated Erk1/2 in GC-2 cells performed by different ways ( A , B ) Expression of GPR30 in GC-2 cells transfected with specific siRNA. GC-2 cells were transfected with 100 nM siRNA against GPR30, non-targeting (control siRNA) siRNA as indicated. At 48 h post transfection, protein was extracted and subjected to a Western blot analysis for GPR30. The levels of b-actin protein were used as loading control. Results are representative of three independent experiments. ( C , D ) Blocking of Erk1/2 signaling with PD, AG and siRNA against Gpr30 . Cells were pretreated with 10 μM of ICI, or10 μM PD+ICI or 10 μM AG+ICI for 30 min or Gpr30 siRNA for 48 h, and then treated with 0.1 μM BPA for 15 min. The activation of Erk1/2 was examined as shown in Figure 4 . D. The figure of Western blot is a representative of three independent experiments. The density of the band for p-Erk1/2 was normalized with that of total Erk1/2 and the value is shown in the graph (right). ** P

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: The expression of activated Erk1/2 in GC-2 cells performed by different ways ( A , B ) Expression of GPR30 in GC-2 cells transfected with specific siRNA. GC-2 cells were transfected with 100 nM siRNA against GPR30, non-targeting (control siRNA) siRNA as indicated. At 48 h post transfection, protein was extracted and subjected to a Western blot analysis for GPR30. The levels of b-actin protein were used as loading control. Results are representative of three independent experiments. ( C , D ) Blocking of Erk1/2 signaling with PD, AG and siRNA against Gpr30 . Cells were pretreated with 10 μM of ICI, or10 μM PD+ICI or 10 μM AG+ICI for 30 min or Gpr30 siRNA for 48 h, and then treated with 0.1 μM BPA for 15 min. The activation of Erk1/2 was examined as shown in Figure 4 . D. The figure of Western blot is a representative of three independent experiments. The density of the band for p-Erk1/2 was normalized with that of total Erk1/2 and the value is shown in the graph (right). ** P

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Expressing, Transfection, Western Blot, Blocking Assay, Activation Assay

    Inhibition of GC-2 cell proliferation by BPA went though EGFR-MAPK pathway mediated by GPR30 GC-2 cells were planted in 96-well plates with a density of 2,000 per well. Incubated with 0.1 μM BPA and examined the cell activity every 24 h using MTT assay. After 72 h treatment, the account of cells number was less compare to the basal, and 96 h later, this number disparity was more apparent ( P

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: Inhibition of GC-2 cell proliferation by BPA went though EGFR-MAPK pathway mediated by GPR30 GC-2 cells were planted in 96-well plates with a density of 2,000 per well. Incubated with 0.1 μM BPA and examined the cell activity every 24 h using MTT assay. After 72 h treatment, the account of cells number was less compare to the basal, and 96 h later, this number disparity was more apparent ( P

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Inhibition, Incubation, Activity Assay, MTT Assay

    Expression of estrogen receptors at mRNA and protein levels in the mouse GC-2 cells ( A ) ERα, ERβ and GPR30 mRNA expression in GC-2 cells was analyzed by real-time PCR. The PCR products were resolved on 1% agarose gel electrophoresis and visualized by ethidium bromide staining. β-actin was used as control gene. ( B ) Western blot analysis of ERs was performed on 30 μg of total proteins extracted from GC-2 cells. Specific antibody for ERα, ERβ and GPR30 are representative of three independent experiments with similar results. GAPDH was used as a loading control.

    Journal: Oncotarget

    Article Title: Low concentration of BPA induces mice spermatocytes apoptosis via GPR30

    doi: 10.18632/oncotarget.16923

    Figure Lengend Snippet: Expression of estrogen receptors at mRNA and protein levels in the mouse GC-2 cells ( A ) ERα, ERβ and GPR30 mRNA expression in GC-2 cells was analyzed by real-time PCR. The PCR products were resolved on 1% agarose gel electrophoresis and visualized by ethidium bromide staining. β-actin was used as control gene. ( B ) Western blot analysis of ERs was performed on 30 μg of total proteins extracted from GC-2 cells. Specific antibody for ERα, ERβ and GPR30 are representative of three independent experiments with similar results. GAPDH was used as a loading control.

    Article Snippet: Cell cultures and treatments GC-2 cells (a mouse spermatocyte-derived cell line; American Type Culture Collection, Manassas, VA) were cultured in DMEM growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (pen/strep) (Invitrogen, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Western Blot

    Effects of DES on GC-2 cell viability and proliferation. a. GC-2 cells were treated with 0~10 −4 M DES for 24, 48 or 72 h. Cell viability was measured by CCK8 assay. b. GC-2 cells were treated with the indicated concentrations of DES for 48 h. The fluorescent thymidine analog EdU was used to identify GC-2 cells by the labeling of their DNA. Hoechst-labeled nuclei was shown in blue, and EdU-labeled newborn cells were shown in red.

    Journal: PLoS ONE

    Article Title: Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

    doi: 10.1371/journal.pone.0143143

    Figure Lengend Snippet: Effects of DES on GC-2 cell viability and proliferation. a. GC-2 cells were treated with 0~10 −4 M DES for 24, 48 or 72 h. Cell viability was measured by CCK8 assay. b. GC-2 cells were treated with the indicated concentrations of DES for 48 h. The fluorescent thymidine analog EdU was used to identify GC-2 cells by the labeling of their DNA. Hoechst-labeled nuclei was shown in blue, and EdU-labeled newborn cells were shown in red.

    Article Snippet: 2 Cell Culture Mouse spermatocyte-derived GC-2 cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: CCK-8 Assay, Labeling

    Effect of DES on GC-2 cell cycle progression. GC-2 cells were treated with the indicated concentrations of DES for 48 h. Cell cycle distribution was assessed by the propidium iodide method using flow cytometry.

    Journal: PLoS ONE

    Article Title: Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

    doi: 10.1371/journal.pone.0143143

    Figure Lengend Snippet: Effect of DES on GC-2 cell cycle progression. GC-2 cells were treated with the indicated concentrations of DES for 48 h. Cell cycle distribution was assessed by the propidium iodide method using flow cytometry.

    Article Snippet: 2 Cell Culture Mouse spermatocyte-derived GC-2 cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: Flow Cytometry, Cytometry

    Effects of DES on global DNA methylation in GC-2 cells. The DNA 5-mC level was estimated by dot blot analysis. The gray values indicated DNA methylation levels.

    Journal: PLoS ONE

    Article Title: Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

    doi: 10.1371/journal.pone.0143143

    Figure Lengend Snippet: Effects of DES on global DNA methylation in GC-2 cells. The DNA 5-mC level was estimated by dot blot analysis. The gray values indicated DNA methylation levels.

    Article Snippet: 2 Cell Culture Mouse spermatocyte-derived GC-2 cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: DNA Methylation Assay, Dot Blot

    Chromosomal distributions of hypomethylated and hypermethylated genes in GC-2 cells exposed to 2×10 −5 M DES. The red indicated the promoter of some genes was hypermethylation, and the blue showed that was hypomethylation in GC-2 cells were exposed to 2×10 −5 M DES.

    Journal: PLoS ONE

    Article Title: Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

    doi: 10.1371/journal.pone.0143143

    Figure Lengend Snippet: Chromosomal distributions of hypomethylated and hypermethylated genes in GC-2 cells exposed to 2×10 −5 M DES. The red indicated the promoter of some genes was hypermethylation, and the blue showed that was hypomethylation in GC-2 cells were exposed to 2×10 −5 M DES.

    Article Snippet: 2 Cell Culture Mouse spermatocyte-derived GC-2 cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques:

    DES induces apoptosis in GC-2 cells. a. GC-2 cells were treated with the indicated concentrations of DES for 48 h. Apoptosis assay was also carried out using Hoechst 33258 staining. b. GC-2 cells were treated with the indicated DES concentrations for 48 h. Apoptosis assay was performed using flow cytometry after Annexin V-FITC/PI staining. Viable cells are shown in the lower left quadrant, early apoptotic cells are shown in the lower right quadrant, late apoptotic and necrotic cells are presented in the upper right quadrant, and nonviable necrotic cells are shown in the upper left quadrant. The data represent the mean ± SD; * P

    Journal: PLoS ONE

    Article Title: Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

    doi: 10.1371/journal.pone.0143143

    Figure Lengend Snippet: DES induces apoptosis in GC-2 cells. a. GC-2 cells were treated with the indicated concentrations of DES for 48 h. Apoptosis assay was also carried out using Hoechst 33258 staining. b. GC-2 cells were treated with the indicated DES concentrations for 48 h. Apoptosis assay was performed using flow cytometry after Annexin V-FITC/PI staining. Viable cells are shown in the lower left quadrant, early apoptotic cells are shown in the lower right quadrant, late apoptotic and necrotic cells are presented in the upper right quadrant, and nonviable necrotic cells are shown in the upper left quadrant. The data represent the mean ± SD; * P

    Article Snippet: 2 Cell Culture Mouse spermatocyte-derived GC-2 cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: Apoptosis Assay, Staining, Flow Cytometry, Cytometry

    Effects of DES on the protein expression of DNMTs in GC-2 cells. * P

    Journal: PLoS ONE

    Article Title: Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

    doi: 10.1371/journal.pone.0143143

    Figure Lengend Snippet: Effects of DES on the protein expression of DNMTs in GC-2 cells. * P

    Article Snippet: 2 Cell Culture Mouse spermatocyte-derived GC-2 cells were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: Expressing

    Overexpressed miR-26b-5p inhibits the expression of CCND2 following 50 Hz ELF-EMF exposure at 3 mT in GC-2 cells. (A) The expression of CCND2 was significantly lower than that of the sham group at a magnetic intensity of 2 mT and was significantly

    Journal: Cell Cycle

    Article Title: Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields

    doi: 10.1080/15384101.2015.1120924

    Figure Lengend Snippet: Overexpressed miR-26b-5p inhibits the expression of CCND2 following 50 Hz ELF-EMF exposure at 3 mT in GC-2 cells. (A) The expression of CCND2 was significantly lower than that of the sham group at a magnetic intensity of 2 mT and was significantly

    Article Snippet: Mouse spermatocyte-derived GC-2 cells (GC-2 cells) were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: Expressing

    50 Hz ELF-EMF exposure alters the expression of miR-26b-5p. (A) MicroRNA-gene network in GC-2 cells between the sham and exposure groups at magnetic field intensity of 3 mT. The vertical axis corresponds to changed miRNAs, and the horizontal axis

    Journal: Cell Cycle

    Article Title: Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields

    doi: 10.1080/15384101.2015.1120924

    Figure Lengend Snippet: 50 Hz ELF-EMF exposure alters the expression of miR-26b-5p. (A) MicroRNA-gene network in GC-2 cells between the sham and exposure groups at magnetic field intensity of 3 mT. The vertical axis corresponds to changed miRNAs, and the horizontal axis

    Article Snippet: Mouse spermatocyte-derived GC-2 cells (GC-2 cells) were purchased from the American Tissue Culture Collection (ATCC, Rockville, MD, USA).

    Techniques: Expressing

    The phosphorylation level of PLK1 and inhibition assay of the PLKs in GC2 cells. A , The expression and pT210 levels of PLK1 in testes and mixed tissues of different developmental stages were analyzed by Western blot, with β actin as a loading control. Mixed tissues is the pooling of equal amount of brain, brown fat, heart, liver, lung, kidney, pancreas, and spleen from 3-week or 8-week mice. The distribution patterns of GC2 cells in different phases of the cell cycle were compared after treatment with BI2536 and/or OA for 2 h B , 4 h C , and 6 h D .

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis *

    doi: 10.1074/mcp.M114.039073

    Figure Lengend Snippet: The phosphorylation level of PLK1 and inhibition assay of the PLKs in GC2 cells. A , The expression and pT210 levels of PLK1 in testes and mixed tissues of different developmental stages were analyzed by Western blot, with β actin as a loading control. Mixed tissues is the pooling of equal amount of brain, brown fat, heart, liver, lung, kidney, pancreas, and spleen from 3-week or 8-week mice. The distribution patterns of GC2 cells in different phases of the cell cycle were compared after treatment with BI2536 and/or OA for 2 h B , 4 h C , and 6 h D .

    Article Snippet: Mouse GC2 cells (ATCC catalog number CRL-2196, Manassas, VA, USA) were cultured for 16 h at 37 °C in 5% CO2 in DMEM culture medium supplemented with 10% fetal bovine serum, 0.5% penicillin and streptomycin.

    Techniques: Inhibition, Expressing, Western Blot, Mouse Assay

    Differentially methylated genes verified with methylation-specific PCR and real-time PCR in 50 Hz ELF-EMF exposure. (a) Representative MSP results of the three genes ( Fut11 , Olfr969A , and Tagln ) methylation in GC-2 cells at magnetic field intensity of 1.0 mT. M: methylated primers; U: unmethylated primers. (b) Validation of mRNA expression of the three genes ( Fut11 , Olfr969A , and Tagln ) by real-time PCR. (c) Representative MSP results of the three genes ( Fut11 , Olfr969B , and Lrrc9 ) methylation in GC-2 cells at magnetic intensity of 3.0 mT. (d) Validation of mRNA expression of the three genes ( Fut11 , Olfr969B , and Lrrc9 ) by real-time PCR.

    Journal: BioMed Research International

    Article Title: Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2

    doi: 10.1155/2015/237183

    Figure Lengend Snippet: Differentially methylated genes verified with methylation-specific PCR and real-time PCR in 50 Hz ELF-EMF exposure. (a) Representative MSP results of the three genes ( Fut11 , Olfr969A , and Tagln ) methylation in GC-2 cells at magnetic field intensity of 1.0 mT. M: methylated primers; U: unmethylated primers. (b) Validation of mRNA expression of the three genes ( Fut11 , Olfr969A , and Tagln ) by real-time PCR. (c) Representative MSP results of the three genes ( Fut11 , Olfr969B , and Lrrc9 ) methylation in GC-2 cells at magnetic intensity of 3.0 mT. (d) Validation of mRNA expression of the three genes ( Fut11 , Olfr969B , and Lrrc9 ) by real-time PCR.

    Article Snippet: These results showed that the mRNA expression of selected gene was consistent with the Affymetrix Array in GC-2 cells ( ).

    Techniques: Methylation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing

    Clustering of differentially expressed genes across 50 Hz ELF-EMF exposure and control group (a) and the differentially regulated genes in GC-2 cells at magnetic field intensity of 1 mT and 3 mT were validated by real-time PCR (b). Gene expression data are presented in a matrix format. Each row represents an individual gene, and each column corresponds to an exposure group, with red indicating upregulation and green indicating downregulation. Black and gray indicate unchanged expression and missing value, respectively.

    Journal: BioMed Research International

    Article Title: Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2

    doi: 10.1155/2015/237183

    Figure Lengend Snippet: Clustering of differentially expressed genes across 50 Hz ELF-EMF exposure and control group (a) and the differentially regulated genes in GC-2 cells at magnetic field intensity of 1 mT and 3 mT were validated by real-time PCR (b). Gene expression data are presented in a matrix format. Each row represents an individual gene, and each column corresponds to an exposure group, with red indicating upregulation and green indicating downregulation. Black and gray indicate unchanged expression and missing value, respectively.

    Article Snippet: These results showed that the mRNA expression of selected gene was consistent with the Affymetrix Array in GC-2 cells ( ).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Network analysis of dynamic gene expression in GC-2 cells at magnetic field intensity of 1 mT (a) and 3 mT (b). The red dot stands for upregulated genes, the blue dot is downregulated genes, and the lilac dot stands for the connection gene.

    Journal: BioMed Research International

    Article Title: Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2

    doi: 10.1155/2015/237183

    Figure Lengend Snippet: Network analysis of dynamic gene expression in GC-2 cells at magnetic field intensity of 1 mT (a) and 3 mT (b). The red dot stands for upregulated genes, the blue dot is downregulated genes, and the lilac dot stands for the connection gene.

    Article Snippet: These results showed that the mRNA expression of selected gene was consistent with the Affymetrix Array in GC-2 cells ( ).

    Techniques: Expressing

    Quantitative analysis of global DNA methylation in GC-2 cells. DNA methylation in GC-2 cells was lower than the sham-exposure group at magnetic field intensity of 1 mT and was higher than the sham-exposure group at 2 mT and 3 mT exposure to 50 Hz ELF-EMF.

    Journal: BioMed Research International

    Article Title: Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2

    doi: 10.1155/2015/237183

    Figure Lengend Snippet: Quantitative analysis of global DNA methylation in GC-2 cells. DNA methylation in GC-2 cells was lower than the sham-exposure group at magnetic field intensity of 1 mT and was higher than the sham-exposure group at 2 mT and 3 mT exposure to 50 Hz ELF-EMF.

    Article Snippet: These results showed that the mRNA expression of selected gene was consistent with the Affymetrix Array in GC-2 cells ( ).

    Techniques: DNA Methylation Assay

    Effect of ELF-EMF electromagnetic field exposures on the mRNA and protein of DNMT1, DNMT3a, and DNMT3b in GC-2 cells. (a) The expression of DNMT1 was significantly lower than the sham-exposure group at magnetic field intensity of 1 mT and 2 mT and was significantly higher than the sham-exposure group at 3 mT. (b) The expression of DNMT3a decreased exposure to 50 Hz ELF-EMF exposure compared with the sham-exposure group at magnetic field intensity of 2 mT. (c) DNMT3b expression was significantly lower than the sham-exposure group at magnetic intensity of 1 mT and 2 mT and significantly higher than the sham-exposure group at 3 mT. (d) The protein expression of DNMT1 decreased at magnetic field intensity of 1 mT and increased at 3 mT.

    Journal: BioMed Research International

    Article Title: Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2

    doi: 10.1155/2015/237183

    Figure Lengend Snippet: Effect of ELF-EMF electromagnetic field exposures on the mRNA and protein of DNMT1, DNMT3a, and DNMT3b in GC-2 cells. (a) The expression of DNMT1 was significantly lower than the sham-exposure group at magnetic field intensity of 1 mT and 2 mT and was significantly higher than the sham-exposure group at 3 mT. (b) The expression of DNMT3a decreased exposure to 50 Hz ELF-EMF exposure compared with the sham-exposure group at magnetic field intensity of 2 mT. (c) DNMT3b expression was significantly lower than the sham-exposure group at magnetic intensity of 1 mT and 2 mT and significantly higher than the sham-exposure group at 3 mT. (d) The protein expression of DNMT1 decreased at magnetic field intensity of 1 mT and increased at 3 mT.

    Article Snippet: These results showed that the mRNA expression of selected gene was consistent with the Affymetrix Array in GC-2 cells ( ).

    Techniques: Expressing

    DBP induces PTEN promoter demethylation and global decline in DNA methylation. a The schematic diagram and sequence of representative CpG Island of the PTEN promoter region. b Representative bisulfite-sequencing PCR results for the CpG island in the PTEN promoter region in GC-1 and GC-2 cells. Each row represents the result of a clone sequence, each column represents a CpG site of the CpG island (10 clones, 53 CpG sites). The solid spots represent methylated CpG sites, the hollow spots represent unmethylated CpG sites. c – e Percentage of methylation for the CpG island above in GC-1 and GC-2 cells in the treatment of DBP, 5-Aza-CdR, or CpG Methyltransferase. All measurements are shown as the means ± SD from three independent experiments, ** p

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: DBP induces PTEN promoter demethylation and global decline in DNA methylation. a The schematic diagram and sequence of representative CpG Island of the PTEN promoter region. b Representative bisulfite-sequencing PCR results for the CpG island in the PTEN promoter region in GC-1 and GC-2 cells. Each row represents the result of a clone sequence, each column represents a CpG site of the CpG island (10 clones, 53 CpG sites). The solid spots represent methylated CpG sites, the hollow spots represent unmethylated CpG sites. c – e Percentage of methylation for the CpG island above in GC-1 and GC-2 cells in the treatment of DBP, 5-Aza-CdR, or CpG Methyltransferase. All measurements are shown as the means ± SD from three independent experiments, ** p

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: DNA Methylation Assay, Sequencing, Methylation Sequencing, Polymerase Chain Reaction, Methylation

    miR-29b and DNMT3b are required for DBP-induced PTEN demethylation. a DNMT1, DNMT3a and DNMT3b were detected using qRT-PCR in GC-2 cells after treated with DBP. b Representative WB images showing the expression of DNMT1, DNMT3a, and DNMT3b in GC-2 cells after treated with DBP. c , d qRT-PCR ( c ) and WB ( d ) results showed the expression of DNMT3b and PTEN in the treatment of si-NC or si-DNMT3b in GC-2 cells. e , f qRT-PCR ( e ) and WB ( f ) results showed the expression of DNMT3b and PTEN in GC-2 cells after DNMT3b overexpression. g Percentage of methylation for the CpG island of PTEN in GC-2 cells in the treatment of DMSO, DBP, vector, or DNMT3b overexpression. h miR-29b expression was detected by qRT-PCR in GC-2 cells after treated with DBP. i qRT-PCR showed the expression of miR-29b, DNMT3b, and PTEN in the treatment of miR-NC, miR-29b-mimic, or miR-29b-inhibitor in GC-2 cells. j The position of the binding sites was numbered relative to the first nucleotide of the 3′-UTR. Mutations were introduced into DNMT3b 3′-UTR that matched the seed region of miR-29b as shown in DNMT3b Mu. Luciferase activity was detected using dual-luciferase assay in GC-2 cells co-transfected with luciferase constructs containing the DNMT3b Wt or Mu 3′-UTR and miR-29b mimics or scrambled oligonucleotides as the negative control. k qRT-PCR showed the expression of DNMT3b in the treatment of DMSO, DBP, miR-NC, or miR-29b-inhibitor. All measurements are shown as the means ± SD from three independent experiments, **** p

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: miR-29b and DNMT3b are required for DBP-induced PTEN demethylation. a DNMT1, DNMT3a and DNMT3b were detected using qRT-PCR in GC-2 cells after treated with DBP. b Representative WB images showing the expression of DNMT1, DNMT3a, and DNMT3b in GC-2 cells after treated with DBP. c , d qRT-PCR ( c ) and WB ( d ) results showed the expression of DNMT3b and PTEN in the treatment of si-NC or si-DNMT3b in GC-2 cells. e , f qRT-PCR ( e ) and WB ( f ) results showed the expression of DNMT3b and PTEN in GC-2 cells after DNMT3b overexpression. g Percentage of methylation for the CpG island of PTEN in GC-2 cells in the treatment of DMSO, DBP, vector, or DNMT3b overexpression. h miR-29b expression was detected by qRT-PCR in GC-2 cells after treated with DBP. i qRT-PCR showed the expression of miR-29b, DNMT3b, and PTEN in the treatment of miR-NC, miR-29b-mimic, or miR-29b-inhibitor in GC-2 cells. j The position of the binding sites was numbered relative to the first nucleotide of the 3′-UTR. Mutations were introduced into DNMT3b 3′-UTR that matched the seed region of miR-29b as shown in DNMT3b Mu. Luciferase activity was detected using dual-luciferase assay in GC-2 cells co-transfected with luciferase constructs containing the DNMT3b Wt or Mu 3′-UTR and miR-29b mimics or scrambled oligonucleotides as the negative control. k qRT-PCR showed the expression of DNMT3b in the treatment of DMSO, DBP, miR-NC, or miR-29b-inhibitor. All measurements are shown as the means ± SD from three independent experiments, **** p

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Methylation, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Negative Control

    DBP induces reproductive toxicity via AKT pathway and PTEN methylation might be involved in it in male mice offsprings. a Bisulfite sequencing PCR results for the CpG island in the PTEN promoter region in DBP administration male mice offsprings. b Quantitative analysis for the results of global methylation in DBP administration group. c qRT-PCR was used to detect miR-29b, DNMT3b, and PTEN levels in DBP administration group. d Representative WB images showing the protein levels of DNMT3b, PTEN and p-AKT in DBP administration group. e – h Pearson correlation test results showing the correlation between PTEN mRNA level and miR-29b level ( e ) and between PTEN mRNA level and DNMT3b mRNA level ( f ) and between DNMT3b mRNA level and miR-29b level ( g ) and between PTEN methylation and p-AKT protein abundances ( h ). i Representative hematoxylin and eosin (h e) staining showed the testicular morphology after DBP treatment, SC79 treatment, or MK-2206 treatment. Representative immunohistochemistry (IHC) images showing BrdU, TUNEL, 8-OHdG, and γ-H2AX positive areas in testicular tissues after DBP treatment, SC79 treatment or MK-2206 treatment. Scale bar, 50 μm. j Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR (S2448), Bax, Bcl-2, cleaved caspase-3, γ-H2AX in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, or MK-2206 treatment. Protein loading is indicated by β-actin. All measurements are shown as the means ± SD from three independent experiments, **** p

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: DBP induces reproductive toxicity via AKT pathway and PTEN methylation might be involved in it in male mice offsprings. a Bisulfite sequencing PCR results for the CpG island in the PTEN promoter region in DBP administration male mice offsprings. b Quantitative analysis for the results of global methylation in DBP administration group. c qRT-PCR was used to detect miR-29b, DNMT3b, and PTEN levels in DBP administration group. d Representative WB images showing the protein levels of DNMT3b, PTEN and p-AKT in DBP administration group. e – h Pearson correlation test results showing the correlation between PTEN mRNA level and miR-29b level ( e ) and between PTEN mRNA level and DNMT3b mRNA level ( f ) and between DNMT3b mRNA level and miR-29b level ( g ) and between PTEN methylation and p-AKT protein abundances ( h ). i Representative hematoxylin and eosin (h e) staining showed the testicular morphology after DBP treatment, SC79 treatment, or MK-2206 treatment. Representative immunohistochemistry (IHC) images showing BrdU, TUNEL, 8-OHdG, and γ-H2AX positive areas in testicular tissues after DBP treatment, SC79 treatment or MK-2206 treatment. Scale bar, 50 μm. j Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR (S2448), Bax, Bcl-2, cleaved caspase-3, γ-H2AX in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, or MK-2206 treatment. Protein loading is indicated by β-actin. All measurements are shown as the means ± SD from three independent experiments, **** p

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: Methylation, Mouse Assay, Methylation Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemistry, TUNEL Assay

    DBP induces apoptosis, proliferation inhibition and DNA damage in germs. a , b Cell viability was detected by CCK-8 proliferation assay in GC-1 ( a ) and GC-2 ( b ) cells after treated with different concentrations of DBP. c – e Early apoptotic percentage was determined by flow cytometry in GC-1 ( d ) and GC-2 ( e ) cells after treated with DBP. f – i Representative immunofluorescence (IF) images showing the number of EdU- ( f ), TUNEL- ( g ), 8-OHdG- ( h ), and γ-H2AX ( i ) -positive cells in GC-1 and GC-2 cells after treated with DBP. Scale bar, 20 μm. All measurements are shown as the means ± SD from three independent experiments, ** p

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: DBP induces apoptosis, proliferation inhibition and DNA damage in germs. a , b Cell viability was detected by CCK-8 proliferation assay in GC-1 ( a ) and GC-2 ( b ) cells after treated with different concentrations of DBP. c – e Early apoptotic percentage was determined by flow cytometry in GC-1 ( d ) and GC-2 ( e ) cells after treated with DBP. f – i Representative immunofluorescence (IF) images showing the number of EdU- ( f ), TUNEL- ( g ), 8-OHdG- ( h ), and γ-H2AX ( i ) -positive cells in GC-1 and GC-2 cells after treated with DBP. Scale bar, 20 μm. All measurements are shown as the means ± SD from three independent experiments, ** p

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: Inhibition, CCK-8 Assay, Proliferation Assay, Flow Cytometry, Cytometry, Immunofluorescence, TUNEL Assay

    AKT pathway is downregulated in DBP-treated germ cells. a Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR in GC-1, and GC-2 cells after treated with DBP. Protein loading is indicated by β-actin. b Representative WB images showing the protein levels of Bax, Bcl-2, cleaved caspase-3, and γ-H2AX in GC-1 and GC-2 cells after treated with DBP. Protein loading is indicated by β-actin. c Quantitative analysis of Bax/Bcl-2 ratio for Fig. 3b . d Quantitative analysis for Fig. 3a . e Quantitative analysis for Fig. 3b . All measurements are shown as the means ± SD from three independent experiments, *** p

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: AKT pathway is downregulated in DBP-treated germ cells. a Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR in GC-1, and GC-2 cells after treated with DBP. Protein loading is indicated by β-actin. b Representative WB images showing the protein levels of Bax, Bcl-2, cleaved caspase-3, and γ-H2AX in GC-1 and GC-2 cells after treated with DBP. Protein loading is indicated by β-actin. c Quantitative analysis of Bax/Bcl-2 ratio for Fig. 3b . d Quantitative analysis for Fig. 3a . e Quantitative analysis for Fig. 3b . All measurements are shown as the means ± SD from three independent experiments, *** p

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: Western Blot

    DBP-mediated AKT activity by regulating PTEN expression in germ cells. a qRT-PCR was used to detect the efficiency of PTEN knockdown in GC-1 and GC-2 cells. b qRT-PCR was used to detect PTEN expression after treated with DBP in GC-1 and GC-2 cells. c , d GC-1 ( c ) and GC-2 ( d ) cells were transfected with PTEN siRNA or treated with DBP alone, and together, respectively. And then samples were analyzed for PTEN, AKT, p-AKT, PI3K, and p-PI3K expression. All measurements are shown as the means ± SD from three independent experiments, **** p

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: DBP-mediated AKT activity by regulating PTEN expression in germ cells. a qRT-PCR was used to detect the efficiency of PTEN knockdown in GC-1 and GC-2 cells. b qRT-PCR was used to detect PTEN expression after treated with DBP in GC-1 and GC-2 cells. c , d GC-1 ( c ) and GC-2 ( d ) cells were transfected with PTEN siRNA or treated with DBP alone, and together, respectively. And then samples were analyzed for PTEN, AKT, p-AKT, PI3K, and p-PI3K expression. All measurements are shown as the means ± SD from three independent experiments, **** p

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Transfection

    DBP induces cytotoxicity dependent on inhibiting AKT pathway. a Representative IF images showing the number of EdU, TUNEL, 8-OHdG, and γ-H2AX positive cells in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, AKT knockdown, or MK-2206 treatment. Scale bar, 20 μm. b Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR, Bax, Bcl-2, cleaved caspase-3, and γ-H2AX in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, AKT knockdown, or MK-2206 treatment. Protein loading is indicated by β-actin. All measurements are shown as the means ± SD from three independent experiments

    Journal: Cell Death & Disease

    Article Title: Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

    doi: 10.1038/s41419-019-1547-8

    Figure Lengend Snippet: DBP induces cytotoxicity dependent on inhibiting AKT pathway. a Representative IF images showing the number of EdU, TUNEL, 8-OHdG, and γ-H2AX positive cells in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, AKT knockdown, or MK-2206 treatment. Scale bar, 20 μm. b Representative WB images showing the protein levels of AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, p-mTOR, Bax, Bcl-2, cleaved caspase-3, and γ-H2AX in GC-1 and GC-2 cells after DBP treatment, SC79 treatment, AKT knockdown, or MK-2206 treatment. Protein loading is indicated by β-actin. All measurements are shown as the means ± SD from three independent experiments

    Article Snippet: GC-1 and GC-2 cells were pretreated with MK-2206 at 3 μM according to manufacturer’s instructions (Selleck, USA).

    Techniques: TUNEL Assay, Western Blot

    The phosphorylation level of PLK1 and inhibition assay of the PLKs in GC2 cells. A , The expression and pT210 levels of PLK1 in testes and mixed tissues of different developmental stages were analyzed by Western blot, with β actin as a loading control. Mixed tissues is the pooling of equal amount of brain, brown fat, heart, liver, lung, kidney, pancreas, and spleen from 3-week or 8-week mice. The distribution patterns of GC2 cells in different phases of the cell cycle were compared after treatment with BI2536 and/or OA for 2 h B , 4 h C , and 6 h D .

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis *

    doi: 10.1074/mcp.M114.039073

    Figure Lengend Snippet: The phosphorylation level of PLK1 and inhibition assay of the PLKs in GC2 cells. A , The expression and pT210 levels of PLK1 in testes and mixed tissues of different developmental stages were analyzed by Western blot, with β actin as a loading control. Mixed tissues is the pooling of equal amount of brain, brown fat, heart, liver, lung, kidney, pancreas, and spleen from 3-week or 8-week mice. The distribution patterns of GC2 cells in different phases of the cell cycle were compared after treatment with BI2536 and/or OA for 2 h B , 4 h C , and 6 h D .

    Article Snippet: GC2 cells was stained with PI (BD Biosciences, San Diego, CA) and analyzed with a BD FACS Calibur flow cytometry system (BD Biosciences).

    Techniques: Inhibition, Expressing, Western Blot, Mouse Assay