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. 2018 Apr;93(4):903–920. doi: 10.1016/j.kint.2017.11.014

Figure 3.

Figure 3

Notch activation coincides with onset of glomerulosclerosis in in CAGG-CreERTM−/+;Wt1f/ftransgenic mice following tamoxifen administration. (a) Relative mean transcript levels of Notch pathway components in primary podocytes of mutants compared with controls at day (D) 6 post-induction (PI). Bars represent means and error bars represent SDs. CAGG-CreERTM−/−;Wt1f/f (n = 6) versus CAGG-CreERTM+/−;Wt1f/f (n = 10): mean Notch1, 1.1 ± 0.2 versus 2.5 ± 0.5, *P = 0.05; mean Nrarp, 0.9 ± 0.4 versus 3.6 ± 0.4, **P = 0.001; mean Rbpsuh, 1.0 ± 0.1 versus 1.7 ± 0.3, P = 0.09; mean Hes1, 1.0 ± 0.1 versus 1.6 ± 0.2, P = 0.09; mean Hes3, 1.3 ± 0.4 versus 2.1 ± 0.5, P = 0.25; mean Hes5, 1.0 ± 0.2 versus 3.9 ± 1.1, P = 0.07; mean Hey1, 1.1 ± 0.3 versus 2.2 ± 0.4, P = 0.11; mean Hey2, 1.2 ± 0.3 versus 4.0 ± 1, *P = 0.04; mean HeyL, 1.1 ± 0.2 versus 2.3 ± 0.5, P = 0.14; mean MFng, 1.9 ± 0.9 versus 5.0 ± 2.4, P = 0.19; mean FoxC2, 1.2 ± 0.4 versus 0.3 ± 0.2, *P = 0.04, Student t-test. (b) Shown are representative images of glomeruli following double immunofluorescence labeling of D6 PI mouse kidney sections with anticleaved Notch1 (Val1744) and anti-Nestin of CAGG-CreERTM−/−;Wt1f/f (controls) and CAGG-CreERTM+/−;Wt1f/f (mutants) transgenic mice following multichannel labeling with Alexa Fluor 488–conjugated secondary antibody (Nestin, demarcates podocytes) and Alexa Fluor 594–conjugated secondary antibody (cleaved Notch1). Sections are counterstained with DAPI. Arrows show positive cleaved Notch1 nuclear staining in mutant glomeruli within Nestin-positive cells that are not evident in control glomeruli. Bars = 50 μm. Inset shows high-power view of same cells. Bar = 10 μm. (c,d) Representative images of Western blot analyses of protein derived from primary podocytes isolated from mutants and control mice. Immunoblots show that cleaved Notch1 (Val1744) (c) and Pofut1 (d) are expressed at D6 PI in mutant podocytes but not in controls. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.