Fig 2: The Glycolysis Pathway, Driven by IL-1ß, Prevents DATPs from Converting into Terminally Mature AT1 Cells(A–C) Violin plots showing the log-transformed (log10(TPM+1)), normalized expression levels of early AT1 (A), late AT1 (B), and DATP (C) marker genes in DATPs or control or IL-1ß-treated AT1 cells, as revealed by scRNA-seq analysis of organoids.(D) Schematic of an AT2 organoid culture treated with or without IL-1ß.(E) qPCR analysis of mature AT1 markers on isolated epithelial cells from AT2 organoids. Date are presented as mean ± SEM of four biological replicates from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.(F) Schematic of an AT2 organoid culture treated with or without 2-deoxy glucose (2-DG) in the presence of IL-1ß.(G) qPCR analysis of mature AT1 markers on isolated epithelial cells from AT2 organoids. Each individual dot represents one experiment, and data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.(H) Representative IF images showing rescued maturation of AT1 cells in 2-DG-treated organoids in the presence of IL-1ß: SPC (white), Hopx (top, red), Cav-1 (bottom, red), and DAPI (blue). Scale bars, 50 µm.(I) Representative IF images of a KRT8+CLDN4+ DATP-like population in lungs from normal donors (n = 3): HTII-280 (red), CLDN4 (white), KRT8 (green) and DAPI (blue). Scale bar, 50 µm. See also Figure S7.(J) Representative IF images of a KRT8+CLDN4+ DATP-like population in lungs from IPF patients (n = 5). HTII-280 (red), CLDN4 (white), KRT8 (green), and DAPI (blue). Scale bars, 50 µm.(K) High-power view of the insets in (J): HTII-280 (red), CLDN4 (white), KRT8 (green), and DAPI (blue). Scale bar, 50 µm.

Fig 3: Validation of the human BNB transcriptome. Merged digital photomicrographs of sural nerve endoneurial microvessels (UEA-1 FITC-positive, green) in situ show BNB endothelial cell expression (yellow-green, yellow or orange co-localization dependent of the relative fluorescent intensity of protein marker in red) of transporters (ABCA8, ABCB1, AQP1, SLC1A1, SLC2A1, SLC3A2, SLC5A6, SLC16A1 and SLC19A2), cytoskeletal proteins (ACTG1, CALD1, FLNA, MYO10), cell membrane proteins (CAV1, CD44, CD63, ESAM), junctional complex proteins (CDH5, CDH6, CLDN4, CLDN5, CTNNA1, GJA1, LIN7A, MPDZ, PCDH1, TJP1, VEZT, ZYX), vascular endothelial cell secreted protein CTGF, and chemokine receptor CXCR4. Positive expression of AQP1, CALD1, CAV1, CD44, CD63, FLNA, MYO10 and SLC2A1 (red) by cells that surround and are in direct contact with endothelial cells (most likely pericytes), and ABCA8 by cells close to but not in direct contact with endothelial microvessels (most likely Schwann cells) are also observed. Nuclei are identified in blue. Original magnification 400X.

Fig 4: 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.

Fig 5: ATF4-dependent essential amino acid uptake is crucial for the cell fate decision of secretory cells Western blot images for the expression of YAP, ATF4, and LAT1/Slc7a5 in control or YAP5SA overexpressed C22 airway epithelial cell line. Knockdown (KD) of ATF4 was induced by using lentiviral-mediated inducible scramble shRNA (shScramble, control) or shATF4.Quantitative PCR (qPCR) analysis of YAP/TAZ targets (Cyr61, Thbs1, Amotl2), ATF4 and the targets of ATF4 (Trib3, Slc7a5, Slc7a11).Concentration of L-amino acids (L-AA, left) or EAA (right; leucine, isoleucine, valine) in cell lines in (A). Data are presented as mean ± SEM (n = 3 biological replicates for each group). *P < 0.05, NS, not significant (Student’s t-test).Experimental designs for culture and viral transduction for KD constructs (shRNA vectors) targeting Atf4 or Slc7a5 and ectopic expression of Atf4 in 3D secretory organoids.Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP marker CLDN4, and AT1 cell marker CAV1 in organoids derived from Lats1/2 dKO secretory cells with shscramble, shAtf4, or shSlc7a5. CC10 (green), CLDN4 (white), CAV1 (white) and DAPI (blue). Scale bars, 100 µm.Quantification of Scgb1a1 + lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATPs (green), and tdTomato+CAV1+ AT1 cells (blue) in (E). Data are presented as mean ± SEM (n = 3 technical replicates for each group). ***P < 0.0001, NS, not significant (Student’s t-test).Representative H&E images and IF images showing the expression of secretory cell marker CC10, DATP marker CLDN4, or AT1 cell marker CAV1 in organoids derived from Scgb1a1 lineage-labeled secretory cells with or without hydroxytamoxifen-induced ATF4 overexpression. CC10 (green), CLDN4 (white), CAV1 (white), and DAPI (blue). Scale bars, 100 µm.Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATPs (green), and tdTomato+CAV1+ AT1 cells (blue) in (G). Data are presented as mean ± SEM (n = 3 technical replicates for each group). ***P < 0.0001 (Student’s t-test).