EMBO/soda-nel huggingface dataset:
Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism (2022)
Figure 1

Figure 1. Lats1/2 deletion induces the differentiation of secretory cells into AT1 cells in the airways

Experimental design for lineage-tracing analysis of secretory cells following genetic depletion of Lats1 and Lats2. Specific time points for tamoxifen induction and tissue analysis are indicated.

Representative H&E (top) and immunofluorescent (IF) images (bottom) of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (for secretory cell, white), Acetylated tubulin (Act-Tub, for ciliated cell, green), and DAPI (blue). Scale bar, 100μm.

Statistical quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (B). Data are presented as mean ± SEM (n= 5 mice for each group,). ***p<0.001 (Student's t-test).

Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (for AT1 cell, white), and DAPI (blue). Scale bar, 100μm.

Quantification of Scgb1a1 lineage-labeled tdTomato+AQP5+ AT1 cells in (D). Data are presented as mean ± SEM (n=5 mice for each group). **p<0.01, ***p<0.001 (Student's t-test).

Experimental design for lineage-tracing analysis of secretory cells following genetic depletion of Lats1/2 (double knockout, dKO) and Lats1/2;Yap;Taz (quadruple knockout, qKO). Specific time points for tamoxifen induction and tissue analysis are indicated.

Representative H&E (top) and IF images (bottom) of control, dKO, and qKO airways at day 21 post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CC10 (green), AQP5 (white), and DAPI (blue). Scale bar, 100μm.

Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells in (G). Data are presented as mean ± SEM (n=3 mice for each genotype). ***p<0.001, NS=not significant (Student's t-test).
Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism (2022)
Figure 2

Figure 2. Sustained activation of YAP/TAZ drives the fate conversion of secretory cells into AT1 cells via DATP cell state

Experimental design for isolating Scgb1a1 lineage-labeled cells from control and Lats1/2 dKO lungs at indicated time points post tamoxifen treatment. Isolated cells were subjected to scRNA-seq analysis.

Clusters of Scgb1a1 lineage-labeled cells from 10XGenomics 3' single-cell RNA-sequencing (scRNA-seq) analysis visualized by Uniform Manifold Approximation and Projection (UMAP), assigned by specific colors at day 5 and 14 post tamoxifen treatment. The number of cells in the individual cluster is depicted in the figure.

Distribution of each cluster across indicated time points after tamoxifen treatment.

Bar graph showing the percentage of secretory, DATP, and AT1 cell clusters in control and Lats1/2 dKO lungs at day 5 and 14 post tamoxifen treatment.

Blob plot depicting selected marker genes in cell clusters. Dot size encodes the percentage of cells expressing the gene, and color encodes the average per cell gene expression level.

Pseudotime ordering of Scgb1a1 lineage-labeled cells colored by cluster assignment (right) according to diffusion pseudotime (DPT, left) order.

Representative IF images of control and Lats1/2 dKO airways at indicated time points post tamoxifen treatment. Tomato (for Scgb1a1 lineage, red), CLDN4 (for DATPs, white), CC10 (G, green), AGER (H, for AT1 cell, green), and DAPI (blue). Scale bar, 100μm.

Quantification of Scgb1a1 lineage-labeled tdTomato+CLDN4+ DATPs in (G and H). Data are presented as mean ± SEM (n=5 mice for each genotype). ***p<0.001 (Student's t-test).
Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism (2022)
Figure 3

Figure 3. The uptake of essential amino acids is required for cell fate conversion of secretory cells mediated by YAP/TAZ activation

Violin plots of DATP cell markers (Cldn4, Ndrg1) and essential amino acid (EAA) transporters (Slc7a5, Slc3a2, Slc6a9) in control and Lats1/2 dKO lung cells at day 5 post tamoxifen treatment.

Flow cytometry sorting scheme for isolation of secretory cells (EpCAM+KDR+) (B) and experimental designs for organoid cultures (C). Secretory cells were isolated from unlabeled Lats1/2 dKO lungs, followed by 3D organoid co-cultures with stromal cells. 3D normal media was changed to experimental media 1 day prior to 4-hydroxytamoxifen (4-OHT) or ethanol (EtOH) treatment. Organoids were analyzed at day 8 post 4-OHT or EtOH treatment.

Representative brightfield and H&E images of 3D organoids. Arrow indicates transitioned 3D organoids retaining DATP and AT1 cells. Scale bars, 1mm for brightfield images, 200μm for H&E images.

Representative IF images of secretory, DATP, or AT1 cells in secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), CC10 (E, green), CLDN4 (E, white), AGER (F, green), and DAPI (blue). Scale bars, 100μm.

Quantification of transitioned 3D organoids in 3D normal media (3D), amino acid-limited Basal MEM media (BME), or affluent repletion of EAA in BME media (BME+EAA) with 4-OHT or EtOH treatment. Data are presented as mean ± SEM (n= 3 technical replicates per group). *p<0.05, NS= not significant (Student's t-test).

Representative IF images of YAP and TAZ expression of secretory organoids in indicated culture conditions. Tomato (for Scgb1a1 lineage, red), YAP (green), TAZ (white), and DAPI (blue). Scale bars, 100μm.
Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism (2022)
Figure 4

Figure 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, 100μm.

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, 100μm.

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, 100μm.

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 essential amino acids (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, 100μm.
Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism (2022)
Figure 5

Figure 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 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 knockdown 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 cell 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, 100μm.

Quantification of Scgb1a1+ lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (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 cell 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, 100μm.

Quantification of Scgb1a1 lineage-labeled tdTomato+CC10+ secretory cells (red), tdTomato+CLDN4+ DATP cells (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).
Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism (2022)
Figure 6

Figure 6. Aberrant activation of YAP/TAZ-mTORC1 signalling 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 cell 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, 100μm.

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, 100μm.

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, 100μm.

Representative IF images showing the expression of p-S6 (white) in the airway cells losing CC10 (green) expression of BO human lungs. Scale bars, 100μm.
Preclinical validation of a novel metastasis-inhibiting Tie1 function-blocking antibody (2020)
Figure 1

Figure 1: Neoadjuvant treatment with AB-Tie1-39 slows tumor growth with no overt vascular defects.

(A) 22 Tie1-binding antibodies were screened for phospho-AKT levels in an in vitro Ang1-stimulation experiment. Only one clone (AB-Tie1-39) reduced AKT phosphorylation indicating suppressed Tie2 signaling (n = 1 experiment).

(B) Experimental outline of spontaneous metastatic breast (4T1) cancer model treated with IgG or AB-Tie1-39 in a presurgical neoadjuvant setting.

(C) Tumor growth curves show delayed primary tumor growth upon treatment with AB-Tie1-39 antibody as compared to IgG treatment (mean ± SEM, n = 9 mice). *, P<0.05; ***, P<0.001 (2-way ANNOVA test).

(D) Representative immunofluorescence images of tumor sections stained with CD31 (EC-specific marker) and Desmin (mural cell-specific marker). Scale bars = 200 μm.

(E) Dot plots show quantitation of intratumoral vessel density, mural cell coverage using Desmin and aSMA co-staining, and tumor necrosis (mean ± SD, n = 9 mice). All comparisons were rendered non-significant according to two-tailed Mann-Whitney U test.
Preclinical validation of a novel metastasis-inhibiting Tie1 function-blocking antibody (2020)
Figure 3

Figure 3: AB-Tie1-39 treatment stabilizes lung vessels to restrict extravasation of disseminated tumor cells.

(A) Mice were pretreated with IgG or AB-Tie1-39. Thereafter, B16F10 cells were intravenously injected to initiate experimental metastasis independent of the primary tumor.

(B) Representative images of the whole lungs are shown on the left. Scale bars = 5 mm. Quantitation of the number of lung metastatic foci is plotted on the right (mean ± SD, nIgG = 9, nAB-Tie1-39 = 8 mice). *, P<0.05 (two-tailed Mann-Whitney U test).

(C) EC were seeded on gelatin-coated Transwell inserts. RFP-labeled LLC cells were allowed to transmigrate for 8 h through the endothelial layer. Thereafter, transmigrated tumor cells were counted. The dot plot presents the normalized data (mean ± SD, n = 9 inserts from three independent experiments). ***, P<0.001 (two-tailed Mann-Whitney U test).

(D) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung tissue lysates were immunoblotted with anti-phospho-TIE2, total-TIE2 and ACTB (upper panel; n = 6 mice). Densitometric quantitation of the blots presented in the upper panel is shown (mean ± SD, n = 6 mice). **, P<0.01 (two-tailed Mann-Whitney U test).

(E) LLC tumor-bearing mice were pretreated with IgG or AB-Tie1-39. On day 14, lung EC were FACS-sorted and used for global gene expression (n = 4 mice). On the left, a heatmap shows significantly- regulated genes (red outline = AB-Tie1-39-treated; black outline = IgG-treated). On the right, top significantly regulated bio-functions in AB-Tie1-39-treated as compared to IgG-treated mice in Ingenuity Pathway Analysis.
Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter (2022)
Figure 1

Figure 1. Engineering a GAT1 binding site into dDAT.

A, Sequence alignment between hGAT1, hSERT, hNET, dDATWT and dDATGAT in the vicinity of the primary binding site. GAT1 like substitutions (11 mutations) were carried out in dDAT.

B, Chemical structures of GABA and substrate analogues, nipecotic acid and guvacine. Structures of specific inhibitors of GAT1 tiagabine, NO711 and SKF89976a.

C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711.

D, dDATmfc did not show any interactions with NO711 in the MST experiments.

E, SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM.

F, MST experiments display no interactions of purified dDATmfc with SKF89976a. The MST profiles shown are one of two independent measurements.

G, Michaelis-Menten uptake kinetics of 3H-GABA by the GAT1WT used in the study displaying a KM value of 11.4 μM.

H, Inhibition of 3H-GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14.4 μM.

I, Inhibition of 3H-GABA uptake through GAT1WT by tiagabine (Ki= 725 nM), NO711 (1.07 μM) and SKF89976a (7.3 μM).

Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m. Control data is included in Appendix figure S6, S7.
Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter (2022)
Figure 4

Figure 4. TM6 linker plays a vital role in GABA uptake.

A, Side view of structural overlaps between dDATGAT and hGlyT1 in its inward-open state (PDB id. 6ZBV) with a Cα rmsd of 2.73 Å and hGlyT1 AlphaFold2 model in outward-open state with a Cα rmsd of 1.43 Å. Insets show the presence of W376 in the position of L300 that occludes the substrate binding within the primary binding site and restrict it to smaller substrates like glycine.

B, Substitution of L300 to other hydrophobic side chains and the consequences on transport activity. L300W has the maximal effect on the transport activity. The histogram represents mean of a total 6 measurements (n=6) done as two independent measurements. Individual datapoints are represented as black dots around the error bar that represents s.e.m.

C-G, Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. A near 10-fold loss of nipecotic acid inhibition potency was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake.

Data information: (C-G) All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows; nipecotic acid, panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a, panel H-0.0009). GAT1WT transport kinetics and nipecotic acid inhibition potency are redisplayed from figure panels 1G, 1H and 1I for comparison. Control data is represented as Appendix figures S6, S7.
Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter (2022)
Figure 6

Figure 6. EL4 is involved in neurotransmitter gating.

A, Water mediated H-bond interactions displayed in SKF89976a interactions with E384S substitution.

B, Alignment shows the presence of acidic side chains in biogenic amine transporters in the EL4.

C, Michaelis-Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value (28.54 ± 2.3 μM). The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC.

D, 3H-GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30.1 μM

E, The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT.

Data information: (C-E) The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.
The role of autophagy during the early neonatal starvation period (2004)
figf1

a, Autophagosome formation in the heart indicated by GFP–LC3. Hearts were isolated from GFP–LC3 transgenic mice at multiple stages, including 18.5 day embryos and 0.5 h, 3 h, 6 h, 24 h and 2.5 day neonates, immediately fixed, cryosectioned, and analysed by fluorescence microscopy. Scale bar, 10 µm. b, Quantification of GFP–LC3 dots in neonatal tissues. Cryosections were prepared from tissues isolated at the indicated times. The ratio of the total area of GFP–LC3 dots to the total cellular area is shown as a percentage. Values represent mean ± s.d. of three mice. Diaph., diaphragm; Panc., pancreas; Muscle; gastrocnemius muscle. c, Electron microscopic analysis of the heart from wild-type neonates. Typical autophagosomes (arrows) and autolysosomes (arrowhead) were observed in the heart at 10 h after birth. Scale bar, 1 µm. d, Conversion of LC3-I to LC3-II in neonatal hearts. Heart homogenates from five embryos/neonates were pooled and analysed by immunoblotting using an anti-LC3 antibody. The bands representing LC3-I and LC3-II are marked with arrows.
The role of autophagy during the early neonatal starvation period (2004)
figf2

a, Detection of wild-type (WT) and targeted alleles. Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis. Restriction enzymes used were HpaI for the 5′ probe and SpeI for the 3′ probe (see Supplementary Fig.S2 for the restriction map). KO, knockout (targeted) allele. b, Photograph of a representative Atg5-/- neonate compared to a wild-type littermate. c, Immunoblot analysis of Atg5 and LC3. Heart homogenates were prepared from neonates 10 h after birth. MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10% FBS (unstarved) or Hanks’ solution for 2 h (starved), and total cell lysates were prepared. Immunoblot analysis was performed using anti-Atg5 and anti-LC3 antibodies. The position of the Atg12–Atg5 conjugate on the blot is indicated. d, Absence of GFP–LC3 dot formation in Atg5-/- mouse heart. Various tissues from Atg5-/- mice expressing GFP–LC3 were analysed for dot formation by fluorescence microscopy. A representative result from a neonatal heart at 3 h after birth is shown.
The role of autophagy during the early neonatal starvation period (2004)
figf4

a, Plasma and tissue amino acid concentrations. Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions. ‘Total’ indicates the sum of the Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg concentrations; ‘Essential’ indicates the sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg concentrations; ‘BCAA’ indicates the sum of the Val, Ile and Leu concentrations. Tissue amino acid concentrations are expressed as mmol kg-1 of wet weight. Bars represent mean ± s.d. of three mice. Single and double asterisks indicate a significant difference between mutant and control (wild-type plus heterozygous) mice at P 0.05 and P 0.01, respectively. b, Activation of AMPK in Atg5-/- mice. Hearts were isolated from unfed neonates at 0 h (lanes 1, 3, 5) and 10 h (lanes 2, 4, 6), and force-fed mice at 10 h (lane 7) after the caesarean delivery. Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti-AMPK and anti-phospho-AMPK antibodies. c, ECGs from neonatal mice at the indicated time after caesarean delivery. ST elevation was observed in all Atg5-/- mice (n = 4) at 8–9 h after delivery.