e11 murine kidney podocyte cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11 murine kidney podocyte cells
    E11 Murine Kidney Podocyte Cells, supplied by CLS Cell Lines Service GmbH, 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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    e11 murine kidney podocyte cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11 murine kidney podocyte cells
    E11 Murine Kidney Podocyte Cells, supplied by CLS Cell Lines Service GmbH, 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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    e11  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11
    E11, supplied by CLS Cell Lines Service GmbH, 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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    e11 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11 cells
    E11 Cells, supplied by CLS Cell Lines Service GmbH, 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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    murine podocyte cell line  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH murine podocyte cell line
    Murine Podocyte Cell Line, supplied by CLS Cell Lines Service GmbH, 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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    e11 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11 cells
    E11 Cells, supplied by CLS Cell Lines Service GmbH, 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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    e11  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11
    AM095 inhibits LPA-induced pyroptosis in <t>E11</t> cells. ( A , B ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 3 ( A ) or 6 ( B ) h, or treated with either AM095 or LPA alone. Protein levels of NEK7, NLRP3, ASC ( A ), cleaved-caspase-1 (Cle-cas-1), N-GSDMD, and IL-1β ( B ) were analyzed via Western blotting, quantified using ImageJ software and normalized to those of β-actin, pro-caspase-1 (Pro-cas-1), and GSDMD, respectively ( n = 3 or 4). ( C , D ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 4.5 h, or treated with either AM095 or LPA alone. ( C ) Cells were stained with FAM FLICA caspase-1 (green), propidium iodide (PI) (red), and Hoechst 33342 (blue). The white arrowheads indicate the pyroptotic cells (FLICA + PI + cells). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 4). ( D ) Cells were stained with FAM FLICA caspase-1 and analyzed via flow cytometry immediately after adding PI. Representative flow cytometry scatter plots (left panel) and the percentage dotted plot of pyroptotic cells (Q2) (right panel) are shown ( n = 4). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.
    E11, supplied by CLS Cell Lines Service GmbH, 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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    1) Product Images from "Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2"

    Article Title: Lysophosphatidic Acid Induces Podocyte Pyroptosis in Diabetic Nephropathy by an Increase of Egr1 Expression via Downregulation of EzH2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms24129968

    AM095 inhibits LPA-induced pyroptosis in E11 cells. ( A , B ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 3 ( A ) or 6 ( B ) h, or treated with either AM095 or LPA alone. Protein levels of NEK7, NLRP3, ASC ( A ), cleaved-caspase-1 (Cle-cas-1), N-GSDMD, and IL-1β ( B ) were analyzed via Western blotting, quantified using ImageJ software and normalized to those of β-actin, pro-caspase-1 (Pro-cas-1), and GSDMD, respectively ( n = 3 or 4). ( C , D ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 4.5 h, or treated with either AM095 or LPA alone. ( C ) Cells were stained with FAM FLICA caspase-1 (green), propidium iodide (PI) (red), and Hoechst 33342 (blue). The white arrowheads indicate the pyroptotic cells (FLICA + PI + cells). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 4). ( D ) Cells were stained with FAM FLICA caspase-1 and analyzed via flow cytometry immediately after adding PI. Representative flow cytometry scatter plots (left panel) and the percentage dotted plot of pyroptotic cells (Q2) (right panel) are shown ( n = 4). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.
    Figure Legend Snippet: AM095 inhibits LPA-induced pyroptosis in E11 cells. ( A , B ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 3 ( A ) or 6 ( B ) h, or treated with either AM095 or LPA alone. Protein levels of NEK7, NLRP3, ASC ( A ), cleaved-caspase-1 (Cle-cas-1), N-GSDMD, and IL-1β ( B ) were analyzed via Western blotting, quantified using ImageJ software and normalized to those of β-actin, pro-caspase-1 (Pro-cas-1), and GSDMD, respectively ( n = 3 or 4). ( C , D ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 4.5 h, or treated with either AM095 or LPA alone. ( C ) Cells were stained with FAM FLICA caspase-1 (green), propidium iodide (PI) (red), and Hoechst 33342 (blue). The white arrowheads indicate the pyroptotic cells (FLICA + PI + cells). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 4). ( D ) Cells were stained with FAM FLICA caspase-1 and analyzed via flow cytometry immediately after adding PI. Representative flow cytometry scatter plots (left panel) and the percentage dotted plot of pyroptotic cells (Q2) (right panel) are shown ( n = 4). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.

    Techniques Used: Western Blot, Software, Staining, Flow Cytometry, MANN-WHITNEY

    Egr1 is required for LPA-induced pyroptosis in E11 cells. ( A ) Differentiated E11 cells were treated with 5 µM LPA for 10, 30, 60, 120, and 180 min. ( B , C ) Cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 1 h, or with either AM095 or LPA alone. ( A , B ) Egr1 mRNA level was determined via RT-qPCR. ( n = 4) ( C ) The Egr1 protein level was analyzed via Western blotting, quantified using the ImageJ software, and normalized to that of β-actin ( n = 3). ( D , E ) Differentiated E11 cells were transfected with scrambled siRNA (siCon) or Egr1 siRNA (siEgr1) for 6 h, starved for 18 h, and then treated with 5 µM LPA for 4.5 ( E ) or 6 ( D ) h. ( D ) Protein levels of Egr1, cleaved-caspase-1 (Cle-cas-1), and IL-1β were measured using Western blotting, quantified using ImageJ software, and normalized to those of β-actin or pro-caspase-1 (Pro-cas-1), respectively ( n = 3–5). ( E ) Cells were stained with FAM FLICA caspase-1 (green), propidium iodide (PI) (red), and Hoechst 33342 (blue). The white arrowheads indicate pyroptotic cells (FLICA + PI + cells). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 5). Data are represented as the mean ± SD. ns, statistically non-significant. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.
    Figure Legend Snippet: Egr1 is required for LPA-induced pyroptosis in E11 cells. ( A ) Differentiated E11 cells were treated with 5 µM LPA for 10, 30, 60, 120, and 180 min. ( B , C ) Cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 1 h, or with either AM095 or LPA alone. ( A , B ) Egr1 mRNA level was determined via RT-qPCR. ( n = 4) ( C ) The Egr1 protein level was analyzed via Western blotting, quantified using the ImageJ software, and normalized to that of β-actin ( n = 3). ( D , E ) Differentiated E11 cells were transfected with scrambled siRNA (siCon) or Egr1 siRNA (siEgr1) for 6 h, starved for 18 h, and then treated with 5 µM LPA for 4.5 ( E ) or 6 ( D ) h. ( D ) Protein levels of Egr1, cleaved-caspase-1 (Cle-cas-1), and IL-1β were measured using Western blotting, quantified using ImageJ software, and normalized to those of β-actin or pro-caspase-1 (Pro-cas-1), respectively ( n = 3–5). ( E ) Cells were stained with FAM FLICA caspase-1 (green), propidium iodide (PI) (red), and Hoechst 33342 (blue). The white arrowheads indicate pyroptotic cells (FLICA + PI + cells). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 5). Data are represented as the mean ± SD. ns, statistically non-significant. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.

    Techniques Used: Quantitative RT-PCR, Western Blot, Software, Transfection, Staining, MANN-WHITNEY

    Egr1 knockdown decreases LPA-induced NF-κB activation and desmin expression in E11 cells. ( A , B ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 1 (for p-NF-κB, ( A )) or 6 (for desmin, ( B )) h, or treated with either AM095 or LPA alone. ( C – E ) Differentiated E11 cells were transfected with siCon or siEgr1 for 6 h, starved for 18 h, and then treated with 5 µM LPA for 1 ( C ) or 6 ( D , E ) h. Protein levels of p-NF-κB, Egr1, and desmin were analyzed by Western blotting, quantified using ImageJ software, and normalized to those of NF-κB or β-actin, respectively ( n = 4). ( E ) Cells were stained with desmin (green), Egr1 (red), and DAPI (blue). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 5). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.
    Figure Legend Snippet: Egr1 knockdown decreases LPA-induced NF-κB activation and desmin expression in E11 cells. ( A , B ) Differentiated E11 cells were treated with 5 µM LPA and 10 µM AM095 simultaneously for 1 (for p-NF-κB, ( A )) or 6 (for desmin, ( B )) h, or treated with either AM095 or LPA alone. ( C – E ) Differentiated E11 cells were transfected with siCon or siEgr1 for 6 h, starved for 18 h, and then treated with 5 µM LPA for 1 ( C ) or 6 ( D , E ) h. Protein levels of p-NF-κB, Egr1, and desmin were analyzed by Western blotting, quantified using ImageJ software, and normalized to those of NF-κB or β-actin, respectively ( n = 4). ( E ) Cells were stained with desmin (green), Egr1 (red), and DAPI (blue). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 5). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.

    Techniques Used: Activation Assay, Expressing, Transfection, Western Blot, Software, Staining, MANN-WHITNEY

    LPA increased Egr1expression by downregulation of EzH2 in E11 cells. ( A ) Differentiated E11 cells were treated with 5 µM LPA for 1 h, and chromatin immunoprecipitation (ChIP)-qPCR assay was performed to evaluate the enrichment of H3K27me3 at the Egr1 promoter region. A normal IgG antibody was used as a negative control ( n = 3). ( B , C ) Differentiated E11 cells were treated with a combination of 5 µM LPA and 10 µM AM095 for 1 h, or treated with either AM095 or LPA alone. ( B ) Cells were stained with Egr1 (green), H3K27me3 (red), and DAPI (blue). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 5). ( D ) Differentiated E11 cells were transfected with siCon or EzH2 siRNA (siEzH2) for 6 h, starved for 18 h, and then treated with 5 µM LPA for 1 h. ( C , D ) Protein levels of H3K27me3, EzH2, and Egr1 were analyzed via Western blotting, quantified using ImageJ software, and normalized to those of Histone H3 (H3) or β-actin, respectively ( n = 3 or 4). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.
    Figure Legend Snippet: LPA increased Egr1expression by downregulation of EzH2 in E11 cells. ( A ) Differentiated E11 cells were treated with 5 µM LPA for 1 h, and chromatin immunoprecipitation (ChIP)-qPCR assay was performed to evaluate the enrichment of H3K27me3 at the Egr1 promoter region. A normal IgG antibody was used as a negative control ( n = 3). ( B , C ) Differentiated E11 cells were treated with a combination of 5 µM LPA and 10 µM AM095 for 1 h, or treated with either AM095 or LPA alone. ( B ) Cells were stained with Egr1 (green), H3K27me3 (red), and DAPI (blue). Scale bar: 20 μm. Representative images (left) and quantitative analysis scatter dot plot (right) are shown ( n = 5). ( D ) Differentiated E11 cells were transfected with siCon or EzH2 siRNA (siEzH2) for 6 h, starved for 18 h, and then treated with 5 µM LPA for 1 h. ( C , D ) Protein levels of H3K27me3, EzH2, and Egr1 were analyzed via Western blotting, quantified using ImageJ software, and normalized to those of Histone H3 (H3) or β-actin, respectively ( n = 3 or 4). Data are represented as the mean ± SD. *: p < 0.05, **: p < 0.01 via Kruskal–Wallis test followed by Dunn’s test for multiple comparisons and Mann–Whitney U test for two specific groups.

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Staining, Transfection, Western Blot, Software, MANN-WHITNEY

    murine kidney podocyte cell line e11  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH murine kidney podocyte cell line e11
    Murine Kidney Podocyte Cell Line E11, supplied by CLS Cell Lines Service GmbH, 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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    e11 murine podocyte cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11 murine podocyte cells
    LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured <t>podocytes.</t> Representative immunoblots of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin in cultured murine <t>E11</t> podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to β-Actin from three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells
    E11 Murine Podocyte Cells, supplied by CLS Cell Lines Service GmbH, 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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    1) Product Images from "Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin"

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-022-00884-6

    LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured podocytes. Representative immunoblots of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin in cultured murine E11 podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to β-Actin from three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells
    Figure Legend Snippet: LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured podocytes. Representative immunoblots of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin in cultured murine E11 podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to β-Actin from three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells

    Techniques Used: Expressing, Cell Culture, Western Blot, Injection, Isolation

    PKM2 podocyte deletion ameliorates LPS induced proteinuria and kidney injury. A Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in whole kidney lysates from wild type (Ctrl) and podocyte PKM2 knockout mice (KO). mRNA levels (right panel) of Pkm2 in total kidney lysates of Ctrl and KO mice (n = 6 group). B Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in primary podocytes isolated from Ctrl and KO mice. The right panel is the mRNA level of Pkm2 and Pkm1 C in primary podocytes (n = 12; 4 animals/lane). D Body weights, E kidney weights, and F kidney to body weight ratio of wild type (Ctrl) and podocyte PKM2 knockout mice (KO) mice, under PBS and LPS treated states. Assessment of G total urinary proteins levels, H serum albumin levels, I urine albumin levels, J albumin to creatinine ratios (ACR), and K blood urea nitrogen (BUN) levels of Ctrl and KO mice after PBS and LPS injection (n = 6–12 per group). L PAS staining of kidney sections in Ctrl and KO mice under PBS and LPS conditions; the arrow is pointing to the expansion of Bowman space in response to LPS treatment. Scale bar = 50 μM. M Immunofluorescence of Nephrin (green), PKM2 (red), and nuclear DNA using DAPI (blue) in kidney sections obtained from both genotypes injected with PBS or LPS. Scale bar = 50 μM. In D–K, * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice
    Figure Legend Snippet: PKM2 podocyte deletion ameliorates LPS induced proteinuria and kidney injury. A Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in whole kidney lysates from wild type (Ctrl) and podocyte PKM2 knockout mice (KO). mRNA levels (right panel) of Pkm2 in total kidney lysates of Ctrl and KO mice (n = 6 group). B Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in primary podocytes isolated from Ctrl and KO mice. The right panel is the mRNA level of Pkm2 and Pkm1 C in primary podocytes (n = 12; 4 animals/lane). D Body weights, E kidney weights, and F kidney to body weight ratio of wild type (Ctrl) and podocyte PKM2 knockout mice (KO) mice, under PBS and LPS treated states. Assessment of G total urinary proteins levels, H serum albumin levels, I urine albumin levels, J albumin to creatinine ratios (ACR), and K blood urea nitrogen (BUN) levels of Ctrl and KO mice after PBS and LPS injection (n = 6–12 per group). L PAS staining of kidney sections in Ctrl and KO mice under PBS and LPS conditions; the arrow is pointing to the expansion of Bowman space in response to LPS treatment. Scale bar = 50 μM. M Immunofluorescence of Nephrin (green), PKM2 (red), and nuclear DNA using DAPI (blue) in kidney sections obtained from both genotypes injected with PBS or LPS. Scale bar = 50 μM. In D–K, * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice

    Techniques Used: Western Blot, Knock-Out, Isolation, Injection, Staining, Immunofluorescence

    Histology assessment of kidneys injury in control and PKM2 KO mice. We examined the tubules of the cortical and corticomedullary areas using PAS-stained renal sections obtained from PBS or LPS-treated wild type (Ctrl) and  podocyte  PKM2 knockout (KO) mice and scored the degree of injury as detailed in the methods section
    Figure Legend Snippet: Histology assessment of kidneys injury in control and PKM2 KO mice. We examined the tubules of the cortical and corticomedullary areas using PAS-stained renal sections obtained from PBS or LPS-treated wild type (Ctrl) and podocyte PKM2 knockout (KO) mice and scored the degree of injury as detailed in the methods section

    Techniques Used: Knock-Out

    Podocyte specific deletion of PKM2 attenuates LPS-Induced inflammation and apoptosis in Vivo. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of major signal transduction molecules involved in the NF-κB (pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65), MAPK (pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38) signaling pathways, and β-Actin as a loading control in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantification (right panel) of apoptotic markers: Caspase 12, 7, and 3, their cleaved forms, and CHOP in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice
    Figure Legend Snippet: Podocyte specific deletion of PKM2 attenuates LPS-Induced inflammation and apoptosis in Vivo. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of major signal transduction molecules involved in the NF-κB (pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65), MAPK (pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38) signaling pathways, and β-Actin as a loading control in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantification (right panel) of apoptotic markers: Caspase 12, 7, and 3, their cleaved forms, and CHOP in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice

    Techniques Used: In Vivo, Western Blot, Transduction, Knock-Out, Injection

    PKM2 deficiency in cultured E11 podocytes alleviates LPS-induced nephrin loss. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of PKM2, PKM1, and PKM1/2 levels in undifferentiated (day 1; D1) and differentiated (D15) E11 murine podocytes infected with lentivirus particles carrying scramble-shRNA (SCR), shRNA targeting PKM2 (M2KD) or an open reading frame of the human DNA (M2R). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between differentiated and undifferentiated cells. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell and control E11 cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, and Nephrin in differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment
    Figure Legend Snippet: PKM2 deficiency in cultured E11 podocytes alleviates LPS-induced nephrin loss. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of PKM2, PKM1, and PKM1/2 levels in undifferentiated (day 1; D1) and differentiated (D15) E11 murine podocytes infected with lentivirus particles carrying scramble-shRNA (SCR), shRNA targeting PKM2 (M2KD) or an open reading frame of the human DNA (M2R). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between differentiated and undifferentiated cells. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell and control E11 cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, and Nephrin in differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment

    Techniques Used: Cell Culture, Western Blot, Infection, shRNA

    PKM2 deficiency ameliorates LPS-induced inflammation and apoptosis in cultured E11 podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65, pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38, and PKM2 in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of CHOP and cleaved-caspase 3 (C-Cas3) in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Caspase 3 activity in M2R and M2KD cells treated (LPS) or non-treated (Ctrl) with LPS for 24 h. ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. D Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes treated with LPS for 24 h. Scale bar: 100 μm. Images are representative of at least three independent experiments
    Figure Legend Snippet: PKM2 deficiency ameliorates LPS-induced inflammation and apoptosis in cultured E11 podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65, pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38, and PKM2 in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of CHOP and cleaved-caspase 3 (C-Cas3) in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Caspase 3 activity in M2R and M2KD cells treated (LPS) or non-treated (Ctrl) with LPS for 24 h. ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. D Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes treated with LPS for 24 h. Scale bar: 100 μm. Images are representative of at least three independent experiments

    Techniques Used: Cell Culture, Western Blot, Activity Assay, Staining

    PKM2 depletion suppress LPS-induced-β-catenin activation in kidneys and E11 cultured podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin S33 , pβ-Catenin Y333 , β-Catenin and its downstream targets (WT1 and c-Myc), and nephrin in total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n ≥ 6/group). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y33 , β-Catenin, WT1, and c-Myc in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y333 , β-Catenin, GFP, WT1, nephrin, C-Caspase 3, and PKM2 in in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA), or the pLHCX vector expressing the K433E mutant of PKM2. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. D Caspase 3 activity in M2R, M2KD cells in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA) plasmids, or the pLHCX vector expressing the K433E mutant of PKM2. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. E Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes transfected with empty pCS2 + vector (Ctrl) or a pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA). Scale bar: 100 μm. Images are representative of at least three independent experiments
    Figure Legend Snippet: PKM2 depletion suppress LPS-induced-β-catenin activation in kidneys and E11 cultured podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin S33 , pβ-Catenin Y333 , β-Catenin and its downstream targets (WT1 and c-Myc), and nephrin in total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n ≥ 6/group). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y33 , β-Catenin, WT1, and c-Myc in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y333 , β-Catenin, GFP, WT1, nephrin, C-Caspase 3, and PKM2 in in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA), or the pLHCX vector expressing the K433E mutant of PKM2. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. D Caspase 3 activity in M2R, M2KD cells in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA) plasmids, or the pLHCX vector expressing the K433E mutant of PKM2. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. E Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes transfected with empty pCS2 + vector (Ctrl) or a pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA). Scale bar: 100 μm. Images are representative of at least three independent experiments

    Techniques Used: Activation Assay, Cell Culture, Western Blot, Injection, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Activity Assay, Staining

    e11 cells  (CLS Cell Lines Service GmbH)


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    CLS Cell Lines Service GmbH e11 cells
    In vitro analysis of surface expression of podocalyxin and Rho-GTPase activity using a podocyte cell line. ERM protein expression levels in <t>E11</t> cells were investigated by immunoblotting. Knockdown of ezrin was performed by siRNA. The siRNA targeting ezrin gene effectively down-regulated expression of ezrin in E11 cells ( a ). A surface biotinylation assay was performed using negative control siRNA- and ezrin siRNA-treated E11 cells. Surface expression of podocalyxin was examined and no contamination of cytosolic proteins was identified by immunoblotting for GAPDH. Densitometry analysis was performed (n = 5 for each group) ( b ). RhoA and Rac1 activity in siRNA-treated E11 cells was investigated by Rho-GTPase activity assay (n = 5 for each group). * P < 0.05, vs. Negative siRNA-treated E11 cells ( c ).
    E11 Cells, supplied by CLS Cell Lines Service GmbH, 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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    1) Product Images from "Loss of ezrin expression reduced the susceptibility to the glomerular injury in mice"

    Article Title: Loss of ezrin expression reduced the susceptibility to the glomerular injury in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22846-0

    In vitro analysis of surface expression of podocalyxin and Rho-GTPase activity using a podocyte cell line. ERM protein expression levels in E11 cells were investigated by immunoblotting. Knockdown of ezrin was performed by siRNA. The siRNA targeting ezrin gene effectively down-regulated expression of ezrin in E11 cells ( a ). A surface biotinylation assay was performed using negative control siRNA- and ezrin siRNA-treated E11 cells. Surface expression of podocalyxin was examined and no contamination of cytosolic proteins was identified by immunoblotting for GAPDH. Densitometry analysis was performed (n = 5 for each group) ( b ). RhoA and Rac1 activity in siRNA-treated E11 cells was investigated by Rho-GTPase activity assay (n = 5 for each group). * P < 0.05, vs. Negative siRNA-treated E11 cells ( c ).
    Figure Legend Snippet: In vitro analysis of surface expression of podocalyxin and Rho-GTPase activity using a podocyte cell line. ERM protein expression levels in E11 cells were investigated by immunoblotting. Knockdown of ezrin was performed by siRNA. The siRNA targeting ezrin gene effectively down-regulated expression of ezrin in E11 cells ( a ). A surface biotinylation assay was performed using negative control siRNA- and ezrin siRNA-treated E11 cells. Surface expression of podocalyxin was examined and no contamination of cytosolic proteins was identified by immunoblotting for GAPDH. Densitometry analysis was performed (n = 5 for each group) ( b ). RhoA and Rac1 activity in siRNA-treated E11 cells was investigated by Rho-GTPase activity assay (n = 5 for each group). * P < 0.05, vs. Negative siRNA-treated E11 cells ( c ).

    Techniques Used: In Vitro, Expressing, Activity Assay, Western Blot, Surface Biotinylation Assay, Negative Control

    RhoA and Rac1 activity in E11 cells transfected with ezrin mutants. E11 cells were transfected with different kinds of ezrin mutants (Full length ezrin: FL-ezrin; N-terminal (FERM) domain (aa:1-310): N term-ezrin; C-terminal deletion mutant (aa: 1-533): ΔC term-ezrin; constitutively inactive ezrin: T567A-ezrin; and constitutively active ezrin: T567D-ezrin). RhoA ( a ) and Rac1 ( b ) activity was measured in these E11 cells. * P < 0.05, vs. control. (n = 3–5).
    Figure Legend Snippet: RhoA and Rac1 activity in E11 cells transfected with ezrin mutants. E11 cells were transfected with different kinds of ezrin mutants (Full length ezrin: FL-ezrin; N-terminal (FERM) domain (aa:1-310): N term-ezrin; C-terminal deletion mutant (aa: 1-533): ΔC term-ezrin; constitutively inactive ezrin: T567A-ezrin; and constitutively active ezrin: T567D-ezrin). RhoA ( a ) and Rac1 ( b ) activity was measured in these E11 cells. * P < 0.05, vs. control. (n = 3–5).

    Techniques Used: Activity Assay, Transfection, Mutagenesis

    Morphological analysis of FL-ezrin and T567D-ezrin transfected E11 cells. E11 cells transfected with FL-ezrin ( a ) and T567D-ezrin ( b ) were double-stained with an anti-flag antibody (green) and rhodamine-phalloidin (red). Both FL-ezrin and T567D-ezrin were detected at the cellular cortex. Some protrusions were observed in FL-ezrin-transfected E11 cells ( a ), while enhanced membrane ruffling was observed in T567D-ezrin transfected E11 cells (white arrow) ( b ). The number of lamellipodia per cell was counted. Lamellipodia were defined as 3- to 10-μm regions located at the cell periphery, as previously described by Raucher et al .
    Figure Legend Snippet: Morphological analysis of FL-ezrin and T567D-ezrin transfected E11 cells. E11 cells transfected with FL-ezrin ( a ) and T567D-ezrin ( b ) were double-stained with an anti-flag antibody (green) and rhodamine-phalloidin (red). Both FL-ezrin and T567D-ezrin were detected at the cellular cortex. Some protrusions were observed in FL-ezrin-transfected E11 cells ( a ), while enhanced membrane ruffling was observed in T567D-ezrin transfected E11 cells (white arrow) ( b ). The number of lamellipodia per cell was counted. Lamellipodia were defined as 3- to 10-μm regions located at the cell periphery, as previously described by Raucher et al .

    Techniques Used: Transfection, Staining

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    CLS Cell Lines Service GmbH e11 murine kidney podocyte cells
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    CLS Cell Lines Service GmbH e11 cells
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    CLS Cell Lines Service GmbH murine podocyte cell line
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    CLS Cell Lines Service GmbH e11 murine podocyte cells
    LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured <t>podocytes.</t> Representative immunoblots of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin in cultured murine <t>E11</t> podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to β-Actin from three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells
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    LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured podocytes. Representative immunoblots of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin in cultured murine E11 podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to β-Actin from three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: LPS increases PKM2 phosphorylation and expression levels in renal tissue and cultured podocytes. Representative immunoblots of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin as a loading control in A total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 6/group) and B primary podocytes isolated from C57bl6/J wild type mice 24 h after PBS or LPS injection (n = 12; 4 animals/lane). C Representative immunoblots (left panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, nephrin, and β-Actin in cultured murine E11 podocytes in response to PBS or LPS treatment for the indicated duration. Bar graphs (right panel) displays changes in PKM2 and nephrin levels normalized to β-Actin from three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with LPS and non-treated cells

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: Expressing, Cell Culture, Western Blot, Injection, Isolation

    PKM2 podocyte deletion ameliorates LPS induced proteinuria and kidney injury. A Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in whole kidney lysates from wild type (Ctrl) and podocyte PKM2 knockout mice (KO). mRNA levels (right panel) of Pkm2 in total kidney lysates of Ctrl and KO mice (n = 6 group). B Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in primary podocytes isolated from Ctrl and KO mice. The right panel is the mRNA level of Pkm2 and Pkm1 C in primary podocytes (n = 12; 4 animals/lane). D Body weights, E kidney weights, and F kidney to body weight ratio of wild type (Ctrl) and podocyte PKM2 knockout mice (KO) mice, under PBS and LPS treated states. Assessment of G total urinary proteins levels, H serum albumin levels, I urine albumin levels, J albumin to creatinine ratios (ACR), and K blood urea nitrogen (BUN) levels of Ctrl and KO mice after PBS and LPS injection (n = 6–12 per group). L PAS staining of kidney sections in Ctrl and KO mice under PBS and LPS conditions; the arrow is pointing to the expansion of Bowman space in response to LPS treatment. Scale bar = 50 μM. M Immunofluorescence of Nephrin (green), PKM2 (red), and nuclear DNA using DAPI (blue) in kidney sections obtained from both genotypes injected with PBS or LPS. Scale bar = 50 μM. In D–K, * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: PKM2 podocyte deletion ameliorates LPS induced proteinuria and kidney injury. A Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in whole kidney lysates from wild type (Ctrl) and podocyte PKM2 knockout mice (KO). mRNA levels (right panel) of Pkm2 in total kidney lysates of Ctrl and KO mice (n = 6 group). B Representative immunoblots (left panel) of PKM2, PKM1, PKM1/2, and β-Actin in primary podocytes isolated from Ctrl and KO mice. The right panel is the mRNA level of Pkm2 and Pkm1 C in primary podocytes (n = 12; 4 animals/lane). D Body weights, E kidney weights, and F kidney to body weight ratio of wild type (Ctrl) and podocyte PKM2 knockout mice (KO) mice, under PBS and LPS treated states. Assessment of G total urinary proteins levels, H serum albumin levels, I urine albumin levels, J albumin to creatinine ratios (ACR), and K blood urea nitrogen (BUN) levels of Ctrl and KO mice after PBS and LPS injection (n = 6–12 per group). L PAS staining of kidney sections in Ctrl and KO mice under PBS and LPS conditions; the arrow is pointing to the expansion of Bowman space in response to LPS treatment. Scale bar = 50 μM. M Immunofluorescence of Nephrin (green), PKM2 (red), and nuclear DNA using DAPI (blue) in kidney sections obtained from both genotypes injected with PBS or LPS. Scale bar = 50 μM. In D–K, * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: Western Blot, Knock-Out, Isolation, Injection, Staining, Immunofluorescence

    Histology assessment of kidneys injury in control and PKM2 KO mice. We examined the tubules of the cortical and corticomedullary areas using PAS-stained renal sections obtained from PBS or LPS-treated wild type (Ctrl) and  podocyte  PKM2 knockout (KO) mice and scored the degree of injury as detailed in the methods section

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: Histology assessment of kidneys injury in control and PKM2 KO mice. We examined the tubules of the cortical and corticomedullary areas using PAS-stained renal sections obtained from PBS or LPS-treated wild type (Ctrl) and podocyte PKM2 knockout (KO) mice and scored the degree of injury as detailed in the methods section

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: Knock-Out

    Podocyte specific deletion of PKM2 attenuates LPS-Induced inflammation and apoptosis in Vivo. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of major signal transduction molecules involved in the NF-κB (pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65), MAPK (pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38) signaling pathways, and β-Actin as a loading control in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantification (right panel) of apoptotic markers: Caspase 12, 7, and 3, their cleaved forms, and CHOP in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: Podocyte specific deletion of PKM2 attenuates LPS-Induced inflammation and apoptosis in Vivo. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of major signal transduction molecules involved in the NF-κB (pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65), MAPK (pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38) signaling pathways, and β-Actin as a loading control in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantification (right panel) of apoptotic markers: Caspase 12, 7, and 3, their cleaved forms, and CHOP in whole kidney lysates of control (Ctrl) and podocyte PKM2 knockout mice (KO) mice harvested 24 h after PBS or LPS injection (n ≥ 6/group). * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: In Vivo, Western Blot, Transduction, Knock-Out, Injection

    PKM2 deficiency in cultured E11 podocytes alleviates LPS-induced nephrin loss. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of PKM2, PKM1, and PKM1/2 levels in undifferentiated (day 1; D1) and differentiated (D15) E11 murine podocytes infected with lentivirus particles carrying scramble-shRNA (SCR), shRNA targeting PKM2 (M2KD) or an open reading frame of the human DNA (M2R). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between differentiated and undifferentiated cells. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell and control E11 cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, and Nephrin in differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: PKM2 deficiency in cultured E11 podocytes alleviates LPS-induced nephrin loss. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of PKM2, PKM1, and PKM1/2 levels in undifferentiated (day 1; D1) and differentiated (D15) E11 murine podocytes infected with lentivirus particles carrying scramble-shRNA (SCR), shRNA targeting PKM2 (M2KD) or an open reading frame of the human DNA (M2R). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between differentiated and undifferentiated cells. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell and control E11 cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pPKM2 Y105 , pPKM2 S37 , PKM2, PKM1, PKM1/2, and Nephrin in differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: Cell Culture, Western Blot, Infection, shRNA

    PKM2 deficiency ameliorates LPS-induced inflammation and apoptosis in cultured E11 podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65, pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38, and PKM2 in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of CHOP and cleaved-caspase 3 (C-Cas3) in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Caspase 3 activity in M2R and M2KD cells treated (LPS) or non-treated (Ctrl) with LPS for 24 h. ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. D Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes treated with LPS for 24 h. Scale bar: 100 μm. Images are representative of at least three independent experiments

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: PKM2 deficiency ameliorates LPS-induced inflammation and apoptosis in cultured E11 podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pIKKα S178/S180 , IKKα, pIκBα S32 , IκBα, pNF-κBp65 S36 , NF-κBp65, pJNK1/2 T183/Y185 , JNK, pP38 T180/Y182 , P38, and PKM2 in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of CHOP and cleaved-caspase 3 (C-Cas3) in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Caspase 3 activity in M2R and M2KD cells treated (LPS) or non-treated (Ctrl) with LPS for 24 h. ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. D Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes treated with LPS for 24 h. Scale bar: 100 μm. Images are representative of at least three independent experiments

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: Cell Culture, Western Blot, Activity Assay, Staining

    PKM2 depletion suppress LPS-induced-β-catenin activation in kidneys and E11 cultured podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin S33 , pβ-Catenin Y333 , β-Catenin and its downstream targets (WT1 and c-Myc), and nephrin in total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n ≥ 6/group). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y33 , β-Catenin, WT1, and c-Myc in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y333 , β-Catenin, GFP, WT1, nephrin, C-Caspase 3, and PKM2 in in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA), or the pLHCX vector expressing the K433E mutant of PKM2. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. D Caspase 3 activity in M2R, M2KD cells in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA) plasmids, or the pLHCX vector expressing the K433E mutant of PKM2. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. E Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes transfected with empty pCS2 + vector (Ctrl) or a pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA). Scale bar: 100 μm. Images are representative of at least three independent experiments

    Journal: Cell Communication and Signaling : CCS

    Article Title: Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

    doi: 10.1186/s12964-022-00884-6

    Figure Lengend Snippet: PKM2 depletion suppress LPS-induced-β-catenin activation in kidneys and E11 cultured podocytes. A Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin S33 , pβ-Catenin Y333 , β-Catenin and its downstream targets (WT1 and c-Myc), and nephrin in total kidney lysates harvested from C57bl6/J wild type mice 24 h after PBS or LPS injection (n ≥ 6/group). β-Actin was used as a loading control. * p < 0.05, ** p < 0.01 indicate a significant difference between PBS- and LPS-injected mice. & p < 0.05, && p < 0.01 indicate a significant difference between Ctrl and KO mice. B Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y33 , β-Catenin, WT1, and c-Myc in total cell lysates from differentiated M2R and M2KD podocytes treated with LPS for the indicated durations. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. C Representative immunoblots (left panel) and bar graph quantitative assessment (right panel) of pβ-Catenin Y333 , β-Catenin, GFP, WT1, nephrin, C-Caspase 3, and PKM2 in in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA), or the pLHCX vector expressing the K433E mutant of PKM2. β-Actin was used as a loading control. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. D Caspase 3 activity in M2R, M2KD cells in total cell lysates from LPS treated or non-treated M2R, M2KD transfected with empty pCS2 + vector (Ctrl), pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA) plasmids, or the pLHCX vector expressing the K433E mutant of PKM2. Data is representative of at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between LPS-treated or non-treated cells. & p < 0.05, && p < 0.01 indicate a significant difference between M2R and M2KD cells exposed to the same treatment. # p < 0.05, ## p < 0.01 indicate a significant difference between the indicated cell overexpressing the constitutively active mutant of β-catenin (β-CA) or the K433E mutant of PKM2 and M2KD cells exposed to the same treatment. E Representative images of chromatin condensation in Hoechst-stained M2R and M2KD podocytes transfected with empty pCS2 + vector (Ctrl) or a pCS2 plasmid expressing the constitutively active β-catenin mutant (β-CA). Scale bar: 100 μm. Images are representative of at least three independent experiments

    Article Snippet: E11 murine podocyte cells were obtained from Cell Lines Service (Eppelheim, Germany) and cultured at 33ºC in a humidified atmosphere of 10% CO 2 .

    Techniques: Activation Assay, Cell Culture, Western Blot, Injection, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Activity Assay, Staining