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. Author manuscript; available in PMC: 2016 May 19.
Published in final edited form as: Nat Med. 2016 Jan 18;22(2):154–162. doi: 10.1038/nm.4035

Figure 2.

Figure 2

Pro-fibrotic role of the Notch ligand Jag1 in mouse PCECs after bleomycin injury. (a) Jag1 expression in lung cryosections of mice at day 10 after the fourth injection of bleomycin (Bleo) or PBS. Other Notch ligand expression is shown in Supplementary Fig. 3b–c; Scale bar, 50 μm in Figure 2. (b) Inducible endothelial cell–specific deletion of Jag1 in adult mice (Jag1iΔEC/iΔEC) was generated by breeding mice expressing VE-cadherin (Cdh5)-driven tamoxifen-responsive Cre (VE-Cad-CreERT2) with Jag1loxP/loxP mice. Mice with endothelial cell-specific haplodeficiency of Jag1 (Jag1iΔEC/+) served as a control. (c) Sirius red staining of control and Jag1iΔEC/iΔEC mouse lungs at day 35 after the sixth bleomycin injection. (d,e) Levels of collagen I, SMA, and hydroxyproline in injured mouse lungs at day 35 after the sixth bleomycin injection; *P< 0.05; n = 8 mice in the Jag1iΔEC/iΔEC group and n = 9 animals in the control group. Error bars denote s.e.m. in Figure 2. Quantification of protein levels is shown in Supplementary Fig. 3f,g. (f) Smad3 activity in lung fibroblasts of the indicated groups of mice was analyzed at day 35 after the sixth bleomycin injection by electrophoretic mobility shift assay (EMSA). Quantification is shown in Supplementary Fig. 5c. (g) GFP expression in WT mouse lungs after intravenous injection of endothelial cell–targeted virus encoding GFP (GfpEC). GFP localization was examined in VE-cadherin+ PCECs and E–cadherin+ AECs. The generation and characterization of endothelial cell–targeted virus are shown in Supplementary Fig. 6a–f. (h–j) After injection of EC–targeted Jag1 shRNA (shJag1EC), levels of Jag1, SMA, and hydroxyproline were examined in mouse lungs, and Hes1 protein was detected by immunoblotting in isolated lung fibroblasts. Antibody Mec13.3 recognizing endothelial-enriched antigen CD31 was coupled with pseudotyped virus expressing scrambled sequence (SrbEC) or different Jag1 shRNA clones (shJag1EC, C1–C5). Anti-CD31 mAb and Rat IgG lanes indicate mouse groups that received equal amounts of uncoupled mAb Mec13.3 or rat IgG isotype control. Assays were performed at day 35 after the sixth bleomycin injection. *P < 0.05; n = 8 mice per group. Immunoblot quantification is shown in Supplementary Fig. 6h.