Fig 2: Minimal impact of secreted IGFBP-3 on cell migration. (a) Concentration of secreted IGFBP-3 determined by ELISA. SAS-Fucci cells were transfected with non-targeted control (Cont) or IGFBP3 siRNA #1 for 48 h (left) and treated under either normoxia or hypoxia (right) for 24 h. After each treatment, conditioned media were collected and applied for ELISA. Means ± S.D. (n = 3 independent experiments). (b) Representative anti-IGFBP-3 immunofluorescence images of control (Cont) and IGFBP-3 knockdown (IGFBP3 siRNA #1) cells. Scale bar: 10 μm. (c) Representative images of individual cell tracks (top) and average distance traveled (bottom) by IGFBP-3 knockdown (IGFBP3 siRNA #1) and control (Cont) cells with or without recombinant human IGFBP-3 (rhIGFBP-3) derived from mouse or human cells and normoxic- or hypoxic-conditioned medium (CM). rhIGFBP-3 or CM was added 48 h after siRNA treatment, with which cells were pre-treated for 90 min before time-lapse imaging. Each colored line indicates individual cell tracks during the observation period. Each average distance is represented as a box-whisker plot showing outliers, distribution intervals, interquartile range (box), and median. Cell numbers in each group ranged between 80 and 160. Either two or three independent experiments were performed, and representatives are shown in (b, c). *p < 0.05, **p < 0.01; two-tailed Student’s t-test (a) or Kruskal–Wallis test with Dunn’s multiple comparisons test (c).

Fig 3: Gene expression analysis in 8 vs 12 pcw human embryonic lungs and adult lung samples. a Hierarchical cluster analysis showing how the different samples group according to their expression pattern. b Principal component analysis showing that samples group according to their age using Dimension 1 (47.8% of variability) and Dimension 2 (24.7%). In the small box in the right bottom the PCA have been represented using Dimension 1 and Dimension 3 (6.5%) and sows similar aggrupation clusters. c Heat map with the 88 differentially expressed genes (DEG) identified between 8 vs 12 pcw in human embryonic lungs. The hierarchical cluster showed that using this gene signature samples grouped in 3 groups: adult; 8–9 pcw; and 12 pcw. Arrows indicate the location of the genes that we have further analyzed in the following analysis, including IGFBP3 which is highlighted in red. d String analysis (https://string-db.org) showing the relation between the identified genes. Color nodes are based on the results of the k-means clustering analysis (n = 2). e Enrichment analysis for Gene Ontology (GO) terms using the DEG identified. The numbers indicated in the bars indicate the number of genes included in each GO term. f Hierarchical cluster analysis by gene showing 4 gene clusters with different expression trends. The expression trend from 8 pcw to adult of each cluster is shown in the bottom of the graph and the red line is showing the mean expression of all genes included in the cluster. g Expression analysis by real time PCR of selected genes of each cluster in embryonic samples from 7–8 (n = 4), 9 (n = 4), 10 (n = 5), 11 (n = 4) and 12 (n = 5) pcw human lung embryonic tissues

Fig 4: Cryo-EM reconstruction of PAPP-A2 at 3.13 Å resolution.a Schematic domain organization of PAPP-A2. Numbering shown here and discussed in the text refers to mature PAPP-A2 after removal of the signal secretion and pro-peptide sequences. Domains corresponding to observed density are color-coded while unresolved domains are shown in white. b Cryo-EM map density of PAPP-A2 monomer with domains colored according to Fig. 1a. c Alignment of PAPP-A2 (colored domains) with one copy of PAPP-ABP5 (PDB 7ufg). PAPP-A is shown in transparent gray and olive cartoon and one copy of the anchor peptide is shown as orange cartoon. d PAPP-A2 active site. PAPP-A2 MP domain residues are shown as green sticks. Zinc and water are in gray and red spheres, respectively. Zinc coordination bonds are shown as yellow dashes and hydrogen bonds are black dashes. Aligned PAPP-A residues (from PDB 7ufg) are shown as plum sticks and IGFBP5 anchor peptide is shown as transparent orange cartoon with sticks. Residues for both PAPP-A and IGFBP5 are labeled in parentheses to differentiate them from PAPP-A2 residues. e Cleavage assays for wildtype and active site PAPP-A2 mutants using IGFBP5 as the substrate. For assays containing IGFBP5 as the substrate shown here and in the remainder of the report, unless indicated otherwise, a concentration of 500 nM was used and reactions were incubated for 4 h at 37 °C (please see METHODS section for further details). Data shown in graphs here and in the rest of the report, unless indicated otherwise, represent a concentration of 30 nM PAPP-A2 in assays. Protein quality and representative, primary data are shown in Supplementary Fig. 4a and Supplementary Fig. 4b, respectively. Additional data for all experiments is included in Supplementary Data 2. f Cleavage assays for wild type and active site PAPP-A2 mutants using IGFBP3 as a substrate. Representative, primary data is shown in Supplementary Fig. 4c. Assays shown here and elsewhere in the manuscript used 500 nM of IGFBP3. IGFBP3 was observed to be cleaved less efficiently than IGFBP5. Therefore, for assays containing IGFBP3 as a substrate shown here and in the remainder of the report, reactions were incubated for 18 h at 37 °C. g Cleavage assays for wild type and anchor peptide binding deficient mutant PAPP-A2 or PAPP-A using IGFBP5 as a substrate. Protein quality is shown in Supplementary Fig. 5a and Supplementary Fig. 5b, and representative data is in Supplementary Fig. 5c. In assay results shown in Fig. 1.e–g, error bars represent the standard deviation of experiments done in triplicate.

Fig 5: Silencing of IGFBP3 in tip-derived organoids induces alveolar-like differentiation. a IGFBP3 mRNA levels in tip-derived organoids after siRNA silencing (n = 6 per group). b Time-lapse images of control and IGFBP3 silenced tip-derived organoids during 25 h after transfection. c Representative images of Control and IGFBP3 silenced tip-derived organoids at 48 h post-transfection showed differentiation in alveolar-like structures in the IGFBP3 silenced group. d Expression analysis of stem cell markers in control and IGFBP3 siRNA-transfected organoids (n = 4 per group). e Expression analysis of AT2 cell markers in control and IGFBP3 siRNA-transfected organoids (n = 4 per group). f Expression analysis of AT1 cell markers in control and IGFBP3 siRNA-transfected organoids (n = 4 per group). g Immunofluorescence analysis of SFTPC and E-CAD in control and IGFBP3 silenced organoids. h Hematoxylin staining of tissue sections of lung explants from 10 pcw embryos cultured for 48 h with or without IGFBP3 peptide. A distinctive epithelial morphology can be observed between both groups: the control group showed a cubic epithelium (black arrows) while the IGFBP3 group remained in a more undifferentiated stage (red arrows). i Representative images of one of the 3 independent replicates of TGFβ protein arrays showing differences in p-SMAD1, p-SMAD2 and p-SMAD5 between control and IGFBP3 siRNA-transfected organoids. j Protein quantification in 3 independent replicates of TGFβ related phosphorylated proteins using the protein Arrays in control and IGFBP3 siRNA-transfected organoids (n = 3 per group). k Schematic representation of the effect of IGFBP3 silencing in tip-derived organoid on TGFβ and BMP signaling. *p < 0.05; **p < 0.01; ***p < 0.001