Primary supplement

Topography-dependent gene expression and function of common cell archetypes in large and small porcine airways

Pezzulo et al. 2021

Supplemental figure 1: Large and small porcine airway epithelia share same common cell types. Immunofluorescence images showing ciliated (α-acetyl tubulin), secretory (Muc5AC, SCGB3A2), and basal (KRT5) cell markers in large and small porcine airways. Merge images show F actin in red (except for small airway KRT5) and DAPI in blue. Scale bar = 25μm.

Supplemental figure 2: Expression profiles of airway surface epithelia from CFTR+/+ and CFTR-/- pigs are similar. Volcano plots of CFTR+/+ vs. CFTR-/- (A: basal, B: secretory, and C: ciliated) cells in large and small airways. n = CFTR+/+ pigs (5 large and 4 small airway) and CFTR-/- pigs (4 large and 3 small airway)

Supplemental figure 3: Differential gene expression in non-epithelial cells: Cells that more directly interact with epithelial cells (fibroblasts and immune cells) had more site-specific differentially expressed genes compared to endothelial and smooth muscle cells. n = 10 large airway, 7 small airway. CFTR+/+ and CFTR -/- pig samples were grouped together as they had comparable gene expression.

MUC19 MUC16 MUC13 MUC4 MucinsMUC20 MUC5B MUC5AC MUC15 MUC1 cell_type Secretory Ciliated region 3 region region 3 region -3 0 3 Large Small Large Small cell_type Lg cell_type Lg − − − 0 1 2 3 3 2 1 2 cell_type 2 Sm Sm MUC1 Secretory Ciliated Basal MUC1 1 cell_type 1 cell_type MUC15 Basal MUC15 Basal 0 0 Secretory Secretory MUC5AC −1 Ciliated MUC5AC −1 Ciliated

−2 MUC5B −2 MUC5B −3 −3 MUC20 MUC20

MUC4 MUC4

MUC13 MUC13

MUC16 MUC16

MUC19 MUC19

Supplemental figure 4: Mucin genes expression in small and large airway secretory and ciliated cells at the subject level: Each column is the normalized/centered average expression per cell type (basal, secretory, or ciliated) per subject. n = 10 large airway, 7 small airway. CFTR+/+ and CFTR -/- pig samples were grouped together as they had comparable gene expression.

Ion transport MUC19 MUC16 MUC13 MUC4 MUC20 MUC5B MUC5AC MUC15 MUC1 cell_type -3 0 3 − − − 0 1 2 3

Basal Secretory Ciliated 3 2 1 region cell_type region

Secretory Ciliated Basal 4 Large Small Large Small Large Small cell_type Lg SERPINE2 Sm FLNA 2 SLC9B2 cell_type Basal SCNN1B 0 SCNN1G Secretory CLIC4 Ciliated SHROOM2 −2 FXYD6 DMD −4 SGK1 ADRB2 SLC45A4 PKP2 P2RX4 SLC11A2 CHP1 KLHL24 ATP6V1B1 SLC4A11 KEL SLC9A7 SLC12A8 KCNH8 TPCN1 PTPN3 ATP6V0E1 SLC5A8 DRD2 COX7A1 SLC15A2 SLC1A4 HBB CLCA2 SLC5A5 FXYD3 KCNE3 BPIFA1 TRPM4 SLC5A1 GABRP SLC26A2 CLCA1

Supplemental figure 5: Ion transporter and ion transporter regulator genes expression in small and large airway basal, secretory, and ciliated cells at the subject level: Each column is the normalized/centered average expression per cell type (basal, secretory, or ciliated) per subject. n = 10 large airway, 7 small airway. CFTR+/+ and CFTR -/- pig samples were grouped together as they had comparable gene expression.