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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Nat Metab. 2020 Nov 9;2(11):1350–1367. doi: 10.1038/s42255-020-00306-2

Extended Data Fig. 6. Validation of epithelial specific deletion of c-Rel in kidney and lung fibrosis models.

Extended Data Fig. 6

(a) Flow cytometry gating strategy to identify immune cells (CD45+), epithelial cells (EPCAM+) and endothelial cells (CD31+) isolated from the kidney or lungs of Rel flfl, Rel ΔTEC or Rel ΔAEC mice respectively. (b-c) Ex vivo images of GFP fluorescence signal in the left and right kidneys, heart, liver, lungs and spleen of Rel ΔTEC mice (b) or Rel ΔAEC mice (c) imaged using an In Vivo Imaging System (IVIS). Graph and flow cytometry histograms show the Mean Fluorescence Intensity (MFI) of GFP in EpCAM+, CD31+, CD45+ and EpCAM-CD31-CD45- cells from the kidney of Rel ΔTEC and Relfl/flcontrol mice (b) or from the lung of Rel ΔAEC and Relfl/flcontrol mice (c), n=5 mice/group. (d) Heatmap showing mRNA levels of Tgfb1, Col1a1, Col1a2, Acta2 and Timp1 in control versus UUO kidney of Rel flfl, Rel ΔTEC and Rel ΔLysM mice (left) or control versus bleomycin lung of Rel flfl, Rel ΔAEC and Rel ΔLysM mice (right). Heatmap data are from 2 mice/group in control kidney or lung and 5 mice/group in the injured kidney or lung. (e) Quantification of hydroxyproline levels in nM per left lobe of lung tissue from bleomycin injured in Rel flfl, Rel ΔAEC (p=0.0105), and Rel ΔLysM (p=0.0006) mice. (f) Histological assessment and representative images of TUNEL stained lung sections in day 3 bleomycin injured in Rel flfl, Rel ΔAEC, and Rel ΔLysM mice. Scale bar is 50 microns. Data are mean ± s.e.m. in n=5 mice/group. (g) Quantification of neutrophil numbers in UUO injured kidneys of Rel flfl, Rel ΔTEC (p=0.0014) and Rel ΔLysM (p<0.0001) mice and macrophage numbers in UUO injured kidneys of Rel flfl, Rel ΔTEC (p=0.0045) and Rel ΔLysM (p=0.0004) mice. (h) Quantification of neutrophil numbers in Bleomycin injured lungs of Rel flfl, Rel ΔAEC and Rel ΔLysM mice and macrophage numbers in Bleomycin injured lungs of Rel flfl, Rel ΔAEC (p=0.0018) and Rel ΔLysM (p=0.0327) mice. (e,g,h) Data are mean ± s.e.m. in a minimum of 7 mice/group for the kidney and 10 mice/group for the lung. P values were calculated using one-way ANOVA with Tukey post-hoc t-test (* P <0.05, **P <0.01 and ***P <0.001).