Figure 7.

Fibrogenic changes are mediated by alveolar epithelial cell-derived TGFβ1 signaling in BLM-treated FD-AOs

(A) Heatmap indicating log₂(fold change) of TGFB1 and TGFBR1 in each population calculated from RNA-seq counts using DESeq2. DESeq2 adjusted: ^∗p < 0.05, ^∗∗∗p < 0.001.

(B) Cell-surface levels of integrin αVβ6 in EpCAM⁺ and EpCAM⁻ cell populations were measured by FCM.

(C) GSEA analysis of “Verrecchia_early/delayed response to TGFB1.” Data from EpCAM⁻ cells separated from FD-AOs were used and ranked based on the p value of DESeq2.

(D) Whole-well imaging of cultivation matrices at day 17. Each well was treated with BLM from day 11 to day 14 and with 1 μM SB525334 or 3 ng/mL active TGFβ1 from day 14 to day 17. Scale bars, 2 mm.

(E) Quantification of matrix area. Data are presented as mean ± SEM (n = 3 or 4 independent experiments).

(F) Expression of contractile genes in fibroblasts separated from FD-AOs measured by qRT-PCR and normalized to each control. Data are presented as mean ± SEM (n = 8 independent experiments).

(G) Pathway enrichment analysis using the Reactome software of 427 proteins upregulated by BLM. Proteomic analysis was performed on whole cultivation matrices including cells, and the threshold for upregulation was set to log₂(fold change) > 0.4.

(H) Efficacy of SB525334 on all 30 proteins annotated with “Extracellular matrix organization” that were upregulated by BLM. Heatmap indicating Z score of each protein. The log₂(fold change) was used to calculate the Z scores.

One-way ANOVA with Tukey’s multiple comparisons test: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; ns, not significant.