a, Schematic representation of Jag2 inhibition using lentiviruses (LV) carrying shRNAs. Infected GFP+ cells were cultured in ALI culture system for 23 days, when they were collected, sorted and analysed. b, Relative mRNA expression of Jag2 in tracheal epithelial cells infected with mock vector (control) or with vectors carrying 4 different shRNAs targeting Jag2 72 h after infection. c, Relative mRNA expression of Jag2 in tracheal epithelial basal cells infected with mock vector (control) or with lentivirus targeting Jag2 (shJag2 877) after 23 days in ALI. d, Relative mRNA expression of the secretory genes (Scgb1a1 and Scgb3a2) and the ciliated cell genes (FoxJ1 and c-myb) in mock (black bars) and shJag2 877 (grey bars) infected cells 23 days after ALI initiation. Relative expression is normalized to baseline transcript levels in mock-infected cells. e, Relative mRNA expression of Jag2 on sorted recombined (YFP+) basal cells and unrecombined YFP− basal cells from Tam-treated CK5-Jag2fl/fl mice (n = 3 mice). Relative expression is normalized to baseline transcript levels in YFP− cells. f, Percentage of YFP+ cells per total DAPI+ cells (efficiency of recombination) on either Tam-treated CK5-Jag2+/+ control (black bars) or Tam-treated CK5-Jag2fl/fl (white bars) mice assessed by manual counting (left graph) (n = 5 mice) or by flow cytometry (right graph) (n = 3 mice). g, Immunostaining for SCGB3A2 (left panels) and SSEA-1 (right panels) (red) in combination with YFP (green) in control (top panels) and experimental (bottom panels) mice. h, Immunostaining for AcTub (left panels) and c-MYB (right panels) (red) in combination with YFP (green) in control (top panels) and experimental (bottom panels) mice. i, Flow cytometry analysis of EpCAM+CD24+ ciliated cells and EpCAM+SSEA-1+ secretory cells in control and experimental mice. j, Percentage of epithelial (EpCAM+) basal, secretory and ciliated cells from both groups assessed by flow cytometry (n = 3 mice). k, Immunostaining for p63 (red) on control (top panel) and experimental mice (bottom panel). l, Percentage of p63+ cells per total DAPI+ cells on both groups. m, Immunostaining for FOXJ1 (green), N2ICD (red) and SCGB1A1 (cyan). n, Immunostaining for BrdU (green), p63 (red) and Ki67 (cyan) in either control (upper panels) or experimental mice (lower panels). o, Percentage of ciliated FOXJ1+ cells that incorporate BrdU after continuous administration of BrdU to Tam-treated CK5-Jag2fl/fl mice (n = 3 mice). p, Immunostaining for FOXJ1 (green) and BrdU (red) on Tam-treated CK5-Jag2fl/fl mice that received continuous BrdU (n = 3 mice). q, Immunostaining to detect apoptotic cells by TUNEL assay (green) on both groups. r, Immunostaining for YFP (green) in combination with activated caspase3 (red) on control (upper panel) or experimental mice (lower panel). f–r, Analysis performed 10 days after induction. Images are representative of n = 5 mice per condition (biological replicates) repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. Data shown in the graphs are means ± s.e.m. Nuclei, DAPI (blue). Scale bar, 20 μm.