Extended Data Figure 9 : Loss of Notch ligands in basal stem cells promotes secretory cell differentiation into ciliated cells without affecting proliferation or apoptosis.

From: Parent stem cells can serve as niches for their daughter cells

Extended Data Figure 9

a, Quantification of the percentage of basal PDPN+ cells that express Mib1 (left graph) on either Dox-treated CK5-Mib1+/+ control mice or Dox-treated CK5-Mib1fl/fl mice (n = 4 mice). Right graph, percentage of basal cells in which Mib1 was deleted in Dox-treated CK5-Mib1fl/fl mice (n = 4 mice). b, Immunostaining for Mib1 (red) and the basal cell marker CK5 (green). White arrowheads point to Mib1+ basal cells. c, Immunostaining for the secretory cell markers SCGB3A2 (left panels) and SSEA-1 (right panels) (red) in control (top panels) and experimental (bottom panels) mice. d, Immunostaining for the ciliated cell markers AcTub (left panels) and c-MYB (right panels) (green) in control (top panels) and experimental (bottom panels) mice. e, Flow cytometry analysis of EpCAM+ CD24+ ciliated cells and EpCAM+ SSEA-1+ secretory cells from control and experimental mice. f, Percentage of epithelial (EpCAM+) basal, secretory and ciliated cells on both groups by flow cytometry (n = 3 mice). g, Immunostaining for Ki67 (green) and the secretory cell marker SCGB1A1 (red) on control (top panel) or Dox-treated CK5-Mib1fl/fl mice (bottom panel). h, Immunostaining for BrdU (green) in combination with the basal cell transcription factor p63 (red) on both groups. i, Immunostaining for FOXJ1 (green) and BrdU (red) on Dox-treated CK5-Mib1fl/fl mice that received continuous BrdU. j, Percentage of ciliated FOXJ1+ cells that incorporate BrdU after continuous BrdU administration to Dox-treated CK5-Mib1fl/fl mice (n = 4 mice). k, Immunostaining to detect apoptotic cells by TUNEL assay (green) on either control (upper panel) or experimental mice (lower panel). l, Immunostaining for activated caspase3 (green) on both groups). m, Immunostaining for N2ICD (red), SCGB1A1 and SCGB3A2 (red), or FOXJ1 and AcTub (green) in control (top panels) or experimental mice (bottom panels) after five weeks of continuous doxycycline treatment (n = 4 mice). al, Analysis performed 2 weeks after the beginning of Dox induction. Images are representative of n = 4 mice per condition (biological replicates) repeated twice. *P < 0.05; ***P < 0.001. Data shown in the graphs are means ± s.e.m. Nuclei, DAPI (blue). Scale bar, 20 μm.