Journal: Diabetes

Article Title: Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabetic cardiomyopathy.

doi: 10.2337/db06-1662

Figure Lengend Snippet: FIG. 3. Representative histological pictures of CD68 and CD11b cells (magnification 200 and 1,000, marked by black arrows) of an STZ animal. CD11b cell with 1,000 magnification demonstrating an intravascular leukocyte before undergoing transendothelial migration. Bars representing data from histological evaluation of immunocompetent cells positive for the macrophage marker CD68 and for the leukocyte markers CD18, CD11a, and CD11b in cardiac tissue of control, STZ-induced diabetes, and STZ treated with the ICEI (STZICEI). *Significantly different compared with control and STZICEI, with P < 0.05. §Significantly different compared with control and STZ.

Article Snippet: Staining was performed with the following primary antibodies at the dilutions given (45 min, room temperature): for mouse anti-rat: ICAM-1 (clone 1A29, 1:100; Serotec, Munich, Germany), VCAM-1 (clone 5F10, 1:50; BabCO, Richmond, VA), CD18 (clone WT3, 1:50; Serotec), CD11a (clone WT1, 1:100; Serotec), CD11b (clone MRC OX-42, 1:100; Biozol, Eching, Germany), COL 1 (1:200; Abcam Limited, Cambridge, U.K.), Col3 (clone FH-7A, 1:40.000; Abcam Limited), and CD68 (clone ED1, 1:2,000; Serotec); for goat anti-rat: TNF- (1:25; R&D Systems, Wiesbaden, Germany) and IL1- (1:25; R&D Systems); and for rabbit anti-rat: IL-18 (1:25; R&D Systems).

Techniques: Migration, Marker, Control

Journal: Molecular Metabolism

Article Title: P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

doi: 10.1016/j.molmet.2020.101089

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Wilms Tumor-1 (WT1) , Boster Biological Technology , #M00199-1 , Rabbit , 1 μg/ml (IHC).

Techniques:

Journal: Molecular Metabolism

Article Title: P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

doi: 10.1016/j.molmet.2020.101089

Figure Lengend Snippet: P2Y2R deficiency protects against podocyte loss and glomerular injury in DN. (A) Relative mRNA levels of Nphs1 (nephrin) and Nphs2 (podocin) were determined by real-time PCR analysis (n = 3–5). (B) Localisation of WT1, a podocyte marker, was analysed by immunohistochemistry, and representative images are shown. The number of stained podocytes per glomeruli was counted by using ImageJ software (n = 3). (C) Kidney sections were stained with PAS staining and glomerular morphological changes were scored as described in the method section (n = 3). Data are presented as mean ± SEM. One-way ANOVA was used for statistical analysis followed by Bonferroni's multiple comparisons test. ∗∗∗p < 0.001 vs WT control mice; and ### p < 0.001 vs WT DN mice. Scale bar, 50 μm.

Article Snippet: Wilms Tumor-1 (WT1) , Boster Biological Technology , #M00199-1 , Rabbit , 1 μg/ml (IHC).

Techniques: Real-time Polymerase Chain Reaction, Marker, Immunohistochemistry, Staining, Software, Control

Journal: Cell reports

Article Title: Cdc42 is required for male germline niche development in mice.

doi: 10.1016/j.celrep.2021.109550

Figure Lengend Snippet: Figure 6. Downregulation of DMRT1 and SOX8/9 in Cdc42-deficient testis (A) Western blot analysis of DMRT1 (n = 6), SOX8/9 (n = 5), and WT1 (n = 5) in busulfan-treated testes from Cdc42-deficient mice. (B) Immunostaining of Cdc42-deficient testes with GATA4 and DMRT1, SOX8/9, or WT1 antibodies. At least 12 tubules were counted. Arrows indicate Sertoli cells lacking candidate proteins. Arrowheads indicate GATA4+ Sertoli cells with candidate protein expression. (C) Western blot analysis of GDNF and candidate proteins following in vitro PD treatment (n = 7). (D) Immunostaining of DMRT1 and SOX8/9 in WT testes following in vitro PD treatment. At least 12 tubules were counted. Arrows indicate Sertoli cells lacking candidate proteins. Arrowheads indicate GATA4+ Sertoli cells with candidate protein expression. Bar, 20 mm (B and D). Stain, Hoechst 33342 (B and D). Asterisk indicates statistical significance (p < 0.05).

Article Snippet: Reagent or resource Source Identifier Antibodies Rat anti-mouse CD117 (KIT) eBioscience Cat#14-1171; RRID:AB_467434; Lot E03982-1632 Rabbit anti-mouse CDC42 Abcam Cat#64533; RRID:AB_1310067; Lot GR3200090-2 Rat anti-mouse CDH1 Gift from Dr. Masatoshi Takeichi, RIKEN CDB RRID: CVCL_A6WX Rabbit anti-mouse CLDN11 Gift from Dr. Sachiko Tsukita, Teikyo University N/A Rabbit anti-mouse non-phospho (active) CTNNB1 (Ser33/37/Thr41) Cell Signaling Technology Cat#8814 (clone: D13A1); Lot 3 Rabbit anti-mouse CXCL12 Torrey Pines Biolabs Cat#201; RRID:AB_2335896; Lot 010718 Goat anti-mouse DMRT1 Santa Cruz Cat#104885X; Lot J2610 Goat anti-mouse FGF2 Santa Cruz Cat#1390; RRID:AB_631496; Lot F1113 Goat anti-mouse GATA4 Santa Cruz Cat#1237; Lot K1611 Rabbit anti-mouse GATA4 Abcam Cat#84593; RRID:AB_10670538; Lot GR275402-1 Rabbit anti-mouse GDNF Santa Cruz Cat#328; RRID:AB_631570; Lot F2713 Rabbit anti-GFP Medical & Biological Laboratories Cat#598; RRID:AB_591819; Lot 081 Goat anti-mouse GFRA1 R&D Systems Cat#AF560; RRID:AB_2110307; Lot BQE0514081 Rabbit anti-human phospho-histone H2A.X (gH2AX) Cell Signaling Technology Cat#2577; RRID:AB_2118010; Lot 4 Rabbit anti-mouse LAMININ Sigma Cat#L9393; RRID:AB_477163; Lot 054K4780 Rat anti-Occludin Gift from Dr. Sachiko Tsukita, Teikyo University N/A Mouse anti-mouse PAFAH1B1 Santa Cruz, Dallas, TX Cat#374586 (clone: H-7); RRID:AB_11008596; Lot H0217 Mouse anti-mouse PEA15A Santa Cruz, Dallas, TX Cat#166678 (clone H-3); RRID:AB_2236964; Lot B1418 Rabbit anti-mouse phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Cell Signaling Technology Cat#9101S; RRID:AB_331646; Lot 26 Rabbit anti-mouse SOX8 Thermo Fisher Scientific Cat#PA1-28072; RRID:AB_2196251; Lot UE2770328F Goat anti-mouse SOX9 Santa Cruz Cat#17341; RRID:AB_661281; Lot F1311 Rabbit anti-mouse SOX9 Santa Cruz Cat#20095; RRID:AB_661282; Lot l1897 Rabbit anti-mouse SYCP1 Abcam Cat#15090; RRID:AB_371671; Lot GR3184119-1 Rabbit anti-mouse SYCP3 Thermo Fisher Scientific Cat#PA1-16764; RRID:AB_568727; Lot WD3244703A Mouse anti-mouse TUBB3 Sigma Cat#T5076; RRID:AB_532291; Lot 047K4852 Rabbit anti-mouse Vimentin Cell Signaling Technology Cat#5741 (clone: D21H3); RRID:AB_10695459; Lot 1 Rabbit anti-mouse WT1 Santa Cruz Cat#192; RRID:AB_632611; Lot K0811 Alexa Fluor 488-conjugated donkey anti-goat IgG Thermo Fisher Scientific Cat#A-11055; RRID:AB_2534102; Lot 2059218 (Continued on next page) e1 Cell Reports 36, 109550, August 17, 2021

Techniques: Western Blot, Immunostaining, Expressing, In Vitro, Staining

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

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

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