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mouse anti wt1 monoclonal antibody  (Novus Biologicals)


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    Novus Biologicals mouse anti wt1 monoclonal antibody
    Figure 1. Generation of podocyte-specific Pfn1-KO mice results in severe proteinuria and kidney failure. (A) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1-KO mice (age P7). (B) Representative immunofluorescence images of profilin1 (green) and <t>WT1</t> (red) in control and Pfn1-KO primary podocytes. Scale bar: 10 μm. (C) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1-KO mice (age 3 weeks). Scale bars: 10 μm. (D) Pfn1-KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. *P < 0.05 vs. control. (E) The survival curve of Pfn1-KO mice (red) demon- strates approximately 90% death by 12 weeks of age. n = 8 mice. (F) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1-KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. (G) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. *P < 0.05 vs. control. (H) Elevated plasma creatinine in Pfn1-KO mice at 3, 6, and 10 weeks of age. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.
    Mouse Anti Wt1 Monoclonal Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti wt1 monoclonal antibody - by Bioz Stars, 2026-02
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    Images

    1) Product Images from "Profilin1 is required to prevent mitotic catastrophe in murine and human glomerular diseases"

    Article Title: Profilin1 is required to prevent mitotic catastrophe in murine and human glomerular diseases

    Journal: Journal of Clinical Investigation

    doi: 10.1172/jci171237

    Figure 1. Generation of podocyte-specific Pfn1-KO mice results in severe proteinuria and kidney failure. (A) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1-KO mice (age P7). (B) Representative immunofluorescence images of profilin1 (green) and WT1 (red) in control and Pfn1-KO primary podocytes. Scale bar: 10 μm. (C) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1-KO mice (age 3 weeks). Scale bars: 10 μm. (D) Pfn1-KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. *P < 0.05 vs. control. (E) The survival curve of Pfn1-KO mice (red) demon- strates approximately 90% death by 12 weeks of age. n = 8 mice. (F) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1-KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. (G) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. *P < 0.05 vs. control. (H) Elevated plasma creatinine in Pfn1-KO mice at 3, 6, and 10 weeks of age. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.
    Figure Legend Snippet: Figure 1. Generation of podocyte-specific Pfn1-KO mice results in severe proteinuria and kidney failure. (A) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1-KO mice (age P7). (B) Representative immunofluorescence images of profilin1 (green) and WT1 (red) in control and Pfn1-KO primary podocytes. Scale bar: 10 μm. (C) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1-KO mice (age 3 weeks). Scale bars: 10 μm. (D) Pfn1-KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. *P < 0.05 vs. control. (E) The survival curve of Pfn1-KO mice (red) demon- strates approximately 90% death by 12 weeks of age. n = 8 mice. (F) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1-KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. (G) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. *P < 0.05 vs. control. (H) Elevated plasma creatinine in Pfn1-KO mice at 3, 6, and 10 weeks of age. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Techniques Used: Western Blot, Expressing, Isolation, Control, Immunofluorescence, SDS Page, Staining, Clinical Proteomics

    Figure 3. Loss of podocyte Pfn1 results in morphologic MC appearance, chromosomal instability, and dsDNA damage. (A) Representative images of transmission electron micrography demonstrate abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control glomeruli at 4 weeks of age. Scale bar: 1 μm. (B) Immunofluorescence images of primary podocytes isolated from control and Pfn1-KO mice at P7 stained with WT1 (red, podocyte marker) and Hoechst (blue, DNA marker) showing abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control podocytes. Scale bar: 20 μm. (C) Immunofluorescence images of primary culture podocytes isolated from control and Pfn1-KO mice stained with Hoechst and anti-tubulin antibody showing the chromosome bridge (arrow) in a MC Pfn1-KO podocyte. Scale bar: 20 μm. (D) Quantification of the percentage of MC podocytes per field of view in B. Total of 100 fields of view in 5 independent experiments. (E) Quantification of the percentage of chromosome bridge per field of view in C. n = 5 independent experiments. *P < 0.05 vs. control. (F) Immunofluorescence images of primary culture podocytes stained with γ-H2AX (green, double-strand breaks [DSBs] marker) and WT1 (red) showing abnormal MC podocytes in Pfn1-KO mice, as indicated by the arrows. Scale bar: 20 μm. (G) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in F. Total of 400 cells in 5 independent experiments. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.
    Figure Legend Snippet: Figure 3. Loss of podocyte Pfn1 results in morphologic MC appearance, chromosomal instability, and dsDNA damage. (A) Representative images of transmission electron micrography demonstrate abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control glomeruli at 4 weeks of age. Scale bar: 1 μm. (B) Immunofluorescence images of primary podocytes isolated from control and Pfn1-KO mice at P7 stained with WT1 (red, podocyte marker) and Hoechst (blue, DNA marker) showing abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control podocytes. Scale bar: 20 μm. (C) Immunofluorescence images of primary culture podocytes isolated from control and Pfn1-KO mice stained with Hoechst and anti-tubulin antibody showing the chromosome bridge (arrow) in a MC Pfn1-KO podocyte. Scale bar: 20 μm. (D) Quantification of the percentage of MC podocytes per field of view in B. Total of 100 fields of view in 5 independent experiments. (E) Quantification of the percentage of chromosome bridge per field of view in C. n = 5 independent experiments. *P < 0.05 vs. control. (F) Immunofluorescence images of primary culture podocytes stained with γ-H2AX (green, double-strand breaks [DSBs] marker) and WT1 (red) showing abnormal MC podocytes in Pfn1-KO mice, as indicated by the arrows. Scale bar: 20 μm. (G) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in F. Total of 400 cells in 5 independent experiments. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Techniques Used: Transmission Assay, Control, Immunofluorescence, Isolation, Staining, Marker

    Figure 5. Loss of podocyte Pfn1 results in podocytopenia and cell cycle entry. (A) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1-KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). (B) Representative immunofluorescence images of podocytes in control-FUCCI and Pfn1-KO-FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. (C) Quantification of the distribution of podocytes in cell cycle phase in B. Total of 100 glomeruli in 5 different mice. (D) Primary podocytes isolated from control and Pfn1-KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. (E) Representative immunofluorescence images of podocytes in control and Pfn1-KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. (F) Quantification of podocyte density in glomeruli in E. Total of 40 glomeruli from 5 different mice. (G) Representative immunofluorescence images of urinary podocytes from control and Pfn1-KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.
    Figure Legend Snippet: Figure 5. Loss of podocyte Pfn1 results in podocytopenia and cell cycle entry. (A) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1-KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). (B) Representative immunofluorescence images of podocytes in control-FUCCI and Pfn1-KO-FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. (C) Quantification of the distribution of podocytes in cell cycle phase in B. Total of 100 glomeruli in 5 different mice. (D) Primary podocytes isolated from control and Pfn1-KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. (E) Representative immunofluorescence images of podocytes in control and Pfn1-KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. (F) Quantification of podocyte density in glomeruli in E. Total of 40 glomeruli from 5 different mice. (G) Representative immunofluorescence images of urinary podocytes from control and Pfn1-KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Techniques Used: Immunofluorescence, Control, Staining, Isolation



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    Image Search Results


    Figure 1. Generation of podocyte-specific Pfn1-KO mice results in severe proteinuria and kidney failure. (A) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1-KO mice (age P7). (B) Representative immunofluorescence images of profilin1 (green) and WT1 (red) in control and Pfn1-KO primary podocytes. Scale bar: 10 μm. (C) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1-KO mice (age 3 weeks). Scale bars: 10 μm. (D) Pfn1-KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. *P < 0.05 vs. control. (E) The survival curve of Pfn1-KO mice (red) demon- strates approximately 90% death by 12 weeks of age. n = 8 mice. (F) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1-KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. (G) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. *P < 0.05 vs. control. (H) Elevated plasma creatinine in Pfn1-KO mice at 3, 6, and 10 weeks of age. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: Journal of Clinical Investigation

    Article Title: Profilin1 is required to prevent mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/jci171237

    Figure Lengend Snippet: Figure 1. Generation of podocyte-specific Pfn1-KO mice results in severe proteinuria and kidney failure. (A) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1-KO mice (age P7). (B) Representative immunofluorescence images of profilin1 (green) and WT1 (red) in control and Pfn1-KO primary podocytes. Scale bar: 10 μm. (C) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1-KO mice (age 3 weeks). Scale bars: 10 μm. (D) Pfn1-KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. *P < 0.05 vs. control. (E) The survival curve of Pfn1-KO mice (red) demon- strates approximately 90% death by 12 weeks of age. n = 8 mice. (F) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1-KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. (G) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. *P < 0.05 vs. control. (H) Elevated plasma creatinine in Pfn1-KO mice at 3, 6, and 10 weeks of age. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti– mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817- 31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Western Blot, Expressing, Isolation, Control, Immunofluorescence, SDS Page, Staining, Clinical Proteomics

    Figure 3. Loss of podocyte Pfn1 results in morphologic MC appearance, chromosomal instability, and dsDNA damage. (A) Representative images of transmission electron micrography demonstrate abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control glomeruli at 4 weeks of age. Scale bar: 1 μm. (B) Immunofluorescence images of primary podocytes isolated from control and Pfn1-KO mice at P7 stained with WT1 (red, podocyte marker) and Hoechst (blue, DNA marker) showing abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control podocytes. Scale bar: 20 μm. (C) Immunofluorescence images of primary culture podocytes isolated from control and Pfn1-KO mice stained with Hoechst and anti-tubulin antibody showing the chromosome bridge (arrow) in a MC Pfn1-KO podocyte. Scale bar: 20 μm. (D) Quantification of the percentage of MC podocytes per field of view in B. Total of 100 fields of view in 5 independent experiments. (E) Quantification of the percentage of chromosome bridge per field of view in C. n = 5 independent experiments. *P < 0.05 vs. control. (F) Immunofluorescence images of primary culture podocytes stained with γ-H2AX (green, double-strand breaks [DSBs] marker) and WT1 (red) showing abnormal MC podocytes in Pfn1-KO mice, as indicated by the arrows. Scale bar: 20 μm. (G) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in F. Total of 400 cells in 5 independent experiments. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: Journal of Clinical Investigation

    Article Title: Profilin1 is required to prevent mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/jci171237

    Figure Lengend Snippet: Figure 3. Loss of podocyte Pfn1 results in morphologic MC appearance, chromosomal instability, and dsDNA damage. (A) Representative images of transmission electron micrography demonstrate abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control glomeruli at 4 weeks of age. Scale bar: 1 μm. (B) Immunofluorescence images of primary podocytes isolated from control and Pfn1-KO mice at P7 stained with WT1 (red, podocyte marker) and Hoechst (blue, DNA marker) showing abnormal MC podocytes in Pfn1-KO mice (arrow) compared with control podocytes. Scale bar: 20 μm. (C) Immunofluorescence images of primary culture podocytes isolated from control and Pfn1-KO mice stained with Hoechst and anti-tubulin antibody showing the chromosome bridge (arrow) in a MC Pfn1-KO podocyte. Scale bar: 20 μm. (D) Quantification of the percentage of MC podocytes per field of view in B. Total of 100 fields of view in 5 independent experiments. (E) Quantification of the percentage of chromosome bridge per field of view in C. n = 5 independent experiments. *P < 0.05 vs. control. (F) Immunofluorescence images of primary culture podocytes stained with γ-H2AX (green, double-strand breaks [DSBs] marker) and WT1 (red) showing abnormal MC podocytes in Pfn1-KO mice, as indicated by the arrows. Scale bar: 20 μm. (G) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in F. Total of 400 cells in 5 independent experiments. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti– mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817- 31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Transmission Assay, Control, Immunofluorescence, Isolation, Staining, Marker

    Figure 5. Loss of podocyte Pfn1 results in podocytopenia and cell cycle entry. (A) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1-KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). (B) Representative immunofluorescence images of podocytes in control-FUCCI and Pfn1-KO-FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. (C) Quantification of the distribution of podocytes in cell cycle phase in B. Total of 100 glomeruli in 5 different mice. (D) Primary podocytes isolated from control and Pfn1-KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. (E) Representative immunofluorescence images of podocytes in control and Pfn1-KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. (F) Quantification of podocyte density in glomeruli in E. Total of 40 glomeruli from 5 different mice. (G) Representative immunofluorescence images of urinary podocytes from control and Pfn1-KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: Journal of Clinical Investigation

    Article Title: Profilin1 is required to prevent mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/jci171237

    Figure Lengend Snippet: Figure 5. Loss of podocyte Pfn1 results in podocytopenia and cell cycle entry. (A) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1-KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). (B) Representative immunofluorescence images of podocytes in control-FUCCI and Pfn1-KO-FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. (C) Quantification of the distribution of podocytes in cell cycle phase in B. Total of 100 glomeruli in 5 different mice. (D) Primary podocytes isolated from control and Pfn1-KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. (E) Representative immunofluorescence images of podocytes in control and Pfn1-KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. (F) Quantification of podocyte density in glomeruli in E. Total of 40 glomeruli from 5 different mice. (G) Representative immunofluorescence images of urinary podocytes from control and Pfn1-KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. *P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti– mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817- 31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Immunofluorescence, Control, Staining, Isolation

    ( A ) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1 -KO mice (age P7). ( B ) Representative immunofluorescence images of profilin1 (green) and WT1 (red) in control and Pfn1 -KO primary podocytes. Scale bar: 10 μm. ( C ) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1- KO mice (age 3 weeks). Scale bars: 10 μm. ( D ) Pfn1- KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. * P < 0.05 vs. control. ( E ) The survival curve of Pfn1- KO mice (red) demonstrates approximately 90% death by 12 weeks of age. n = 8 mice. ( F ) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1 -KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. ( G ) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. * P < 0.05 vs. control. ( H ) Elevated plasma creatinine in Pfn1- KO mice at 3, 6, and 10 weeks of age. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: The Journal of Clinical Investigation

    Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/JCI171237

    Figure Lengend Snippet: ( A ) Representative immunoblot images of profilin1 expression in primary podocytes freshly isolated from littermate control (Ctrl) and Pfn1 -KO mice (age P7). ( B ) Representative immunofluorescence images of profilin1 (green) and WT1 (red) in control and Pfn1 -KO primary podocytes. Scale bar: 10 μm. ( C ) Representative immunofluorescence images of profilin1 (green) and nephrin (red) on kidney sections of control and Pfn1- KO mice (age 3 weeks). Scale bars: 10 μm. ( D ) Pfn1- KO mice (red) failed to gain body weight by 8 weeks of age compared with control mice (green). n = 9 mice. * P < 0.05 vs. control. ( E ) The survival curve of Pfn1- KO mice (red) demonstrates approximately 90% death by 12 weeks of age. n = 8 mice. ( F ) SDS-PAGE (Coomassie blue staining) of standard BSA and of urine samples from Pfn1 -KO mice at 4 weeks of age demonstrates albuminuria. Equal volumes of standard BSA and urine (4 μL) were loaded in each lane. ( G ) Quantification of urine albumin normalized to creatinine at 2, 6, and 10 weeks of age. n = 6 mice. * P < 0.05 vs. control. ( H ) Elevated plasma creatinine in Pfn1- KO mice at 3, 6, and 10 weeks of age. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Western Blot, Expressing, Isolation, Control, Immunofluorescence, SDS Page, Staining, Clinical Proteomics

    ( A ) Representative images of transmission electron micrography demonstrate abnormal MC podocytes in Pfn1 -KO mice (arrow) compared with control glomeruli at 4 weeks of age. Scale bar: 1 μm. ( B ) Immunofluorescence images of primary podocytes isolated from control and Pfn1 -KO mice at P7 stained with WT1 (red, podocyte marker) and Hoechst (blue, DNA marker) showing abnormal MC podocytes in Pfn1- KO mice (arrow) compared with control podocytes. Scale bar: 20 μm. ( C ) Immunofluorescence images of primary culture podocytes isolated from control and Pfn1 -KO mice stained with Hoechst and anti-tubulin antibody showing the chromosome bridge (arrow) in a MC Pfn1- KO podocyte. Scale bar: 20 μm. ( D ) Quantification of the percentage of MC podocytes per field of view in B . Total of 100 fields of view in 5 independent experiments. ( E ) Quantification of the percentage of chromosome bridge per field of view in C . n = 5 independent experiments. * P < 0.05 vs. control. ( F ) Immunofluorescence images of primary culture podocytes stained with γ-H2AX (green, double-strand breaks [DSBs] marker) and WT1 (red) showing abnormal MC podocytes in Pfn1- KO mice, as indicated by the arrows. Scale bar: 20 μm. ( G ) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in F . Total of 400 cells in 5 independent experiments. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: The Journal of Clinical Investigation

    Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/JCI171237

    Figure Lengend Snippet: ( A ) Representative images of transmission electron micrography demonstrate abnormal MC podocytes in Pfn1 -KO mice (arrow) compared with control glomeruli at 4 weeks of age. Scale bar: 1 μm. ( B ) Immunofluorescence images of primary podocytes isolated from control and Pfn1 -KO mice at P7 stained with WT1 (red, podocyte marker) and Hoechst (blue, DNA marker) showing abnormal MC podocytes in Pfn1- KO mice (arrow) compared with control podocytes. Scale bar: 20 μm. ( C ) Immunofluorescence images of primary culture podocytes isolated from control and Pfn1 -KO mice stained with Hoechst and anti-tubulin antibody showing the chromosome bridge (arrow) in a MC Pfn1- KO podocyte. Scale bar: 20 μm. ( D ) Quantification of the percentage of MC podocytes per field of view in B . Total of 100 fields of view in 5 independent experiments. ( E ) Quantification of the percentage of chromosome bridge per field of view in C . n = 5 independent experiments. * P < 0.05 vs. control. ( F ) Immunofluorescence images of primary culture podocytes stained with γ-H2AX (green, double-strand breaks [DSBs] marker) and WT1 (red) showing abnormal MC podocytes in Pfn1- KO mice, as indicated by the arrows. Scale bar: 20 μm. ( G ) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in F . Total of 400 cells in 5 independent experiments. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Transmission Assay, Control, Immunofluorescence, Isolation, Staining, Marker

    ( A ) Representative immunoblot images of cyclin D1, cyclin B1, P21, profilin1, and WT1 as loading control in control and Pfn1 -KO mouse primary podocytes. ( B ) Quantification of immunoblots in A . n = 5 independent experiments. ( C ) Representative immunofluorescence image of P21 expression in control and Pfn1- KO mouse glomeruli at 5 weeks of age stained with P21 (green) and WT1 (red). Scale bar: 20 μm. ( D ) Quantification of immunofluorescence intensity of nuclear P21 per podocyte in C . Total of 310 podocytes in 5 mice. ( E ) Representative immunofluorescence images of P53 in control and Pfn1- KO mouse glomeruli at 5 weeks of age stained with P53 (green) and WT1 (red). Scale bar: 20 μm. ( F ) Quantification of immunofluorescence intensity of nuclear P53 per podocyte in E . Total of 310 podocytes in 5 different mice. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: The Journal of Clinical Investigation

    Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/JCI171237

    Figure Lengend Snippet: ( A ) Representative immunoblot images of cyclin D1, cyclin B1, P21, profilin1, and WT1 as loading control in control and Pfn1 -KO mouse primary podocytes. ( B ) Quantification of immunoblots in A . n = 5 independent experiments. ( C ) Representative immunofluorescence image of P21 expression in control and Pfn1- KO mouse glomeruli at 5 weeks of age stained with P21 (green) and WT1 (red). Scale bar: 20 μm. ( D ) Quantification of immunofluorescence intensity of nuclear P21 per podocyte in C . Total of 310 podocytes in 5 mice. ( E ) Representative immunofluorescence images of P53 in control and Pfn1- KO mouse glomeruli at 5 weeks of age stained with P53 (green) and WT1 (red). Scale bar: 20 μm. ( F ) Quantification of immunofluorescence intensity of nuclear P53 per podocyte in E . Total of 310 podocytes in 5 different mice. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Western Blot, Control, Immunofluorescence, Expressing, Staining

    ( A ) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1 -KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). ( B ) Representative immunofluorescence images of podocytes in control -FUCCI and Pfn1- KO- FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. ( C ) Quantification of the distribution of podocytes in cell cycle phase in B . Total of 100 glomeruli in 5 different mice. ( D ) Primary podocytes isolated from control and Pfn1 -KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. ( E ) Representative immunofluorescence images of podocytes in control and Pfn1- KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. ( F ) Quantification of podocyte density in glomeruli in E . Total of 40 glomeruli from 5 different mice. ( G ) Representative immunofluorescence images of urinary podocytes from control and Pfn1 -KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G 1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G 2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Journal: The Journal of Clinical Investigation

    Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/JCI171237

    Figure Lengend Snippet: ( A ) A schematic representing cell cycle phase visualization by different colors in podocytes from the R26Fucci2aR Pfn1 -KO mice. G1 phase nuclei (mCherry, red); S phase nuclei (both mCherry and mVenus, yellow); and G2 phase nuclei (mVenus, green). ( B ) Representative immunofluorescence images of podocytes in control -FUCCI and Pfn1- KO- FUCCI glomeruli at 4 weeks of age stained with Cherry (red), Venus (green), and WT1 (blue). Scale bar: 20 μm. ( C ) Quantification of the distribution of podocytes in cell cycle phase in B . Total of 100 glomeruli in 5 different mice. ( D ) Primary podocytes isolated from control and Pfn1 -KO mice demonstrated a significant decrease in adhesion after plating for 5 minutes and 10 minutes on the collagen type I–coated plates. n = 6 independent experiments. ( E ) Representative immunofluorescence images of podocytes in control and Pfn1- KO glomeruli at 7 weeks of age stained with WT1 (red). Scale bar: 20 μm. ( F ) Quantification of podocyte density in glomeruli in E . Total of 40 glomeruli from 5 different mice. ( G ) Representative immunofluorescence images of urinary podocytes from control and Pfn1 -KO mice at 4 weeks of age stained with mCherry (red), mVenus (green), and WT1 (blue). G 1 phase podocyte nuclei (red arrow), S phase podocyte nuclei (yellow arrow), and G 2 phase podocyte nuclei (green arrow) were depicted. Scale bar: 20 μm. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Immunofluorescence, Control, Staining, Isolation

    ( A ) Representative immunofluorescence images of podocytes in control and Pfn1- KO glomeruli at 5 weeks of age stained with Rrp8 (green) and WT1 (red). Scale bar: 20 μm. ( B ) Quantification of immunofluorescence intensity of Rrp8 per podocyte in A . Total of 320 podocytes in 6 different mice. ( C ) Representative immunofluorescence images of podocytes isolated from control and Pfn1- KO mice at P7 stained with Rrp8 (red) and Hoechst (blue). Scale bar: 20 μm. ( D ) Quantification of immunofluorescence intensity of Rrp8 per podocyte in C . Total of 320 podocytes in 5 independent experiments. ( E and G ) Representative immunofluorescence images of podocyte isolated from Pfn1- KO mouse at P7 transduced with mouse Rrp8 lentiviral activation particles or control particles (Ctrl), followed by staining with Rrp8 (red) and Hoechst (blue), as shown in E , or γH2AX (green) and WT1 (red), as shown in G . Scale bar: 20 μm. ( F ) Quantification of the percentage of MC podocytes per field of view in E . Total of 100 fields of view in 5 independent experiments. ( H ) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in G . Total of 400 cells in 5 independent experiments. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test. OE, overexpression.

    Journal: The Journal of Clinical Investigation

    Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/JCI171237

    Figure Lengend Snippet: ( A ) Representative immunofluorescence images of podocytes in control and Pfn1- KO glomeruli at 5 weeks of age stained with Rrp8 (green) and WT1 (red). Scale bar: 20 μm. ( B ) Quantification of immunofluorescence intensity of Rrp8 per podocyte in A . Total of 320 podocytes in 6 different mice. ( C ) Representative immunofluorescence images of podocytes isolated from control and Pfn1- KO mice at P7 stained with Rrp8 (red) and Hoechst (blue). Scale bar: 20 μm. ( D ) Quantification of immunofluorescence intensity of Rrp8 per podocyte in C . Total of 320 podocytes in 5 independent experiments. ( E and G ) Representative immunofluorescence images of podocyte isolated from Pfn1- KO mouse at P7 transduced with mouse Rrp8 lentiviral activation particles or control particles (Ctrl), followed by staining with Rrp8 (red) and Hoechst (blue), as shown in E , or γH2AX (green) and WT1 (red), as shown in G . Scale bar: 20 μm. ( F ) Quantification of the percentage of MC podocytes per field of view in E . Total of 100 fields of view in 5 independent experiments. ( H ) Quantification of γH2AX foci per podocyte nucleus (left) and per podocyte nuclear area (right) in G . Total of 400 cells in 5 independent experiments. * P < 0.05 vs. control. Statistics were analyzed via a 2-tailed t test. OE, overexpression.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Immunofluorescence, Control, Staining, Isolation, Transduction, Activation Assay, Over Expression

    ( A ) The percentage of patients with MC podocytes observed in kidney biopsy specimens using transmission electron microscopy (TEM) in patients with nonglomerular disease following nephrectomy (control; 0 of 5 patients, 0%), patients with focal segmental glomerulosclerosis (FSGS; 3 of 20 patients, 15%), patients with diabetic kidney disease (DKD; 3 of 26 patients, 11.5%), patients with primary membranous nephropathy (pMN; 16 of 145 patients, 11%), patients with lupus nephritis (LN; 3 of 33 patients, 9.1%), and patients with IgA nephropathy (IgAN; 7 of 110 patients, 6.4%) hospitalized between September 2019 and August 2022. ( B ) Representative TEM images of kidney biopsy specimens. Mononucleated podocytes in control and MC podocytes in patients with FSGS, DKD, pMN, LN, and IgAN are depicted with arrows. Scale bar: 2 μm. ( C ) Representative immunofluorescence images of glomeruli in control patients and patients with FSGS, DKD, pMN, LN, and IgAN with MC podocytes observed with TEM stained with profilin1 (green) and nephrin (red). Scale bar: 20 μm. ( D ) Quantification of C examining mean immunofluorescence intensity of profilin1 in the glomeruli. Total of 17 glomeruli in 3 patients from each group. * P < 0.05. Statistics were analyzed via a 1-way ANOVA with Dunnett’s correction. ( E ) Representative immunofluorescence images of urine samples in patients with FSGS, DKD, pMN, LN, and IgAN with MC podocytes stained with WT1 (green) and DAPI (blue). Arrows denote multinuclei. Scale bar: 20 μm. ( F ) Quantification of urine protein excretion in patients with FSGS, DKD, pMN, LN, and IgAN at the time of kidney biopsy. MC podocytes and non-MC podocytes were observed with TEM individually in each group.* P < 0.05. Statistics were analyzed via a 2-tailed t test.

    Journal: The Journal of Clinical Investigation

    Article Title: Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases

    doi: 10.1172/JCI171237

    Figure Lengend Snippet: ( A ) The percentage of patients with MC podocytes observed in kidney biopsy specimens using transmission electron microscopy (TEM) in patients with nonglomerular disease following nephrectomy (control; 0 of 5 patients, 0%), patients with focal segmental glomerulosclerosis (FSGS; 3 of 20 patients, 15%), patients with diabetic kidney disease (DKD; 3 of 26 patients, 11.5%), patients with primary membranous nephropathy (pMN; 16 of 145 patients, 11%), patients with lupus nephritis (LN; 3 of 33 patients, 9.1%), and patients with IgA nephropathy (IgAN; 7 of 110 patients, 6.4%) hospitalized between September 2019 and August 2022. ( B ) Representative TEM images of kidney biopsy specimens. Mononucleated podocytes in control and MC podocytes in patients with FSGS, DKD, pMN, LN, and IgAN are depicted with arrows. Scale bar: 2 μm. ( C ) Representative immunofluorescence images of glomeruli in control patients and patients with FSGS, DKD, pMN, LN, and IgAN with MC podocytes observed with TEM stained with profilin1 (green) and nephrin (red). Scale bar: 20 μm. ( D ) Quantification of C examining mean immunofluorescence intensity of profilin1 in the glomeruli. Total of 17 glomeruli in 3 patients from each group. * P < 0.05. Statistics were analyzed via a 1-way ANOVA with Dunnett’s correction. ( E ) Representative immunofluorescence images of urine samples in patients with FSGS, DKD, pMN, LN, and IgAN with MC podocytes stained with WT1 (green) and DAPI (blue). Arrows denote multinuclei. Scale bar: 20 μm. ( F ) Quantification of urine protein excretion in patients with FSGS, DKD, pMN, LN, and IgAN at the time of kidney biopsy. MC podocytes and non-MC podocytes were observed with TEM individually in each group.* P < 0.05. Statistics were analyzed via a 2-tailed t test.

    Article Snippet: Rabbit anti-profilin1 monoclonal antibody (used for immunofluorescence, Thermo Fisher Scientific, catalog MA5-32683); rabbit anti-profilin1 polyclonal antibody (used for Western blotting, Cell Signaling Technology, catalog 3237S); guinea pig anti-nephrin polyclonal antibody (Progen, catalog GP-N2); rabbit anti-Wilms tumor 1 (WT1) monoclonal antibody (Abcam, catalog ab89901); mouse anti-WT1 monoclonal antibody (Novus Biologicals, catalog NB11-60011); rabbit anti-p21 monoclonal antibody (Abcam, catalog ab188224); mouse anti-p53 monoclonal antibody (Cell Signaling Technology, catalog 2524S); rabbit anti-cyclin monoclonal D1 antibody (Cell Signaling Technology, catalog 2978S); mouse anti-cyclin polyclonal B1 antibody (Cell Signaling Technology, catalog 4138S); mouse anti–Ser 319–phosphorylated γH2AX monoclonal antibody (EMD Millipore, catalog 05-636); rat anti-mCherry monoclonal antibody (Invitrogen, catalog M11217); goat anti-mVenus polyclonal antibody (MyBioSource, catalog MBS448126); Hoechst (Thermo Fisher Scientific, catalog 62249); rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, catalog 2118S); mouse anti-GFP monoclonal antibody (Roche, catalog 11814460001); Alexa Fluor 488–conjugated phalloidin (Invitrogen, catalog A12379); Alexa Fluor 594–conjugated phalloidin (Invitrogen, catalog A12381); Alexa Fluor 488–conjugated tubulin (Abcam, catalog ab195883); Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen, catalog A11029); Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen, catalog A11034); Alexa Fluor 488 donkey anti-goat IgG antibody (Invitrogen, catalog A11055); Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, catalog A21207); Alexa Fluor 594 goat anti-mouse (Invitrogen, catalog A11032); Alexa Fluor 594 goat anti-guinea pig (Invitrogen, catalog A11076); Alexa Fluor 594 goat rat (Invitrogen, catalog A11007); Alexa Fluor 647 goat anti-rabbit (Invitrogen, catalog A21245); mouse anti–mouse IgG HRP-conjugated antibody (Rockland, catalog 18-8817-31); and rabbit anti–mouse IgG HRP-conjugated antibody (Millipore Sigma, catalog AP160P) were purchased commercially.

    Techniques: Transmission Assay, Electron Microscopy, Control, Immunofluorescence, Staining

    Quantification of relative mRNA expression in kidney tissues. Nphs1 ( A ) , Nphs2 ( B ) , WT1 ( C ) , and Lrp2 ( D ) mRNA expression in kidney tissues of the control, diabetic, and treated diabetic groups; data are expressed as fold-change vs. control group values, normalized to Gapdh ; CTL, control (n = 11); DB, diabetic (n = 6); INS, insulin (n = 11); INS + AFA, insulin + aqueous extract of P. edulis (n = 9); # P < 0.05 vs. control group; *P < 0.05 vs. diabetic group; § P < 0.05 vs. insulin group.

    Journal: Scientific Reports

    Article Title: The nephroprotective action of Passiflora edulis in streptozotocin-induced diabetes

    doi: 10.1038/s41598-022-21826-9

    Figure Lengend Snippet: Quantification of relative mRNA expression in kidney tissues. Nphs1 ( A ) , Nphs2 ( B ) , WT1 ( C ) , and Lrp2 ( D ) mRNA expression in kidney tissues of the control, diabetic, and treated diabetic groups; data are expressed as fold-change vs. control group values, normalized to Gapdh ; CTL, control (n = 11); DB, diabetic (n = 6); INS, insulin (n = 11); INS + AFA, insulin + aqueous extract of P. edulis (n = 9); # P < 0.05 vs. control group; *P < 0.05 vs. diabetic group; § P < 0.05 vs. insulin group.

    Article Snippet: Western blot analysis was performed using the following antibodies: rabbit monoclonal nephrin (ab216341; Abcam Plc, Cambridge, UK), rabbit monoclonal podocin (ab181143; Abcam Plc), rabbit polyclonal LRP2 (megalin) (PA5-67900; Invitrogen), rabbit polyclonal GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (PA1-987; Invitrogen), horseradish peroxidase-conjugated goat anti-rabbit IgG (ab97051; Abcam Plc), mouse monoclonal WT1 (sc-393498; Santa Cruz Biotechnology, Texas, USA), and horseradish peroxidase-conjugated goat anti-mouse IgG (sc-2005; Santa Cruz Biotechnology).

    Techniques: Expressing, Control

    WT1 expression on EpCAM + cancer cells and detection of memory subsets of WT1-CTLs in MPE samples. ( A ) Expression of WT1 on EpCAM positive or negative cells sorted from whole cells in MPE 1st . PC, phase contrast; WT1 cells were stained with anti-WT1 antibody, followed by Alexa Fluor® Plus 488 secondary antibody; DNA, DAPI staining. The white bar indicates 100 μm. ( B ) The ratio of WT-CTLs is defined as WT1 tetramer + CD8 + T cells to MPE samples 1st or MPE 2nd (upper panel). The memory subsets of WT1-CTLs during disease progression (middle panel). CD62L + CD45RO + : central memory T cells (T CM ); CD62L + CD45RO − : naïve T cells (T N ); CD62L − CD45RO + : effector memory T cells (T EM ), CD62L − CD45RO − : terminal effector T cells (T TE ). The function of WT1-CTLs in MPE was evaluated by ELISpot assay for IFN-γ secretion under WT1 235 peptide stimulation (lower panel). The mean number of spots of WT1 235 peptide-specific was shown. The error bar indicated the mean and standard deviation. Unpaired t -test, * p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

    doi: 10.3390/ijms232012177

    Figure Lengend Snippet: WT1 expression on EpCAM + cancer cells and detection of memory subsets of WT1-CTLs in MPE samples. ( A ) Expression of WT1 on EpCAM positive or negative cells sorted from whole cells in MPE 1st . PC, phase contrast; WT1 cells were stained with anti-WT1 antibody, followed by Alexa Fluor® Plus 488 secondary antibody; DNA, DAPI staining. The white bar indicates 100 μm. ( B ) The ratio of WT-CTLs is defined as WT1 tetramer + CD8 + T cells to MPE samples 1st or MPE 2nd (upper panel). The memory subsets of WT1-CTLs during disease progression (middle panel). CD62L + CD45RO + : central memory T cells (T CM ); CD62L + CD45RO − : naïve T cells (T N ); CD62L − CD45RO + : effector memory T cells (T EM ), CD62L − CD45RO − : terminal effector T cells (T TE ). The function of WT1-CTLs in MPE was evaluated by ELISpot assay for IFN-γ secretion under WT1 235 peptide stimulation (lower panel). The mean number of spots of WT1 235 peptide-specific was shown. The error bar indicated the mean and standard deviation. Unpaired t -test, * p < 0.05.

    Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

    Techniques: Expressing, Staining, Biomarker Discovery, Enzyme-linked Immunospot, Standard Deviation

    Detection of CD8 + T cell response to HLA-A*11:01 restricted SMAD4 P130L neoantigen in MPE. ( A ) WT1 tetramer-CD8 + T cells sorted from MPE 1st were expanded with HPL-IFN-DCs containing each predicted neoantigen peptide for SMAD4 P130L . For ELISpot assay to detect IFN-γ production after in vitro expansion, these cells were stimulated using each SMAD4 P130L peptide. Reactivity was shown in the box plot (per each well in 96 plates). DMSO was used as a negative control. A Wilcoxon signed-rank test was performed, * p < 0.05. ( B ) CD8 + T cells purified from in vitro expanded cells with HPL-IFN-DCs contained SMAD4-Neo1 peptides in A were stimulated by HEV0271 cells (HLA-A*11:01 homozygous) containing SMAD4-Neo1 (SVCVNLYH) or SMAD4-WT1 (SVCVNPYH). The reactivity to IFN-γ was evaluated by ELISpot assay. The error bar indicated the mean and standard deviation. Dunnett’s test, * p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

    doi: 10.3390/ijms232012177

    Figure Lengend Snippet: Detection of CD8 + T cell response to HLA-A*11:01 restricted SMAD4 P130L neoantigen in MPE. ( A ) WT1 tetramer-CD8 + T cells sorted from MPE 1st were expanded with HPL-IFN-DCs containing each predicted neoantigen peptide for SMAD4 P130L . For ELISpot assay to detect IFN-γ production after in vitro expansion, these cells were stimulated using each SMAD4 P130L peptide. Reactivity was shown in the box plot (per each well in 96 plates). DMSO was used as a negative control. A Wilcoxon signed-rank test was performed, * p < 0.05. ( B ) CD8 + T cells purified from in vitro expanded cells with HPL-IFN-DCs contained SMAD4-Neo1 peptides in A were stimulated by HEV0271 cells (HLA-A*11:01 homozygous) containing SMAD4-Neo1 (SVCVNLYH) or SMAD4-WT1 (SVCVNPYH). The reactivity to IFN-γ was evaluated by ELISpot assay. The error bar indicated the mean and standard deviation. Dunnett’s test, * p < 0.05.

    Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

    Techniques: Enzyme-linked Immunospot, In Vitro, Negative Control, Purification, Standard Deviation

    Memory subsets of WT1 tetramer − CD8 + T cells and detection of SMAD4 P130L -specific CD8 + T cell expanded from MPE samples. ( A ) WT1 tetramer − CD8 + T cells in MPE 1st or MPE 2nd were stained with antibodies against CD62L and CD45RO for memory T cell subsets. ( B ) WT1 tetramer − CD8 + T cells were sorted from MPE 1st or MPE 2nd , expanding in vitro with HPL-IFN-DCs containing SMAD4-Neo1 peptide. These cells were stimulated with SMAD4-Neo1 or DMSO to detect IFN-γ-producing cells by ELISpot assay. The ratio of IFN-γ spots of SMAD4-Neo1 to control was shown in the box plot (per each well in 96 plates). Mann–Whitney U test, * p < 0.05.

    Journal: International Journal of Molecular Sciences

    Article Title: The Detection of Immunity against WT1 and SMAD4 P130L of EpCAM + Cancer Cells in Malignant Pleural Effusion

    doi: 10.3390/ijms232012177

    Figure Lengend Snippet: Memory subsets of WT1 tetramer − CD8 + T cells and detection of SMAD4 P130L -specific CD8 + T cell expanded from MPE samples. ( A ) WT1 tetramer − CD8 + T cells in MPE 1st or MPE 2nd were stained with antibodies against CD62L and CD45RO for memory T cell subsets. ( B ) WT1 tetramer − CD8 + T cells were sorted from MPE 1st or MPE 2nd , expanding in vitro with HPL-IFN-DCs containing SMAD4-Neo1 peptide. These cells were stimulated with SMAD4-Neo1 or DMSO to detect IFN-γ-producing cells by ELISpot assay. The ratio of IFN-γ spots of SMAD4-Neo1 to control was shown in the box plot (per each well in 96 plates). Mann–Whitney U test, * p < 0.05.

    Article Snippet: Subsequently, 1 × 10 5 cells were fixed with a 10% formalin neutral buffer solution (Wako Pure Chemicals Ltd., Osaka, Japan) for 30 min and then permeabilized with a 0.1% Triton X-100 (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) solution in PBS at room temperature for 5 min and blocked with UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 60 min. After blocking, the cells were incubated with a mouse monoclonal primary anti-WT1 antibody (1:100, clone 6F-H2, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 16 h. Subsequently, a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor plus 488 (1:200, Thermo Fisher Scientific) was added at room temperature for 30 min.

    Techniques: Staining, In Vitro, Enzyme-linked Immunospot, Control, MANN-WHITNEY

    ( A ) Whole-mount X-gal staining of RARECreER T2 embryos pulsed with tamoxifen at E6.5 and sacrificed at E9.5 reveals strong labeling of venous pole derivatives of the heart (outflow tract (OFT) and atria (AT)). Minimal labeling is detected in the right ventricle (RV) and left ventricle (LV) of hearts (n = 2 embryos analyzed). ( B ) Whole-mount X-gal staining of embryos pulsed with tamoxifen at E10.5 and sacrificed at E13.5 reveals strong ventral (v) and dorsal (d) labeling of the heart ventricles with minimal labeling of the atria and outflow tract (n = 2 embryos analyzed). ( C ) Administration of tamoxifen (TAM) at E9.5 to RARECreER T2 ; mTmG embryos followed by analysis at E11.5 reveals specific labeling of the epicardium (GFP + WT1 + cells) and myocardium (GFP + MF20 + cells) in developing hearts (n = 3 embryos analyzed). ( D ) Tamoxifen administration at E10.5 followed by analysis at E13.5 reveals strong labeling of the compact myocardium (C) and minor labeling of the trabecular layer (T) in developing hearts. Minimal labeling of the epicardium (WT1 + cells, inset) is detected at this time-point. Insets shown are from right ventricle of representative heart (n = 3 embryos analyzed). ( E ) Exogenous supplementation of all-trans Retinoic acid (RA) (10 mg/kg) to pregnant dams 4 hr prior to tamoxifen induction leads to increased labeling of the trabecular myocardium and epicardium (WT1 + cells, inset) in developing hearts at E13.5 when compared to non-RA-treated embryos in ( D ) (n = 3 embryos analyzed). ( F ) Supplementation of the RAR reverse agonist BMS493 (5 mg/kg) to pregnant dams 4 hr before and 4 hr after tamoxifen induction (extra two doses given in 8 hour interval 1 day after TAM induction) drastically reduces the number of GFP + cells in RARECreER T2 hearts (n = 3 embryos analyzed). ( G ) Schematic illustrating strategy for isolating primary cardiomyocytes (CMs) from hearts of E18.5 RARECreER T2 ; mTmG embryos followed by 48 hr treatment with 1 µM RA. ( H ) RARECreER T2 ; mTmG primary cardiomyocytes respond directly to RA treatment as demonstrated by co-IF for GFP and Troponin T. No GFP staining is detected in DMSO-treated control (CTL) cells. ( I ) Dose-response relationship of primary cardiomyocytes to RA. Cells were isolated form neonatal RARECreER T2 ; mTmG hearts using the neonatal cardiomyocyte isolation kit (Miltenyi) and treated for 48 hr with varying doses of RA. Each data point represents the percentage of GFP + cells from a single well. Columns are means ± SEM (at least nine technical replicates per treatment). See also . Data information: WT1 = Wilms’ tumour protein, TRO = Troponin T, MF20 = Myosin heavy chain. Scale bars mosaics: 100 µM, Close ups: 40 µM. See also . Figure 2—source data 1. Numerical source data for .

    Journal: eLife

    Article Title: Retinoic acid signaling is directly activated in cardiomyocytes and protects mouse hearts from apoptosis after myocardial infarction

    doi: 10.7554/eLife.68280

    Figure Lengend Snippet: ( A ) Whole-mount X-gal staining of RARECreER T2 embryos pulsed with tamoxifen at E6.5 and sacrificed at E9.5 reveals strong labeling of venous pole derivatives of the heart (outflow tract (OFT) and atria (AT)). Minimal labeling is detected in the right ventricle (RV) and left ventricle (LV) of hearts (n = 2 embryos analyzed). ( B ) Whole-mount X-gal staining of embryos pulsed with tamoxifen at E10.5 and sacrificed at E13.5 reveals strong ventral (v) and dorsal (d) labeling of the heart ventricles with minimal labeling of the atria and outflow tract (n = 2 embryos analyzed). ( C ) Administration of tamoxifen (TAM) at E9.5 to RARECreER T2 ; mTmG embryos followed by analysis at E11.5 reveals specific labeling of the epicardium (GFP + WT1 + cells) and myocardium (GFP + MF20 + cells) in developing hearts (n = 3 embryos analyzed). ( D ) Tamoxifen administration at E10.5 followed by analysis at E13.5 reveals strong labeling of the compact myocardium (C) and minor labeling of the trabecular layer (T) in developing hearts. Minimal labeling of the epicardium (WT1 + cells, inset) is detected at this time-point. Insets shown are from right ventricle of representative heart (n = 3 embryos analyzed). ( E ) Exogenous supplementation of all-trans Retinoic acid (RA) (10 mg/kg) to pregnant dams 4 hr prior to tamoxifen induction leads to increased labeling of the trabecular myocardium and epicardium (WT1 + cells, inset) in developing hearts at E13.5 when compared to non-RA-treated embryos in ( D ) (n = 3 embryos analyzed). ( F ) Supplementation of the RAR reverse agonist BMS493 (5 mg/kg) to pregnant dams 4 hr before and 4 hr after tamoxifen induction (extra two doses given in 8 hour interval 1 day after TAM induction) drastically reduces the number of GFP + cells in RARECreER T2 hearts (n = 3 embryos analyzed). ( G ) Schematic illustrating strategy for isolating primary cardiomyocytes (CMs) from hearts of E18.5 RARECreER T2 ; mTmG embryos followed by 48 hr treatment with 1 µM RA. ( H ) RARECreER T2 ; mTmG primary cardiomyocytes respond directly to RA treatment as demonstrated by co-IF for GFP and Troponin T. No GFP staining is detected in DMSO-treated control (CTL) cells. ( I ) Dose-response relationship of primary cardiomyocytes to RA. Cells were isolated form neonatal RARECreER T2 ; mTmG hearts using the neonatal cardiomyocyte isolation kit (Miltenyi) and treated for 48 hr with varying doses of RA. Each data point represents the percentage of GFP + cells from a single well. Columns are means ± SEM (at least nine technical replicates per treatment). See also . Data information: WT1 = Wilms’ tumour protein, TRO = Troponin T, MF20 = Myosin heavy chain. Scale bars mosaics: 100 µM, Close ups: 40 µM. See also . Figure 2—source data 1. Numerical source data for .

    Article Snippet: Antibody , Anti-WT1 (mouse monoclonal) , Agilent , Cat#: M3561RRID: AB_2304486 , IF (1:100).

    Techniques: Staining, Labeling, Isolation

    Journal: eLife

    Article Title: Retinoic acid signaling is directly activated in cardiomyocytes and protects mouse hearts from apoptosis after myocardial infarction

    doi: 10.7554/eLife.68280

    Figure Lengend Snippet:

    Article Snippet: Antibody , Anti-WT1 (mouse monoclonal) , Agilent , Cat#: M3561RRID: AB_2304486 , IF (1:100).

    Techniques: Isolation, Sequencing, In Situ, Purification, Plasmid Preparation, SYBR Green Assay, Software