Fig. 4

SOX9^−/− reduces proliferation and promotes apoptosis in lung organoids. A Bright-field images of day 98 SOX9 wild-type (WT) and knockout (SOX9^−/−) alveolar organoids and day 123 WT alveolar organoids. Scale bars, 500 μm. B mRNA expression levels of markers in day 98 WT and SOX9^−/− alveolar organoids (n = 3, representative of 3 separate experiments). C Immunofluorescence labeling of cell proliferation marker Ki67 (green) in day 98 WT and SOX9^−/− alveolar organoids. Scale bars, 100 μm (left); 50 μm (right). D Number of Ki67-positive cells in day 98 WT (n = 6) and SOX9^−/− (n = 5) alveolar organoids. ***p < 0.001 (unpaired 2-tailed Student’s t test). E Immunofluorescence labeling of apoptosis marker cleaved caspase-3 (green) and epithelial marker E-cadherin in day 98 WT and SOX9^−/− alveolar organoids. Scale bars, 100 μm (left); 50 μm (right). F Number of cleaved caspase-3–positive cells in day 98 WT (n = 9) and SOX9^−/− (n = 5) alveolar organoids. *p < 0.05 (unpaired 2-tailed Student’s t test). G Schematic of the protocol for single-cell passaging of day 21 NKX2.1⁺ lung progenitors. (H) Bright-field images of day 41 (single cell passaged once [P1]) and day 59 (single cell passage twice [P2]) airway organoids. Scale bars, 500 μm. I, J mRNA expression levels of TSP1 during alveolar organoid (I) or airway organoid (J) differentiation (n = 3)