Supporting Information Rock et al. 10.1073/pnas.1117988108 Fig. S1. Heterogeneity of stromal cells in normal and fibrotic mouse lungs. Sections of normal mouse lungs (A and D) and fibrotic lungs collected (B and E) 14 or (C and F) 21 d after the intratracheal administration of 1.25 U/kg bleomycin were stained with antibodies against stromal cell markers. One marker (noted in parentheses) is omitted from high-magnification Insets for clarity. Representative images from confocal stacks from three mice are shown for each treatment. (A C) Vimentin (red), S100a4 (green), and asma (purple). (A) Note that asma-positive cells are not present within the interstitium of control lungs, and asmapositive myofibroblasts are more abundant at (B) 14 than (C) 21 d postbleomycin. (D F) asma (red), NG2 (green), and Pdgfrb (purple). NG2-positive, Pdgfrbpositive pericyte-like cells, abundant within the interstitium of control and fibrotic lungs, do not express asma. Dashed boxed region of each image is shown at higher magnification in Insets. (Scale bars: A and D, 50 μm; Insets, 20 μm.) 1of6
Fig. S2. Heterogeneity of stromal cells in UIP/IPF. Frozen sections of normal lung or UIP were stained with hematoxylin and eosin or antibodies against asma and NG2. Representative images are shown. (A) Some perivascular cells in normal lungs express both NG2 (red) and asma (green). A population of interstitial pericytes expresses NG2 but not asma. (A ) H&E stained section adjacent to the section shown in A. (A ) Red and green channels and (A ) red channel only from the boxed region in A. (B) Representative image of NG2 (red) and asma (green) in a fibrotic lung. Some cells, usually associated with vasculature, express both markers. (B ) H&E stained section adjacent to section shown in B. Note that cells within classic fibroblast foci (arrow) do not express high levels of NG2 or asma. (C) NG2 (red) and asma (green) in a fibrotic lung from Imaris colocalization analysis. (C ) Voxels demonstrating colocalization (white). The numeric value is the percentage of NG2 positive volume above the threshold (% red voxels) that is colocalized with asma (green) above the threshold. Most colocalization in control and fibrotic lungs is in cells associated with vasculature. Scale bars: A and B, 200 μm; A and A, 50μm; C and C, 100 μm. Fig. S3. FoxJ1-CreER labels pericyte-like cells that expand in fibrotic regions. FoxJ1-CreER;ROSA-farnesylated GFP (fgfp) double heterozygous mice were given four doses of tamoxifen followed 4 d later by intratracheal (A and B) salineor(c and D) 5 U/kg bleomycin. (A and B) FoxJ1-CreER labels asma-positive, NG2- positive perivascular cells as well as asma-negative, NG2-positive pericyte-like interstitial cells. (C and D) After bleomycin, FoxJ1-CreER lineage-labeled pericytelike cells expand and make up a large proportion of cells in fibrotic regions. These regions remain NG2-positive, and most lineage-labeled cells do not express high levels of asma. Images shown are maximum projections (merged images) of optical sections through the tissue thickness. (Scale bars: 50 μm.) 2of6
Fig. S4. Resident fibroblasts proliferate in response to bleomycin. (A) A Pdgfra-H2B:GFP knock-in allele reports expression of Pdgfra in some asma-positive peribronchiolar and perivascular smooth muscle cells and (C) Pdgfrb-positive stromal cells near airways and vessels. (A C) Interstitial Pdgfra-H2B:GFP-positive interstitial fibroblasts do not express Pdgfrb or asma. (D) Pdgfra-H2B:GFP heterozygotes were given 1.25 U/kg intratracheal bleomycin. BrdU was administered 3 h before animals were killed for histology 6 d later. Sections were stained with antibodies against BrdU (red, proliferative cells) and GFP. Arrows show Pdgfra- H2B:GFP-positive cells that have incorporated BrdU. br, bronchiole; bv, blood vessel; ven, venule. (Scale bars: A D, 50μm.) Fig. S5. The proportion of Sftpc-CreERT2 lineage-labeled type 2 alveolar epithelial cells (AEC2) cells is decreased in fibrotic regions. Adult SftpcCreER T2 ;ROSA- Tomato mice were given four doses of tamoxifen to label AEC2 followed 4 d later with 1.25 U/kg intratracheal bleomycin. After 21 d, we imaged endogenous Tomato lineage label and immunofluorescence against Sftpc (green, AEC2 cells). Note that many Sftpc-positive cells are not lineage labeled, especially in fibrotic regions, suggesting that they are derived from an unlabeled progenitor. (Scale bar: 100 μm.) 3of6
Fig. S6. AEC2 cells do not directly contribute to pulmonary fibrosis by epithelial to mesenchymal transition (EMT). Sftpc-CreER T2 ;ROSA-Tomato double heterozygous mice were given four doses of tamoxifen followed 4 10 d later with 1.25 U/kg intratracheal bleomycin. After (A and C) 10, (B) 14, or (D) 21d,we imaged endogenous Tomato lineage label and immunofluorescence against (A and B) vimentin (green) and asma (purple) or (C and D) S100a4 (green). Note that more asma-positive cells are present within fibrotic regions at 10 and 14 d postbleomycin than at 21 d postbleomycin (Fig. 1 and Fig. S1). Similarly, many more S100a4-positive cells, with morphology and distribution consistent with immune cells, are present within fibrotic regions (C) 10 d postbleomycin compared with (D) 21 d postbleomycin. The Tomato lineage label never colocalizes with markers of stromal cells. Dashed boxed regions in A and B are shown at higher magnification in Insets. C Inset shows Tomato and S100a4 staining in another region of the lung at higher magnification and without DAPI for clarity. (Scale bars: A, B, and C Inset, 50μm; A and B Insets, 20μm; C and D, 200μm.) 4of6
Fig. S7. Colocalization analysis of Tomato lineage label with stromal cell markers. Confocal stacks were analyzed with Imaris software to quantify the colocalization of Tomato (red, Sftpc lineage label) with (A) S100a4, (B) vimentin, (C) asma, and (D) Sftpc as a positive control. Images were acquired on a Zeiss 710 confocal microscope (A and B) 21 d postbleomycin, (C) 10 d postbleomycin, or (D) 21 d postsaline. Voxels (i.e., volumetric pixels) showing colocalization (white) are shown in A D. The numeric value is the percentage of red voxels that colocalize with green voxels. Note that this numeric value does not represent the percentage of red cells that colocalize with green cells. The areas of colocalization are typically observed to occur at the interface between red cells and neighboring green cells, an interface that the software cannot clearly distinguish given limitations of image resolution. (Scale bars: A and B, 20μm; C and D, 40 μm.) This analysis shows that 62.6% of Tomato-positive voxels are also Sftpc-positive. However, only 1.15%, 0.94%, and 0.56% of Tomato-positive voxels express S100a4, vimentin, or asma, respectively. Table S1. Patient data for biopsy sample donors Pathological diagnosis Clinical diagnosis Sex Age (y) Smoking history UIP IPF Male 66 Never UIP RA-ILD Male 48 Former UIP? CTD Male 54 Former UIP IPF Male 68 Former UIP RA-ILD Female 59 Never UIP IPF Male 64 Former UIP? CTD Male 61 Former UIP IPF Male 75 Never CTD, connective tissue disease related; IPF, idiopathic pulmonary fibrosis, RA-ILD, rheumatoid arthritis-associated interstitial lung disease; UIP, usual interstitial pneumonia. 5of6
Table S2. Primers used for quantitative PCR Gene Forward Reverse Gapdh 5 -AGGTCGGTGTGAACGGATTTG-3 5 -TGTAGACCATGTAGTTGAGGTCA-3 Tbp 5 -AGAACAATCCAGACTAGCAGCA-3 5 -GGGAACTTCACATCACAGCTC-3 Sftpc 5 -CAAACGCCTTCTCATCGTGGTTGT-3 5 -TTTCTGAGTTTCCGGTGCTCCGAT-3 Pdpn 5 -ACCGTGCCAGTGTTGTTCTG-3 5 -AGCACCTGTGGTTGTTATTTTGT-3 asma 5 -ATTGTGCTGGACTCTGGAGATGGT-3 5 -TGATGTCACGGACAATCTCACGCT-3 Col1a1 5 -GCTCCTCTTAGGGGCCACT-3 5 -ATTGGGGACCCTTAGGCCAT-3 Vimentin 5 -CGGCTGCGAGAGAAATTGC-3 5 -CCACTTTCCGTTCAAGGTCAAG-3 Versican 5 -TTTTACCCGAGTTACCAGACTCA-3 5 -GGAGTAGTTGTTACATCCGTTGC-3 S100a4 5 -TGAGCAACTTGGACAGCAACA-3 5 -CTTCTTCCGGGGCTCCTTATC-3 NG2 5 -ACCATGCTACTCCGCAACAG-3 5 -CCGGTGAACATCTATGTGTACG-3 6of6