Primary alveolar type II cells can be reprogrammed <i>in vitro</i>.

<p>Representative immunofluorescence images showing expression of iSox2 (Red), Cc10 (Green), Sca1(Green) and Trp63 (Green) after culturing the primary type II cells with doxycycline for 3 days (A), 6 days (B) and 9 days (C). AVTII cells cultured for 9 days without doxycycline served as negative controls (D). The results demonstrate that in vitro cultures of AVTII cells have comparable phenotypic plasticity as AVTII cells in vivo. Scale bar 50 µm (A–C), 100 µm (D).</p

Sox2 induces a gradual loss of differentiated type II cells.

<p>Dual immunofluorescence staining of lungs treated with doxycycline for 1 (A), 6 (B) and 9 (C) days shows a gradual loss of colocalization of the type II cell differentiation marker Lpcat1 (green) and the transgenic Sox2 (iSox2, red), indicating that Sox2 induces cellular changes in the type II cells. Scale bar: 25 µm.</p

Significant downregulated genes in the mycHIF3α expressing lungs.

<p>Significant downregulated genes in the mycHIF3α expressing lungs.</p

Sox2 induces Sca1 positive cells by directly transactivating the Sca-1 gene.

<p>Triple immunofluorescence staining with Myc (iSox2, blue), Sca1 (green) and Trp63 (red) on lungs of iSox2^SPC-rtTA animals treated for 28 days with doxycycline demonstrate the emergence of Sca1/Trp63 positive cells (dotted areas; A–D). (E) Luciferase assay shows in vitro transactivation of the Sca1 minimal promoter by the full length Sox2 (WT), but not by deletion constructs lacking the transactivation domain (ΔTAD) or the HMG domain (ΔHMG). The graph represents the average of three independent experiments, and the bars denote the standard error of the mean (SEM). (F) SOX2 specific ChIP analysis showing specific enrichment of the Sca1 promoter region used in the luciferase assay. The SEM is indicated of two independent experiments. (G) Quantification of the expression of the different lung markers in vivo after the indicated time of doxycycline exposure. The graph shows the average of the number of positive cells of five microscopic fields with a 40× objective of three independent experiments, including the corresponding SEM. Scale bar: 25 µm (D).</p

Primers used for ChIP-qPCR.

<p>Primers used for ChIP-qPCR.</p

Sox2 induces proliferation in terminally differentiated alveolar type II pneumocytes.

<p>Lungs of control (A) and iSox2^SPC-rtTA mice treated with doxycycline for 6 (B), 9 (C) or 28 (D) days were analyzed with an antibody against the mitotic cell marker Phh3. Representative images show proliferation in individual type II cells after 6 days of iSox2 induction (B), which gradually develop into proliferative clusters of cells (C, D). (E) Quantification of Phh3 staining indicates the correlation between the increase of Phh3 positive cells and time of doxycycline expression. (F) Colocalization of iSox2 and Phh3 is shown by dual immunofluorescence labeling after 28 days of doxycycline exposure (areas arrows indicate double positive cells). Scale bars: 200 µm (A), 100 µm (B, C, D) and 25 µm (F).</p

Ectopic Sox2 expression induces abnormal cell clusters.

<p>Representative HE staining of a control lung (A) demonstrating the normal lung architecture and of iSox2^SPC-rtTA lungs after 4 weeks doxycycline treatment (B) showing numerous, enlarged emphysematous structures (asterisks) with cuboidal cell clusters (C). Representative IHC staining for the myc-epitope in wild type control (D) and iSox2^SPC-rtTA lungs (E–I) after 1, 3, 6, 9 and 28 days of doxycycline treatment. Transgenic iSox2 positive myc staining is already evident after 1day of dox administration (E), which gradually increased in time (F–I). The positive cells are clearly forming cuboidal clusters (arrows in I). Dual immunofluorescence staining shows the colocalization of the transgenic Sox2 (iSox2, red) with the type II cell marker Prospc (green) after 6 days (J), 9 days (K) and 28 days (L) of doxycycline treatment. Scale bars 200 µm (A, B), 100 µm (C), 50 µm (D–I) and 25 µm (J–L).</p

mycHIF3α induces the expression of proximal differentiation markers.

<p>mycHIF3α induces an expansion of the Foxp2 positive cells in the double transgenic lungs at gestational age E18.5 (A, D), as well as an expansion towards the distal parts of the lungs of Sox2 (B, E) and p63 (C, F). Sox2 was expressed in both proximal airways and alveolar epithelial cells in mycHIF3α transgenic lungs (<i>arrows</i>, E) at PN1. Basal cells are absent in control lungs (C), but are expressed in basal cells of trachea (C, insert). However, p63 is expressed in the proximal airways and alveolar epithelial cells in mycHIF3α transgenic lung (<i>arrows</i>, F). <i>Scale bar</i>: 200 µm (A and D) and 100 µm (B, C, E, F). (G) <i>Foxp2</i> and <i>Rarβ</i> are significantly upregulated in Hif3α transgenic lungs at gestational age E18.5 as shown by quantitative PCR. (Foxp2: 1.25 <u>+</u> 0.1 versus control 0.87 <u>+</u> 0.1, n = 3, P = 0.007; Rarβ: 1.55 <u>+ </u>0.1 versus control 0.87 <u>+</u> 0.1, n = 3, P = 0,009). White bars represent control lung samples, black bars represent mycHIF3α double transgenic lung samples. (H) Hif2α (black bars) and Hif3α (white bars) induce the 9*HRE-Luc (HRE) and Sox2-Luc (Sox2) as measured by the amount of luciferase activity. The fold induction of the HRE promoter is higher with Hif2α (20,3 fold and 24,5 fold under hypoxic conditions-CoCl₂) than with Hif3α (2,4 fold and 13,4 fold under hypoxic conditions-CoCl₂). The induction of the Sox2 promoter is higher with Hif2α than with Hif3α under normoxic conditions (4,8 versus 2,5), but equally strong under hypoxia mimicking conditions (8,8 versus 7,3). Data are presented as the induction (n-fold) relative to cells transfected with the corresponding reporter plasmid and control vector (pcDNA3). The values are the average of two duplicates, and standard deviations are: 0,04 (HRE-Hif2α), 0,02 (Sox2-Hif2α), 0,03 (ΔSox2-Hif2α), 0,08 (HRE-Hif3α), 0,24 (Sox2-Hif3α), 0,06 (ΔSox2-Hif3α), 0,53 (HRE-Hif2α+CoCl2), 0,007 (Sox2-Hif2α+CoCl2), 0,03 (ΔSox2-Hif2α+CoCl2), 0,88 (HRE-Hif3α+CoCl2), 0,02 (Sox2-Hif3α+CoCl2), 0,1 (ΔSox2-Hif3α+CoCl2). (I) Chromatin immunoprecipitation (ChIP) using anti-HIF3α antibody and chromatin isolated from A549 cells. Graph represents the fold enrichment of the HIF3α-specific binding to the conserved HRE of the SOX2 promoter compared to the IgG control ChIP. HIF3α also bound the ARRDC3 HRE region, and the enhancer region D of the EGLN3 gene served as negative control (EGLN3-D).</p

Expression of genes involved in branching morphogenesis.

<p>Analysis of the distribution of mycHIF3α early in lung development in double transgenic animals at E11.5 (A), E12.5 (B), E13.5 (C) and E14.5 (D). Whole mount in situ hybridization to detect the expression and localization of Fgf10 (E and I), FgfR2 (F and J), Bmp4 (G and K) and Shh (H and L) in lungs isolated at gestational age E12.5 from control (E–H) and mycHIF3α double transgenic animals (I–L). Tr: Trachea; Es: Esophagus. Scale bars: 200 µm.</p

Sox2 induces Clara-like cells and BASC cells.

<p>Lungs of control (A, C, E) and iSox2^SPC-rtTA (B, D, F) lungs treated with doxycycline for 3 (A, B), 9 (C, D) and 28 (E, F) days were stained with the Clara cell marker Cc10. Endogenous expression of Cc10 is demonstrated in the conducting airways (A, arrowhead), which also shows the absence of Cc10 positive cells in the distal airways (A, C, E) of control lungs. The progressive increase in number of Clara-like cells (Cc10⁺) in the iSox2^SPC-rtTA lungs with prolonged induction of iSox2 is clearly noticeable (B, D, F). Colocalization of Cc10 (red) and Prospc (green) is demonstrated with dual immunofluorescence staining (G–I), indicating the emergence of BASC cells (arrows). Arrowheads show Prospc positive cells that lack CC10 (and iSox2) expression. Scale bars: 50 µm (B, D, F), 25 µm (G–I).</p