Fig 1: Lack of podocyte Capns1 or Gadd45b-KO in Gak-KO mice improves podocyte numbers and adhesion.(A) Representative immunostaining for phalloidin (green) and WT1 (red) from control, Capns1-KO, Gak-KO, and Gak/Capns1-DKO mice. Scale bar: 10 µm. (B) Quantification of phalloidin staining patterns from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice primary podocytes in A. n = 5. (C) Adhesion of primary podocytes isolated from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice at P14. n = 6. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (D) Adhesion of primary podocytes isolated from control (green), Gadd45b-KO (yellow), Gak-KO (blue), and Gak/Gadd45b-DKO (light blue). n = 5. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (E) Quantification of WT1 staining per glomerulus from control, Capns1-KO, Gak-KO, and Gak/Capns1-DKO mice. n = 5 mouse kidney sections; 100 total glomeruli from each group were evaluated. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (F) Quantification of WT1 staining per glomerulus in control, Gadd45b-KO, Gak-KO, and Gak/Gadd45b-DKO mice. n = 5 mice kidney sections; 120 total glomeruli each group were evaluated. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (B, D, E, and F) Statistically analyzed via a 1-way ANOVA with Dunnett’s correction. In panels E and F, in each group, the large bar represents the mean, the small bar represents the SEM, and each dot represents 1 sample.
Fig 2: RyR2 knockout reveals an increased Calpain-specific fragmentation of human JP2 in 2 months matured hiPSC-CMs. (A) Western blots of WT versus RyR2 knockout hiPSC-CM lysates detecting the proteins RyR2, JP2 versus its Calpain-specific cleavage fragments NT1 and CT1, Calpain-1, Calpain-2, CAPNS1, and the Calpain-specific substrate a-Spectrin versus its 150 kDa a-Spectrin cleavage product SBDP. The complete absence of RyR2-specific band signals confirmed its knockout in hiPSC-CMs. ß-Actin was used as loading control. (B-D) Dot/bar plots summarizing the immunoblot data of WT versus RyR2 knockout hiPSC-CM lysates. (B) In contrast to WT hiPSC-CMs, in RyR2 knockout hiPSC-CMs the relative abundance of FL JP2 was significantly decreased, whereas the abundance of its NT1 and CT1 fragments was significantly increased. (C) Calpain-1, Calpain-2, and CAPNS1 showed significantly decreased levels in RyR2 knockout iPSC-CMs. (D) The abundance of the Calpain-specific substrate a-Spectrin and its breakdown product SBDP were not significantly changed, confirming human FL JP2 as the specific substrate of the increased Calpain activity in RyR2 KO hiPSC-CMs. Data were normalized to REVERT total protein stain (please refer to the methods section for additional details). Data are presented as mean ± SD (n = 4 biological replicates). Student’s t-test; *p < 0.05; ***p < 0.001.
Fig 3: Podocyte deletion of Capns1 improves glomerulosclerosis and kidney failure in Gak-KO mice.(A) Calpain-1/-2 activities in primary podocytes isolated from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice at P14. n = 7. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (B) Calpain-1/-2 activities in urine samples from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice at 3 weeks of age and 5 weeks of age. n = 7. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (C) Quantification of urine albumin/creatinine ratio at 1, 2, 4, and 8 weeks of age from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice. n = 7 as shown on the graph. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (D) Plasma creatinine levels at 3, 5, and 7 weeks of age from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice. n = 6. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (E) Body weight at 1, 2, 3, 4, 6, and 8 weeks of age from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/CapnS1-DKO (black) mice. n = 21. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (F) Survival curves from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice. n = 19. (G) Representative light microscopy images (H&E) from control, Capns1-KO, Gak-KO, and Gak/CAPNS1-DKO mice kidneys at 7 weeks of age. Scale bar: 25 µm. (H) Quantification of glomerulosclerosis in G. n = 4. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (I) Representative trichrome staining from control, Capns1-KO, Gak-KO, and Gak/Capns1-DKO mouse kidneys at 7 weeks of age. Scale bar: 100 µm. (J) Quantification of tubulointerstitial injuries in I. n = 4. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (K) Representative TEM images from control, Capns1-KO, Gak-KO, and Gak/Capns1-DKO mouse kidneys at 5 weeks of age. Scale bar: 1 µm. (L) Quantification of foot processes in K. n = 4 mice per group; 15 capillary tufts of glomeruli for each mouse were evaluated. (A–E, H, J, and L) Statistically analyzed via a 1-way ANOVA with Dunnett’s correction.
Fig 4: Loss of Capns1 results in increased GADD45B expression due to I?Ba cleavage and NF-?B p65 signaling in Gak-KO mice.(A) Representative Western blot images from control, Capns1-KO, Gak-KO, and Gak/CapnsS1-DKO mouse primary podocytes immunoblotted with GADD45B and Wilms tumor 1 (WT1). (B) Quantification of A by densitometry. n = 6. *P < 0.05. (C) Representative immunofluorescence image of Gak-KO mouse glomeruli demonstrates increased GADD45B expression (GADD45B in green and WT1 in red). Scale bar: 50 µm. (D) Representative immunofluorescence image of glomeruli from control and FSGS patient stained with GADD45B (green) and TLE4 (red). Scale bar: 10 µm. (E) Quantification of C. n = 5 mice; 20 glomeruli per mouse were evaluated. (F) Quantification of D. n = 3 patients; 16 glomeruli per group were evaluated. (G) Gak-KO mice podocytes show I?Ba cleavage (upper panel) and talin1 cleavage (middle panel) as determined by Western blots; these cleavage events were reduced by the podocyte-specific loss of Capns1. (H) ChIP assay using phospho-NF-?B p65 and primer sets for Gadd45b promoter in the primary podocytes of control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice. n = 7. (I) Representative Western blot images for phospho-NF-?B p65 (pNF-?B p65) (Ser536) and total NF-?B p65 (NF-?B p65) from primary podocytes in control, Capns1-KO, Gak-KO, and Gak/Capns1-DKO mice. (J) Quantification of the nuclear NF-?B p65 activities of primary podocytes isolated from control (green), Capns1-KO (red), Gak-KO (blue), and Gak/Capns1-DKO (black) mice at the age of P14. n = 6. *P < 0.05 vs. control mice; #P < 0.05 vs. Gak-KO mice. (E, F, H, and J). Statistically analyzed via a 1-way ANOVA with Dunnett’s correction.
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