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path4193-sup-0001-FigureS1.tifTIFF image10251KMDM2 expression in the glomerulus throughout the course of AN. MDM2 immunostaining (red) was assessed by confocal microscopy. In glomeruli the expression mostly co-localized with nephrin (green) which mark podocytes. Original magnification 400×.
path4193-sup-0002-FigureS2.tifTIFF image18562KMDM2 expression in the tubular compartment throughout the course of AN. In the tubulointerstitial compartment MDM2 staining (red) localized to lectin-positive proximal tubules (green) as well as to lectin-negative distal tubules. Original magnification 200×.
path4193-sup-0003-FigureS3.tifTIFF image2686KTNF immunostaining. Kidneys of Balb/c mice 2 weeks after adriamycin injection were stained for TNF-α. Representative images are shown at a magnification of 400×. Note TNF signal in podocytes, which is lacking in nutlin-3a-treated mice.
path4193-sup-0004-FigureS4.tifTIFF image17907KSemi-quantitative assessment of glomerular lesions. A: PAS-stained renal sections were used to quantify normal glomeruli with no lesions, glomeruli with segmental lesions, and globally sclerosed glomeruli for which representative images are shown. Original magnification 400×. B: Using these criteria, Balb/c mice with AN displayed a progressive decrease in the numbers of glomeruli with no lesions in favour of progressively increasing numbers of glomeruli with global lesions. Treatment with nutlin-3a from week 2 to week 4 had no significant effect on these measures. Data are means ± SEM for each time point.
path4193-sup-0005-FigureS5.tifTIFF image1245KMDM2 blockade and proliferation of kidney cells. Renal sections from vehicle- and nutlin-3a- treated kidneys were stained for Ki-67, a cell proliferation marker as described in methods. A: Quantitative assessment of the numbers of proliferating glomerular cells from vehicle- and nutlin-3a-treated kidneys at respective time points, from week 1 to week 2 (left graph) and from week 2 to week 4 (right graph). B: Quantitative assessment of proliferating tubular cells from vehicle- and nutlin-3a-treated kidneys at respective time points, from week 1 to week 2 (left graph) and from week 2 to week 4 (right graph). Data are means ± SEM.
path4193-sup-0006-FigureS6.tifTIFF image12921KMDM2 blockade and tubulointerstitial inflammation in AN. A: Total mRNA was prepared from vehicle- and nutlin-3a-treated kidneys. The mRNA expression levels were determined for the indicated cytokines and chemokines by real-time PCR and expressed as mean of the ratio versus the respective 18s rRNA level ± SEM. p values for the comparison of nutlin-3a- versus vehicle treatment are as indicated. B: Renal sections from vehicle- and nutlin-3a-treated kidneys at week 4 were stained for different leukocyte markers as described in methods. F4/80-positive macrophages were quantified by digital morphometry and data are expressed as percentage of hpf. CD3+ T cells were counted per hpf. Data are means ± SEM of 25 hpf per kidney per group. *p < 0.05 nutlin-3a versus vehicle-treated mice at week 4.
path4193-sup-0007-FigureS7.tifTIFF image11325KMDM2 blockade and interstitial fibrosis. A: Representative images of kidneys at week 4 from α-SMA stains of vehicle- and nutlin-3a-treated groups are shown at a magnification of 200×. B: α-SMA positive area was quantified by digital morphometry and data are expressed as percentage of hpf. C: Total mRNA was prepared from vehicle- and nutlin-3a-treated kidneys. The target mRNA expression levels were determined by real-time PCR and expressed as mean of the ratio versus the respective 18s rRNA level ± SEM. Data are means ± SEM. *p < 0.05 nutlin-3a- versus vehicle-treated mice at week 4.

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