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. 2022 Mar 14;41(8):e109365. doi: 10.15252/embj.2021109365

Figure 4. mTORC1‐ATF4 activity regulates the amino acid uptake for fate conversion of secretory cells.

Figure 4

  1. Heatmap of ATF4‐dependent mTORC1 targets (AA synthesis, 1C metabolism, AA transport, tRNA charging), ATF4 targets, and ATF4‐independent mTORC1 targets (HIF1a, mt tRNA).
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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).
  7. 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).
  8. 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).
  9. 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.