cleaved caspase 3 antibody Search Results


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    Novus Biologicals cleaved caspase 3
    The outcomes of MTX and/or QBS treatments on cleaved <t>caspase-3</t> immunoreactivity in the kidney sections of the treated rats from the indicated groups. Upper panel: Representative immunostaining images showing mean immunoexpression levels of cleaved caspase-3 of kidney tissue sections from 6 rats in each of control ( a ), QBS ( b ), MTX ( c ) & QBS + MTX ( d ) group. Lower panel: Histograms showing the quantitative analysis of the mean area percentage of cleaved caspase-3 immunohistochemical expression in the examined groups. Each bar represents mean ± SEM of 6 animals in each group ( N = 6). Statistical analysis was carried out using chi-squared
    Cleaved Caspase 3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    R&D Systems cleaved caspase 3
    CircHIPK3 is up-regulated in HG-stimulated podocyte and participates in HG-induced podocyte injury. A Summarized data of qRT-PCR showing the relative levels of circHIPK3 in HPC treated with 5.5 mmol/L glucose (NG), 40 mmol/L glucose (HG), or mannitol (34.5 mmol/L mannitol plus 5.5 mmol/L glucose) conditions for indicated time. B qRT-PCR assays were conducted to detect the amount of circHIPK3 and HIPK3 mRNA in podocytes after actinomycin D treatment. C Total RNAs were digested with RNase R followed by qRT-PCR detection of circHIPK3 and HIPK3 mRNA expression levels. D Identification of circHIPK3 cytoplasmic and nuclear distribution by RNA FISH in HPC using a cy3-labled junction specific antisense probe (red), with the nuclei staining with DAPI (blue). The 18S and U6 were applied as positive controls. Scale bar, 10 μm. E Identification of circHIPK3 cytoplasmic and nuclear distribution by qRT-PCR analysis in HPC. GAPDH and U6 were used as cytoplasm and nuclear control, respectively. F Validation of lentivirus-mediated circHIPK3 overexpression and knockdown efficiencies by qRT-PCR analysis in HPC cells. G HPC cells with different treatments were stained with 7-AAD and Annexin V-PE, and analyzed by flow cytometry to evaluate the apoptosis rate. Quantification of the apoptotic cells was showed at right panel (n=3). H Representative Western blot gel images (left panel) and summarized data (right panel) showing the relative protein levels of Cleaved <t>Caspase-3,</t> Bax, as well as Desmin in podocytes with different treatments. GAPDH served as loading control (n=4). I The effect of circHIPK3 knockdown on HG-induced HPC apoptosis and the quantification data (n=3). J Representative Western blot gel images (left panel) and summarized data showing the relative protein levels of the indicated proteins in HPC with different treatments. GAPDH served as loading control (n=3). Data are expressed as mean ± SD of three or four independent experiments. One-way ANOVA was used for comparison among multiple groups. Student's t-test was employed for comparisons between two groups. * P < 0.05; ** P < 0.01; ns means no significant.
    Cleaved Caspase 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cleaved caspase 3/product/R&D Systems
    Average 95 stars, based on 1 article reviews
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    99
    Cell Signaling Technology Inc anti cleaved caspase3
    CircHIPK3 is up-regulated in HG-stimulated podocyte and participates in HG-induced podocyte injury. A Summarized data of qRT-PCR showing the relative levels of circHIPK3 in HPC treated with 5.5 mmol/L glucose (NG), 40 mmol/L glucose (HG), or mannitol (34.5 mmol/L mannitol plus 5.5 mmol/L glucose) conditions for indicated time. B qRT-PCR assays were conducted to detect the amount of circHIPK3 and HIPK3 mRNA in podocytes after actinomycin D treatment. C Total RNAs were digested with RNase R followed by qRT-PCR detection of circHIPK3 and HIPK3 mRNA expression levels. D Identification of circHIPK3 cytoplasmic and nuclear distribution by RNA FISH in HPC using a cy3-labled junction specific antisense probe (red), with the nuclei staining with DAPI (blue). The 18S and U6 were applied as positive controls. Scale bar, 10 μm. E Identification of circHIPK3 cytoplasmic and nuclear distribution by qRT-PCR analysis in HPC. GAPDH and U6 were used as cytoplasm and nuclear control, respectively. F Validation of lentivirus-mediated circHIPK3 overexpression and knockdown efficiencies by qRT-PCR analysis in HPC cells. G HPC cells with different treatments were stained with 7-AAD and Annexin V-PE, and analyzed by flow cytometry to evaluate the apoptosis rate. Quantification of the apoptotic cells was showed at right panel (n=3). H Representative Western blot gel images (left panel) and summarized data (right panel) showing the relative protein levels of Cleaved <t>Caspase-3,</t> Bax, as well as Desmin in podocytes with different treatments. GAPDH served as loading control (n=4). I The effect of circHIPK3 knockdown on HG-induced HPC apoptosis and the quantification data (n=3). J Representative Western blot gel images (left panel) and summarized data showing the relative protein levels of the indicated proteins in HPC with different treatments. GAPDH served as loading control (n=3). Data are expressed as mean ± SD of three or four independent experiments. One-way ANOVA was used for comparison among multiple groups. Student's t-test was employed for comparisons between two groups. * P < 0.05; ** P < 0.01; ns means no significant.
    Anti Cleaved Caspase3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cleaved caspase3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
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    94
    Cell Signaling Technology Inc caspase 3
    Endothelial cells of MTH1-overexpressing tumors are more sensitive to MTH1 inhibition. (A) Endothelial cells from AB1, AE17, and AE17 MTH1–overexpressing tumors (TECs) and normal endothelial cells (NECs) from lung tissue were isolated, and mRNA levels of Mth1 was quantified by real-time PCR. Data are presented as mean ± SEM, n = 3 for each group. *P < 0.05 compared with NECs by 2-tailed Students’ t test. (B) Endothelial cells of AB1, AE17, and AE17 MTH1–overexpressing tumors from mice treated with vehicle or TH1579 were isolated using magnetic beads bearing anti-CD31 antibody. TECs were fixed, permeabilized, and stained <t>for</t> <t>caspase-3</t> in order to measure apoptotic cells using flow cytometry. Data are presented as mean ± SEM, n = 3 for each vehicle and n = 4 for each TH1579 group. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (C) Isolated TECs and NECs from AB1, AE17, and AE17 MTH1–overexpressing mesotheliomas were seeded at 6 × 103 cells/well in 96-well plates and subsequently treated with escalating doses of TH1579 (1–1000 μM). Cell viability was determined by XTT reduction. Data are presented as mean ± SEM, n = 6 for each group. *P < 0.05 compared with vehicle by 2-tailed Students’ t test. #P < 0.05 compared with TECs by 2-tailed Students’ t test. (D) Alternatively, the aforementioned isolated TECs and NECs were serum starved for 4 hours and challenged to migrate toward full medium. Data are presented as mean ± SEM. n = 3 for both groups of AB1 and AE17 NECs, AE17 TECs, AE17 MTH1–overexpressing (AE17mth1over) TECs; n = 4 for each group of AB1 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (E) The aforementioned isolated TECs and NECs were challenged to form capillary-like tubes de novo on Matrigel. Data are presented as mean ± SEM, n = 3 for both groups of AB1 NECs, n = 5 for both groups of AE17 NEC and AE17mth1over TECs, n = 4 for each group of AB1 and AE17 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test.
    Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    caspase 3 - by Bioz Stars, 2024-07
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    99
    Cell Signaling Technology Inc anti cleaved caspase 3
    Endothelial cells of MTH1-overexpressing tumors are more sensitive to MTH1 inhibition. (A) Endothelial cells from AB1, AE17, and AE17 MTH1–overexpressing tumors (TECs) and normal endothelial cells (NECs) from lung tissue were isolated, and mRNA levels of Mth1 was quantified by real-time PCR. Data are presented as mean ± SEM, n = 3 for each group. *P < 0.05 compared with NECs by 2-tailed Students’ t test. (B) Endothelial cells of AB1, AE17, and AE17 MTH1–overexpressing tumors from mice treated with vehicle or TH1579 were isolated using magnetic beads bearing anti-CD31 antibody. TECs were fixed, permeabilized, and stained <t>for</t> <t>caspase-3</t> in order to measure apoptotic cells using flow cytometry. Data are presented as mean ± SEM, n = 3 for each vehicle and n = 4 for each TH1579 group. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (C) Isolated TECs and NECs from AB1, AE17, and AE17 MTH1–overexpressing mesotheliomas were seeded at 6 × 103 cells/well in 96-well plates and subsequently treated with escalating doses of TH1579 (1–1000 μM). Cell viability was determined by XTT reduction. Data are presented as mean ± SEM, n = 6 for each group. *P < 0.05 compared with vehicle by 2-tailed Students’ t test. #P < 0.05 compared with TECs by 2-tailed Students’ t test. (D) Alternatively, the aforementioned isolated TECs and NECs were serum starved for 4 hours and challenged to migrate toward full medium. Data are presented as mean ± SEM. n = 3 for both groups of AB1 and AE17 NECs, AE17 TECs, AE17 MTH1–overexpressing (AE17mth1over) TECs; n = 4 for each group of AB1 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (E) The aforementioned isolated TECs and NECs were challenged to form capillary-like tubes de novo on Matrigel. Data are presented as mean ± SEM, n = 3 for both groups of AB1 NECs, n = 5 for both groups of AE17 NEC and AE17mth1over TECs, n = 4 for each group of AB1 and AE17 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test.
    Anti Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cleaved caspase 3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cleaved caspase 3 - by Bioz Stars, 2024-07
    99/100 stars
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    Image Search Results


    The outcomes of MTX and/or QBS treatments on cleaved caspase-3 immunoreactivity in the kidney sections of the treated rats from the indicated groups. Upper panel: Representative immunostaining images showing mean immunoexpression levels of cleaved caspase-3 of kidney tissue sections from 6 rats in each of control ( a ), QBS ( b ), MTX ( c ) & QBS + MTX ( d ) group. Lower panel: Histograms showing the quantitative analysis of the mean area percentage of cleaved caspase-3 immunohistochemical expression in the examined groups. Each bar represents mean ± SEM of 6 animals in each group ( N = 6). Statistical analysis was carried out using chi-squared

    Journal: Journal of Pharmaceutical Health Care and Sciences

    Article Title: Quillaja saponin mitigates methotrexate-provoked renal injury; insight into Nrf-2/Keap-1 pathway modulation with suppression of oxidative stress and inflammation

    doi: 10.1186/s40780-024-00330-4

    Figure Lengend Snippet: The outcomes of MTX and/or QBS treatments on cleaved caspase-3 immunoreactivity in the kidney sections of the treated rats from the indicated groups. Upper panel: Representative immunostaining images showing mean immunoexpression levels of cleaved caspase-3 of kidney tissue sections from 6 rats in each of control ( a ), QBS ( b ), MTX ( c ) & QBS + MTX ( d ) group. Lower panel: Histograms showing the quantitative analysis of the mean area percentage of cleaved caspase-3 immunohistochemical expression in the examined groups. Each bar represents mean ± SEM of 6 animals in each group ( N = 6). Statistical analysis was carried out using chi-squared "χ 2 " test; a P < 0.05, vs. normal control animals, b P < 0.05, vs. MTX-treated animals

    Article Snippet: After blocking non-specific protein binding, sections were washed by PBS and incubated overnight (at 4 °C) with the primary rabbit antibody against rat; cleaved caspase-3 (NB100-56113) from Novus Biologicals , (dilution 1:1000), or Bcl-2 (ab194583) from Abcam Co (Cambridge, MA, USA), (dilution 1:100).

    Techniques: Immunostaining, Immunohistochemical staining, Expressing

    The postulated mechanisms of Quillaja saponaria bark saponin (QBS) in alleviating methotrexate (MTX)-induced renal toxicity in rats. QBS administration mediates reno-protection by adjusting redox state of the renal microenvironment at a higher reduction potential. This renders a powerful conservancy of the renal tissues against MTX, as confirmed by the improved kidney histology & function profile; significant fall in the serum non-protein-nitrogenous components (BUN & creatinine). Such QBS-mediated protection is suggested to be maintained via attenuation of MTX-induced renal; OS (↑GSH/↓MDA/↓NO x → ↓ROS), & inflammation signaling (↓TNF-α/↑Nrf-2/↓Keap-1), with consequent suppression of cell death signaling (↑Bcl-2/↓ cleaved caspase-3)

    Journal: Journal of Pharmaceutical Health Care and Sciences

    Article Title: Quillaja saponin mitigates methotrexate-provoked renal injury; insight into Nrf-2/Keap-1 pathway modulation with suppression of oxidative stress and inflammation

    doi: 10.1186/s40780-024-00330-4

    Figure Lengend Snippet: The postulated mechanisms of Quillaja saponaria bark saponin (QBS) in alleviating methotrexate (MTX)-induced renal toxicity in rats. QBS administration mediates reno-protection by adjusting redox state of the renal microenvironment at a higher reduction potential. This renders a powerful conservancy of the renal tissues against MTX, as confirmed by the improved kidney histology & function profile; significant fall in the serum non-protein-nitrogenous components (BUN & creatinine). Such QBS-mediated protection is suggested to be maintained via attenuation of MTX-induced renal; OS (↑GSH/↓MDA/↓NO x → ↓ROS), & inflammation signaling (↓TNF-α/↑Nrf-2/↓Keap-1), with consequent suppression of cell death signaling (↑Bcl-2/↓ cleaved caspase-3)

    Article Snippet: After blocking non-specific protein binding, sections were washed by PBS and incubated overnight (at 4 °C) with the primary rabbit antibody against rat; cleaved caspase-3 (NB100-56113) from Novus Biologicals , (dilution 1:1000), or Bcl-2 (ab194583) from Abcam Co (Cambridge, MA, USA), (dilution 1:100).

    Techniques:

    CircHIPK3 is up-regulated in HG-stimulated podocyte and participates in HG-induced podocyte injury. A Summarized data of qRT-PCR showing the relative levels of circHIPK3 in HPC treated with 5.5 mmol/L glucose (NG), 40 mmol/L glucose (HG), or mannitol (34.5 mmol/L mannitol plus 5.5 mmol/L glucose) conditions for indicated time. B qRT-PCR assays were conducted to detect the amount of circHIPK3 and HIPK3 mRNA in podocytes after actinomycin D treatment. C Total RNAs were digested with RNase R followed by qRT-PCR detection of circHIPK3 and HIPK3 mRNA expression levels. D Identification of circHIPK3 cytoplasmic and nuclear distribution by RNA FISH in HPC using a cy3-labled junction specific antisense probe (red), with the nuclei staining with DAPI (blue). The 18S and U6 were applied as positive controls. Scale bar, 10 μm. E Identification of circHIPK3 cytoplasmic and nuclear distribution by qRT-PCR analysis in HPC. GAPDH and U6 were used as cytoplasm and nuclear control, respectively. F Validation of lentivirus-mediated circHIPK3 overexpression and knockdown efficiencies by qRT-PCR analysis in HPC cells. G HPC cells with different treatments were stained with 7-AAD and Annexin V-PE, and analyzed by flow cytometry to evaluate the apoptosis rate. Quantification of the apoptotic cells was showed at right panel (n=3). H Representative Western blot gel images (left panel) and summarized data (right panel) showing the relative protein levels of Cleaved Caspase-3, Bax, as well as Desmin in podocytes with different treatments. GAPDH served as loading control (n=4). I The effect of circHIPK3 knockdown on HG-induced HPC apoptosis and the quantification data (n=3). J Representative Western blot gel images (left panel) and summarized data showing the relative protein levels of the indicated proteins in HPC with different treatments. GAPDH served as loading control (n=3). Data are expressed as mean ± SD of three or four independent experiments. One-way ANOVA was used for comparison among multiple groups. Student's t-test was employed for comparisons between two groups. * P < 0.05; ** P < 0.01; ns means no significant.

    Journal: International Journal of Biological Sciences

    Article Title: Regulation of Podocyte Injury by CircHIPK3/FUS Complex in Diabetic Kidney Disease

    doi: 10.7150/ijbs.75994

    Figure Lengend Snippet: CircHIPK3 is up-regulated in HG-stimulated podocyte and participates in HG-induced podocyte injury. A Summarized data of qRT-PCR showing the relative levels of circHIPK3 in HPC treated with 5.5 mmol/L glucose (NG), 40 mmol/L glucose (HG), or mannitol (34.5 mmol/L mannitol plus 5.5 mmol/L glucose) conditions for indicated time. B qRT-PCR assays were conducted to detect the amount of circHIPK3 and HIPK3 mRNA in podocytes after actinomycin D treatment. C Total RNAs were digested with RNase R followed by qRT-PCR detection of circHIPK3 and HIPK3 mRNA expression levels. D Identification of circHIPK3 cytoplasmic and nuclear distribution by RNA FISH in HPC using a cy3-labled junction specific antisense probe (red), with the nuclei staining with DAPI (blue). The 18S and U6 were applied as positive controls. Scale bar, 10 μm. E Identification of circHIPK3 cytoplasmic and nuclear distribution by qRT-PCR analysis in HPC. GAPDH and U6 were used as cytoplasm and nuclear control, respectively. F Validation of lentivirus-mediated circHIPK3 overexpression and knockdown efficiencies by qRT-PCR analysis in HPC cells. G HPC cells with different treatments were stained with 7-AAD and Annexin V-PE, and analyzed by flow cytometry to evaluate the apoptosis rate. Quantification of the apoptotic cells was showed at right panel (n=3). H Representative Western blot gel images (left panel) and summarized data (right panel) showing the relative protein levels of Cleaved Caspase-3, Bax, as well as Desmin in podocytes with different treatments. GAPDH served as loading control (n=4). I The effect of circHIPK3 knockdown on HG-induced HPC apoptosis and the quantification data (n=3). J Representative Western blot gel images (left panel) and summarized data showing the relative protein levels of the indicated proteins in HPC with different treatments. GAPDH served as loading control (n=3). Data are expressed as mean ± SD of three or four independent experiments. One-way ANOVA was used for comparison among multiple groups. Student's t-test was employed for comparisons between two groups. * P < 0.05; ** P < 0.01; ns means no significant.

    Article Snippet: Primary antibodies included: Desmin (ab32362, abcam), IGF2BP1 (ab184305, abcam), IGF2BP3 (ab225697, abcam), CORO1A (ab203698, abcam), BAX (50599-2-Ig, Proteintech), DYKDDDDK Tag (20543-1-AP, Proteintech), IGF2BP2 (11601-1-AP, Proteintech), FUS (11570-1-AP, Proteintech), Cleaved Caspase-3 (Asp175, R&D), Polyclonal anti-podocin (p0372, Sigma-Aldrich), and EDA2R (sc-377423, Santa).

    Techniques: Quantitative RT-PCR, Expressing, Staining, Over Expression, Flow Cytometry, Western Blot

    CircHIPK3 promotes podocyte damage by upregulating EDA2R. A Clustered heatmap of significant differentially expressed mRNAs in HPC transfected with circHIPK3 overexpression lentivirus versus control lentivirus. Each sample contained a mixture of three repeats. B Schematic flowchart showed the overlapping of circHIPK3-regulated mRNAs identified by RNA-seq in HPC cells and published GSE142025 dataset in human DKD (filtered by fold change > 1.5 or < -1.5 and adjust p-value < 0.05). C Expression levels of indicated mRNAs in HPC cells with circHIPK3 overexpression. D Expression levels of indicated mRNAs in HPC cells under HG (40 mmol/L) stimulation. E Gene set enrichment analysis (GSEA) of GEO datasets (GSE142025) showing that higher EDA2R expression is significantly associated with apoptosis in DKD. F HPC cells with different treatments were stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry to evaluate cell apoptosis rate. Summarized was showed at right panel (n = 3). G, H Western blot analysis of EDA2R, Bax, and Cleaved Caspase-3 in HPC with different treatments (n=3). I Nephroseq expression data for EDA2R in db/m mice glomeruli (n=8) and db/db mice glomeruli (n=9). J qRT-PCR assays were conducted to detect circHIPK3 and EDA2R expression in STZ-induced diabetic mouse glomeruli (n=6) after five months diabetes induction, in comparison with the control mouse glomeruli (n=6). Expression values were normalized to ACTB mRNA. K Correlation analysis revealed positive correlation between the levels of circHIPK3 and EDA2R mRNA from the diabetic mouse glomeruli. L qRT-PCR analysis of circHIPK3 and EDA2R expression levels in renal biopsy samples from patients with DKD (n = 3 for control, n = 3 for patients with DKD). Data are shown as mean ± SD of three independent experiments. Student's t-test was employed for comparisons between two groups. One-way ANOVA was used for comparison among multiple groups. Correlation between circHIPK3 and EDA2R expression was analyzed by Pearson's correlation. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Journal: International Journal of Biological Sciences

    Article Title: Regulation of Podocyte Injury by CircHIPK3/FUS Complex in Diabetic Kidney Disease

    doi: 10.7150/ijbs.75994

    Figure Lengend Snippet: CircHIPK3 promotes podocyte damage by upregulating EDA2R. A Clustered heatmap of significant differentially expressed mRNAs in HPC transfected with circHIPK3 overexpression lentivirus versus control lentivirus. Each sample contained a mixture of three repeats. B Schematic flowchart showed the overlapping of circHIPK3-regulated mRNAs identified by RNA-seq in HPC cells and published GSE142025 dataset in human DKD (filtered by fold change > 1.5 or < -1.5 and adjust p-value < 0.05). C Expression levels of indicated mRNAs in HPC cells with circHIPK3 overexpression. D Expression levels of indicated mRNAs in HPC cells under HG (40 mmol/L) stimulation. E Gene set enrichment analysis (GSEA) of GEO datasets (GSE142025) showing that higher EDA2R expression is significantly associated with apoptosis in DKD. F HPC cells with different treatments were stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry to evaluate cell apoptosis rate. Summarized was showed at right panel (n = 3). G, H Western blot analysis of EDA2R, Bax, and Cleaved Caspase-3 in HPC with different treatments (n=3). I Nephroseq expression data for EDA2R in db/m mice glomeruli (n=8) and db/db mice glomeruli (n=9). J qRT-PCR assays were conducted to detect circHIPK3 and EDA2R expression in STZ-induced diabetic mouse glomeruli (n=6) after five months diabetes induction, in comparison with the control mouse glomeruli (n=6). Expression values were normalized to ACTB mRNA. K Correlation analysis revealed positive correlation between the levels of circHIPK3 and EDA2R mRNA from the diabetic mouse glomeruli. L qRT-PCR analysis of circHIPK3 and EDA2R expression levels in renal biopsy samples from patients with DKD (n = 3 for control, n = 3 for patients with DKD). Data are shown as mean ± SD of three independent experiments. Student's t-test was employed for comparisons between two groups. One-way ANOVA was used for comparison among multiple groups. Correlation between circHIPK3 and EDA2R expression was analyzed by Pearson's correlation. * P < 0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: Primary antibodies included: Desmin (ab32362, abcam), IGF2BP1 (ab184305, abcam), IGF2BP3 (ab225697, abcam), CORO1A (ab203698, abcam), BAX (50599-2-Ig, Proteintech), DYKDDDDK Tag (20543-1-AP, Proteintech), IGF2BP2 (11601-1-AP, Proteintech), FUS (11570-1-AP, Proteintech), Cleaved Caspase-3 (Asp175, R&D), Polyclonal anti-podocin (p0372, Sigma-Aldrich), and EDA2R (sc-377423, Santa).

    Techniques: Transfection, Over Expression, RNA Sequencing Assay, Expressing, Staining, Flow Cytometry, Western Blot, Quantitative RT-PCR

    CircHIPK3 facilitates the expression of EDA2R through FUS. A Summarized data showing the level of cell apoptosis determined by flow cytometric analysis in podocytes with different treatments (n=3). B qRT-PCR analysis of EDA2R mRNA expression levels in HPC cells transfected with Lv-NC or Lv-circHIPK3 and those co-transfected with si-FUS#3 or si-scram. C, D Western blot analysis of EDA2R, Bax, and Cleaved Caspase-3 in HPC with different treatments (n=3). E The predicted sequence of FUS-binding motif as identified by MatrixREDUCE. F The predicted FUS binding site at the promoter of the evolutionarily conserved region of EDA2R in human, mouse and rat, indicated by red and underlined. G Chromatin immunoprecipitation (ChIP) assays using FUS antibody indicating that FUS could bind to promoter of EDA2R gene in HPC cells. IgG was applied as negative control. H, I Schematic model of mutation (H) and sequencing of mutation (I). J Luciferase activity of the reporter vector containing the wide type (WT) or mutant (Mut) promoter of EDA2R was determined after co-transfection with control or FUS expressing plasmids in HPC cells. K ChIP analysis showed that the interaction between EDA2R gene promoter and FUS protein in HPC cells with different treatments. L qRT-PCR analysis of EDA2R mRNA expression levels in HPC cells with indicated treatments. M , N Representative Western blot gel images (M) and summarized data (N) showing the effect of circHIPK3 knockdown on the protein level of EDA2R in HPC cells treated with HG (40mmol/L) (n=3). Values are expressed as mean ± SD of three or four independent experiments. One-way ANOVA was used for comparison among multiple groups. Student's t-test was employed for comparisons between two groups. * P < 0.05, ** P < 0.01; ns represents no significant.

    Journal: International Journal of Biological Sciences

    Article Title: Regulation of Podocyte Injury by CircHIPK3/FUS Complex in Diabetic Kidney Disease

    doi: 10.7150/ijbs.75994

    Figure Lengend Snippet: CircHIPK3 facilitates the expression of EDA2R through FUS. A Summarized data showing the level of cell apoptosis determined by flow cytometric analysis in podocytes with different treatments (n=3). B qRT-PCR analysis of EDA2R mRNA expression levels in HPC cells transfected with Lv-NC or Lv-circHIPK3 and those co-transfected with si-FUS#3 or si-scram. C, D Western blot analysis of EDA2R, Bax, and Cleaved Caspase-3 in HPC with different treatments (n=3). E The predicted sequence of FUS-binding motif as identified by MatrixREDUCE. F The predicted FUS binding site at the promoter of the evolutionarily conserved region of EDA2R in human, mouse and rat, indicated by red and underlined. G Chromatin immunoprecipitation (ChIP) assays using FUS antibody indicating that FUS could bind to promoter of EDA2R gene in HPC cells. IgG was applied as negative control. H, I Schematic model of mutation (H) and sequencing of mutation (I). J Luciferase activity of the reporter vector containing the wide type (WT) or mutant (Mut) promoter of EDA2R was determined after co-transfection with control or FUS expressing plasmids in HPC cells. K ChIP analysis showed that the interaction between EDA2R gene promoter and FUS protein in HPC cells with different treatments. L qRT-PCR analysis of EDA2R mRNA expression levels in HPC cells with indicated treatments. M , N Representative Western blot gel images (M) and summarized data (N) showing the effect of circHIPK3 knockdown on the protein level of EDA2R in HPC cells treated with HG (40mmol/L) (n=3). Values are expressed as mean ± SD of three or four independent experiments. One-way ANOVA was used for comparison among multiple groups. Student's t-test was employed for comparisons between two groups. * P < 0.05, ** P < 0.01; ns represents no significant.

    Article Snippet: Primary antibodies included: Desmin (ab32362, abcam), IGF2BP1 (ab184305, abcam), IGF2BP3 (ab225697, abcam), CORO1A (ab203698, abcam), BAX (50599-2-Ig, Proteintech), DYKDDDDK Tag (20543-1-AP, Proteintech), IGF2BP2 (11601-1-AP, Proteintech), FUS (11570-1-AP, Proteintech), Cleaved Caspase-3 (Asp175, R&D), Polyclonal anti-podocin (p0372, Sigma-Aldrich), and EDA2R (sc-377423, Santa).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Mutagenesis, Luciferase, Activity Assay, Plasmid Preparation, Cotransfection

    Knockdown of circHIPK3 in mouse podocyte attenuates HG-induced apoptosis through regulating EDA2R expression. A Expression levels of circHIPK3 and HIPK3 in MPC-5 cells cultured in media containing 5.5 mmol/L glucose (NG), 40 mmol/L glucose (HG), or mannitol/glucose (34.5 mmol/L mannitol plus 5.5 mmol/L glucose) conditions for 48h. B, C The effects of circHIPK3 knockdown on apoptosis in MPC-5 cells induced by HG treatment, and the quantification data (n=3). D The amino acid sequence of the highly conserved zinc finger (ZnF) domine displaying 100% identity between human and mouse. E RIP (left) and qRT-PCR (right) assays using FUS antibody showing the interaction between circHIPK3 and FUS protein in MPC-5 cells transfected with lentivirus carrying circHIPK3 shRNA or shRNA scram. The IgG-bound RNA was taken as a negative control. F ChIP analysis showing the interaction between EDA2R gene promoter and FUS protein in MPC-5 cells with different treatments. G qRT-PCR showing the expression of EDA2R in MPC-5 cells transfected with Lv-shcircHIPK3 or Lv-shRNA scram under HG (40mmol/L) treatment. Expression values were normalized to ACTB mRNA (n = 3). H, I Representative Western blot gel documents showing the relative protein levels of EDA2R, Desmin, Bax, and Cleaved Caspase-3 in MPC-5 cells with different treatments (n=3). Data are expressed as mean ± SD of three independent experiments. One-way ANOVA was used for comparison among multiple groups. * P < 0.05, ** P < 0.01.

    Journal: International Journal of Biological Sciences

    Article Title: Regulation of Podocyte Injury by CircHIPK3/FUS Complex in Diabetic Kidney Disease

    doi: 10.7150/ijbs.75994

    Figure Lengend Snippet: Knockdown of circHIPK3 in mouse podocyte attenuates HG-induced apoptosis through regulating EDA2R expression. A Expression levels of circHIPK3 and HIPK3 in MPC-5 cells cultured in media containing 5.5 mmol/L glucose (NG), 40 mmol/L glucose (HG), or mannitol/glucose (34.5 mmol/L mannitol plus 5.5 mmol/L glucose) conditions for 48h. B, C The effects of circHIPK3 knockdown on apoptosis in MPC-5 cells induced by HG treatment, and the quantification data (n=3). D The amino acid sequence of the highly conserved zinc finger (ZnF) domine displaying 100% identity between human and mouse. E RIP (left) and qRT-PCR (right) assays using FUS antibody showing the interaction between circHIPK3 and FUS protein in MPC-5 cells transfected with lentivirus carrying circHIPK3 shRNA or shRNA scram. The IgG-bound RNA was taken as a negative control. F ChIP analysis showing the interaction between EDA2R gene promoter and FUS protein in MPC-5 cells with different treatments. G qRT-PCR showing the expression of EDA2R in MPC-5 cells transfected with Lv-shcircHIPK3 or Lv-shRNA scram under HG (40mmol/L) treatment. Expression values were normalized to ACTB mRNA (n = 3). H, I Representative Western blot gel documents showing the relative protein levels of EDA2R, Desmin, Bax, and Cleaved Caspase-3 in MPC-5 cells with different treatments (n=3). Data are expressed as mean ± SD of three independent experiments. One-way ANOVA was used for comparison among multiple groups. * P < 0.05, ** P < 0.01.

    Article Snippet: Primary antibodies included: Desmin (ab32362, abcam), IGF2BP1 (ab184305, abcam), IGF2BP3 (ab225697, abcam), CORO1A (ab203698, abcam), BAX (50599-2-Ig, Proteintech), DYKDDDDK Tag (20543-1-AP, Proteintech), IGF2BP2 (11601-1-AP, Proteintech), FUS (11570-1-AP, Proteintech), Cleaved Caspase-3 (Asp175, R&D), Polyclonal anti-podocin (p0372, Sigma-Aldrich), and EDA2R (sc-377423, Santa).

    Techniques: Expressing, Cell Culture, Sequencing, Quantitative RT-PCR, Transfection, shRNA, Negative Control, Western Blot

    Endothelial cells of MTH1-overexpressing tumors are more sensitive to MTH1 inhibition. (A) Endothelial cells from AB1, AE17, and AE17 MTH1–overexpressing tumors (TECs) and normal endothelial cells (NECs) from lung tissue were isolated, and mRNA levels of Mth1 was quantified by real-time PCR. Data are presented as mean ± SEM, n = 3 for each group. *P < 0.05 compared with NECs by 2-tailed Students’ t test. (B) Endothelial cells of AB1, AE17, and AE17 MTH1–overexpressing tumors from mice treated with vehicle or TH1579 were isolated using magnetic beads bearing anti-CD31 antibody. TECs were fixed, permeabilized, and stained for caspase-3 in order to measure apoptotic cells using flow cytometry. Data are presented as mean ± SEM, n = 3 for each vehicle and n = 4 for each TH1579 group. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (C) Isolated TECs and NECs from AB1, AE17, and AE17 MTH1–overexpressing mesotheliomas were seeded at 6 × 103 cells/well in 96-well plates and subsequently treated with escalating doses of TH1579 (1–1000 μM). Cell viability was determined by XTT reduction. Data are presented as mean ± SEM, n = 6 for each group. *P < 0.05 compared with vehicle by 2-tailed Students’ t test. #P < 0.05 compared with TECs by 2-tailed Students’ t test. (D) Alternatively, the aforementioned isolated TECs and NECs were serum starved for 4 hours and challenged to migrate toward full medium. Data are presented as mean ± SEM. n = 3 for both groups of AB1 and AE17 NECs, AE17 TECs, AE17 MTH1–overexpressing (AE17mth1over) TECs; n = 4 for each group of AB1 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (E) The aforementioned isolated TECs and NECs were challenged to form capillary-like tubes de novo on Matrigel. Data are presented as mean ± SEM, n = 3 for both groups of AB1 NECs, n = 5 for both groups of AE17 NEC and AE17mth1over TECs, n = 4 for each group of AB1 and AE17 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test.

    Journal: JCI Insight

    Article Title: MTH1 favors mesothelioma progression and mediates paracrine rescue of bystander endothelium from oxidative damage

    doi: 10.1172/jci.insight.134885

    Figure Lengend Snippet: Endothelial cells of MTH1-overexpressing tumors are more sensitive to MTH1 inhibition. (A) Endothelial cells from AB1, AE17, and AE17 MTH1–overexpressing tumors (TECs) and normal endothelial cells (NECs) from lung tissue were isolated, and mRNA levels of Mth1 was quantified by real-time PCR. Data are presented as mean ± SEM, n = 3 for each group. *P < 0.05 compared with NECs by 2-tailed Students’ t test. (B) Endothelial cells of AB1, AE17, and AE17 MTH1–overexpressing tumors from mice treated with vehicle or TH1579 were isolated using magnetic beads bearing anti-CD31 antibody. TECs were fixed, permeabilized, and stained for caspase-3 in order to measure apoptotic cells using flow cytometry. Data are presented as mean ± SEM, n = 3 for each vehicle and n = 4 for each TH1579 group. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (C) Isolated TECs and NECs from AB1, AE17, and AE17 MTH1–overexpressing mesotheliomas were seeded at 6 × 103 cells/well in 96-well plates and subsequently treated with escalating doses of TH1579 (1–1000 μM). Cell viability was determined by XTT reduction. Data are presented as mean ± SEM, n = 6 for each group. *P < 0.05 compared with vehicle by 2-tailed Students’ t test. #P < 0.05 compared with TECs by 2-tailed Students’ t test. (D) Alternatively, the aforementioned isolated TECs and NECs were serum starved for 4 hours and challenged to migrate toward full medium. Data are presented as mean ± SEM. n = 3 for both groups of AB1 and AE17 NECs, AE17 TECs, AE17 MTH1–overexpressing (AE17mth1over) TECs; n = 4 for each group of AB1 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test. (E) The aforementioned isolated TECs and NECs were challenged to form capillary-like tubes de novo on Matrigel. Data are presented as mean ± SEM, n = 3 for both groups of AB1 NECs, n = 5 for both groups of AE17 NEC and AE17mth1over TECs, n = 4 for each group of AB1 and AE17 TECs. *P < 0.05 compared with indicated groups by 2-tailed Students’ t test.

    Article Snippet: For immunofluorescence analysis, tumor cryosections were stained for the presence of CD31 (1:50, clone MEC 13.3, BD Biosciences) and caspase-3 (1:50, 9669, Cell Signaling Technology) for endothelial and apoptotic cell staining, respectively.

    Techniques: Inhibition, Isolation, Real-time Polymerase Chain Reaction, Magnetic Beads, Staining, Flow Cytometry