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Additional Supporting Information may be found in the online version of this article.

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SC_12-0197_sm_supplFigure1.TIF708KSupplemental Figure 1. Immunofluorescent staining of lung tissue sections from SFTPC-GFP mice with anti-GFP antibody (green), anti-Scgb1a1 antibody (red) and anti-calcitonin gene related peptide (CGRP) antibody (blue). Panel A: GFP staining. Panel B: merge of GFP, Scgb1a1, and CGRP immunostaining.
SC_12-0197_sm_supplFigure2.TIF2866KSupplemental Figure 2. Immunofluorescent detection of CD24 within isolated lung cells from FoxJ1-GFP mice and sections of wild type mouse lung tissue. Lung cells were isolated from FoxJ1-GFP mice and stained using fluochrome conjugated antibodies. Stained cells were analyzed by flow cytometry (A-C). (A) Dead cells were discriminated by 7-ADD. Stromal, endothelial, and hematopoietic cells were excluded by staining for surface CD34, CD31, and CD45, respectively (Lin+). (B) The live Lin- population was gated to examine expression of EpCAM and GFP. GFP+ ciliated cells were gated and displayed as a dot plot of CD24 vs. Sca-1 (C). Panels D-L: Immunofluorescent colocalization of CD24 with antibodies to acetylated tubulin (ACT; panels D-F), Scgb1a1 (panels G-I) and Pro-SPC (J-L). Only cells showing high levels of CD24 staining are visible in this analysis. High levels of CD24 co-localizes with acetylated tubulin (ACT), a marker for ciliated cells (D-F). Scgb1a1-immunoreactive cells (G-I) and ProSPC-immunoreactive cells (J-L) showed levels of CD24 immunoreactivity that were not significantly different from background.
SC_12-0197_sm_supplFigure3.TIF1393KSupplemental Figure 3. Immunophenotype and purity of fractionated lung cells. Either unsorted dissociated tracheal cells (positive control; A) or freshly sorted GFPneg (B), GFPlow (C), or GFPhi (D) cells from SFTPC-GFP mice, were spotted onto slides and fixed. Slides were stained with Scgb1a1 (Green) or Krt5 (Red) antibodies and counterstained with DAPI (Blue). Abundance of Scgb1a1-immunoreactive cells or Krt5-immunoreactive cells was quantified by counting 4 randomly selected fields/slide/mouse (n = 3 mice). Krt5-immunoreactive basal cells represented 0.7% of the GFPneg population (B) and were absent in GFPlow (C) or GFPhi populations (D). Scgb1a1-immunoreactive cells were highly enriched within each cell fraction, representing 88.9%, 98.9%, and 96.8% of cells present within GFPneg, GFPlow, and GFPhi fractions, respectively.
SC_12-0197_sm_supplFigure4.pdf269KSupplemental Figure 4. GFPneg, GFPlow and GFPhi cells were sorted from SFTPC-GFP mice and seeded in matrigel cultures at a density of 100 cells/well or 3000 cells/well. Cultures are photographed under both bright-field and FITC channels to assess GFP fluorescence of colonies. Representative pictures are presented to show cultures at day 10 after seeding.
SC_12-0197_sm_supplFigure5.TIF649KSupplemental Figure 5. Total RNA was isolated from 14 day cultures of GFPneg, GFPlow and GFPhi cells and the abundance of mRNA's for Sftpc, Scgb1a1, sPlunc, FoxJ1 and Krt5 assessed by qRT-PCR. Expression of β-actin was used as an internal control for normalization of data.
SC_12-0197_sm_supplFigure6.TIF772KSupplemental Figure 6. Total RNA was isolated from freshly isolated GFPneg, GFPlow, GFPhi cell fractions from SFTPC-GFP mice or GFP+ ciliated cells isolated from FoxJ1-GFP mice. Expression of mRNA for the indicated genes was quantified by real-time RT-PCR. Normalized data are presented for total RNA isolated from cell fractions relative to total lung RNA. These data suggest that the indicated genes are expressed by nonciliated cells present within GFPneg, GFPlow and GFPhi cell fractions with minimal expression by ciliated cells.

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