Journal: Nature medicine

Article Title: Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

doi: 10.1038/nm.3901

Figure Lengend Snippet: Figure 2 Renal epithelial cells undergo a partial EMT after UUO. (a) Ksp1.3-Cre;tdTomatofl/fl mice were subjected to UUO and kidneys were collected 15 ds after surgery. Representative images (n = 20) of sections of kidneys from Ksp1.3-Cre;tdTomatofl/fl mice (KCT) 15 d after being subjected to UUO. Images are for obstructed (KCT + UUO) and contralateral non-obstructed kidneys (Control) from KCT mice stained for LTA (Lotus tetragonolobus agglutinin) + PNA (peanut agglutinin) (markers of proximal and distal tubules17), α-SMA and DAPI. tdTomato fluorescent protein expression was directly visualized. Asterisks (*) indicate α-SMA expression in tubular cells that have lost lectin expression 15 d after UUO. dt, dilated tubule; g, glomerulus; t, tubule. (b) Number of dilated and total tubules in kidneys from WT and SFKC mice 15 d after UUO. Ten different fields were counted in 12 mice per group. Data represent mean ± s.e.m. *P < 0.05, NS, nonsignificant; Mann-Whitney test. (c) Representative images (n = 5) of double Snail1 and α-SMA immunolabeling in sections from obstructed kidneys of WT mice euthanized 15 d after surgery. White asterisks indicate Snail1-positive nuclei. Green asterisks indicate double-positive Snail1 and α-SMA tubular cells. (d) Representative images (n = 5) of lectin (LTA) and Snail1 immunofluorescence in sections from the same kidneys used in b. Scale bars, 50 µm.

Article Snippet: The sections were incubated overnight at 4 °C with the following primary antibodies: biotinylated Lotus tetragonolobus agglutinin, LTA (B-1325, Vector Laboratories, Burlingame, CA); biotinylated peanut agglutinin, PNA (B-1075, Vector Laboratories, Burlingame, CA); α smooth muscle actin, (α-SMA) (Sigma-Aldrich, St Louis, MO); vimentin (sc-7557, Santa Cruz Biotechnology, Santa Cruz, CA); Snail1 (Ab1773; Abcam PLC, Cambridge, MA); phosphoNF-κB p65 (Ser276) (3037S, Cell Signaling Technology, Inc., Danvers, MA); phospho-Smad2 (Ser465/467) (3101S, Cell Signaling Technology, Inc., Danvers, MA); F4/80 (MCA497GA, Serotec, Oxford,UK); estrogen receptor alpha, ER-α (sc-543, Santa Cruz Biotechnology, Santa Cruz, CA); CD163 (sc-18796 Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Staining, Expressing, MANN-WHITNEY, Immunolabeling, Immunofluorescence