Fig 2: Sustained YAP/TAZ-mTORC1-ATF4 signaling by chronic injury caused fate conversion of secretory cells into AT1 cells in the airways Experimental schematic diagram of naphthalene-induced acute injury in Scgb1a1-CreERTM /+ ;R26RtdTomato /+ mice post tamoxifen treatment.Representative IF images showing the expression of YAP and TAZ, secretory cell marker CC10, DATP marker CLDN4, mTORC1 activity marker p-S6, and ATF4 in Scgb1a1 lineage-labeled tdTomato+ cells in control and naphthalene-treated lungs. YAP/TAZ (green, 1st panel), CC10 (green, 2nd and 4th panels), p-S6 (white, 2nd panel), CLDN4 (white, 3rd panel), ATF4 (white, 4th and 5th panels). Scale bars, 100 µm.Experimental schematic diagram of repetitive naphthalene-induced chronic injury in Scgb1a1-CreERTM /+ ;R26RtdTomato /+ mice post tamoxifen treatment.Representative IF images showing the expression of AT1 cell marker AGER and DATP marker CLDN4 in Scgb1a1 lineage-labeled tdTomato+ cells in control and naphthalene-treated lungs. AGER (green), CLDN4 (white), and DAPI (blue). Scale bars, 100 µm.

Fig 3: Aberrant activation of YAP/TAZ-mTORC1 signaling in the human lungs of bronchiolitis obliterans syndrome (BO) Representative H&E images of human lung tissues from normal background and bronchiolitis obliterans (BO) with progressive fibrosis.Representative IF images showing the expressions of nuclear YAP, DATP marker CLDN4, and AT1 cell marker AQP5 in the airways of normal background (top) and BO (bottom) human lungs. CLDN4 (white), AQP5 (red), YAP (green), and DAPI (blue). Of note, a flattened airway layer in BO lungs. Scale bars, 100 µm.Representative IF images showing the expressions of secretory cell marker CC10 and mTORC1 activation marker p-S6 in the airways of normal background (top) and BO (bottom) human lungs. CC10 (green), p-S6 (white), and DAPI (blue). Scale bars, 100 µm.Representative IF images showing the expression of nuclear YAP, ATF4, and AQP5 in the airways of normal background (top) and BO (bottom) human lungs. YAP (green), ATF4 (white), AQP5 (red), and DAPI (blue). Of note, co-expressions of nuclear YAP, nuclear ATF4, and AQP5 in the airways of BO lungs. Scale bars, 100 µm.Representative IF images showing the expression of p-S6 (white) in the airway cells losing CC10 (green) expression of BO human lungs. Scale bars, 100 µm.

Fig 4: Ablation of Raptor suppresses YAP/TAZ-induced mTORC1 and ATF4 activation A, BRepresentative H&E and IF images (A), and quantification (B) of secretory cells and mTORC1 activity in control, Lats1/2 dKO (dKO), and Raptor;Lats1/2 triple knockout (tKO) airways at day 5 post tamoxifen treatment. Tomato (A, for Scgb1a1 lineage, red), CC10 (A, green), pS6 (A, white), and DAPI (blue). Scale bars, 100 µm. Data are presented as mean ± SEM (n = 3 mice for each genotype). **P < 0.01, ***P < 0.0001 for pS6+CC10- cells (Student’s t-test).CRepresentative IF images showing the expression of nuclear YAP and ATF4 in Scgb1a1 + lineage-labeled cells of control, Lats1/2 dKO, and Raptor;Lats1/2 tKO airways at day 5 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), YAP (green), ATF4 (white), and DAPI (blue). Scale bars, 100 µm.DExperimental designs for treating mTOR inhibitor (AZD8055) or vehicle to control and Lats1/2 dKO mice twice a day at indicated time points post tamoxifen treatment.ERepresentative H&E and IF images of control and Lats1/2 dKO lungs with vehicle or AZD8055 treatment at day 14 post tamoxifen treatment. Tomato (for secretory lineage, red), CC10 (middle, green), AGER (bottom, green), and DAPI (blue). Scale bars, 100 µm.FQuantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory (top) and AGER+ AT1 cells (bottom) in (E). Data are presented as mean ± SEM (n = 3 mice for each group). **P < 0.005, ***P < 0.001 (Student’s t-test).GRepresentative Sirius red staining for collagen (top) and IF images showing the expression of mesenchymal marker Vimentin (bottom) in control and Lats1/2 dKO lungs with vehicle or AZD8055 treatment. Tomato (for secretory lineage, red), vimentin (VIM, green), and DAPI (blue). Scale bars, 100 µm. Source data are available online for this figure.

Fig 5: mTORC1-ATF4 activity regulates the amino acid uptake for fate conversion of secretory cells Heatmap of ATF4-dependent mTORC1 targets (AA synthesis, 1C metabolism, AA transport, tRNA charging), ATF4 targets, and ATF4-independent mTORC1 targets (HIF1a, mt tRNA).Representative IF images showing the expression of secretory cell marker CC10, mTORC1 activity p-S6, and DATP marker CLDN4 in Scgb1a1 lineage-labeled toTomato+ cells from serial sections of control and Lats1/2 dKO lung tissues. Tomato (for Scgb1a1 lineage, red), CC10 (green), p-S6 (white), CLDN4 (magenta), and DAPI (blue). Scale bars, 100 µm.Representative IF images showing the nuclear expression of YAP and ATF4 in Scgb1a1 lineage-labeled tdTomato+ cells from serial sections of control and Lats1/2 dKO lung tissues. Of note, CLDN4 expression in lineage-labeled cells having the nuclear YAP and TAZ. Tomato (for Scgb1a1 lineage, red), YAP (green), ATF4 (white), CLDN4 (magenta) and DAPI (blue). Scale bars, 100 µm.Experimental designs for lineage-tracing of control, Lats1/2 double knockout (dKO), and Raptor;Lats1/2 homozygous triple knockout (tKO) lungs. Specific time points for tamoxifen treatment and analysis are indicated.Representative H&E and IF images of control, Lats1/2 dKO, and Raptor;Lats1/2 tKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green, upper), AGER (green, lower), and DAPI (blue). Scale bar, 100 µm.Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (left) and tdTomato+AGER+ AT1 cells (right) in (E). Data are presented as mean ± SEM (n = 2–3 mice for each genotype). ***P < 0.001 (Student’s t-test).Quantitative PCR (qPCR) analysis of ATF4-dependent amino acid transporters (Slc7a1, Slc7a3, Slc7a5, Slc34a2, Slc7a11), on freshly isolated Scgb1a1 lineage-labeled cells from control and Lats1/2 dKO lungs at day 2 post tamoxifen treatment. Data are presented as mean ± SEM (n = 2 mice for each group). *P < 0.05, ***P < 0.0001 (Student’s t-test).Measurement of L-amino acid or EAAs (leucine, isoleucine, valine) contents in Scgb1a1 lineage-labeled tdTomato+ organoids derived from control, Lats1/2 dKO or Raptor;Lats1/2 tKO mice. Data are presented as mean ± SEM (n = 3 technical replicates for each genotype). *P < 0.05, **P < 0.001 (Student’s t-test).Representative IF images showing the expression of YAP, p-S6, and ATF4 in serial sections of 3D organoids derived from secretory cells in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), p-S6 (white), ATF4 (magenta), and DAPI (blue). Scale bars, 100 µm. Source data are available online for this figure.