Figure 7. In vitro prostaglandin F2α (PGF_2α) challenge promotes adventitial fibroblast entry into the transitional/inflammatory state.

(A) Sorting strategy for the isolation of adventitial and alveolar fibroblast used in I^ER-Sftpc^I73T/Ptgfr^+/+ and I^ER-Sftpc^I73T/Ptgfr^–/– prior to induction by TAM. Initial gating was performed on the CD45^–CD31^–CD326^–Mcam^–Pdgfra⁺ population. Adventitial fibroblasts are Sca1⁺, and the Sca1^– population is made up of the alveolar fibroblasts. After sorting, cells were seeded for 48 hours on tissue culture plastic with either 10 ng/mL TGF-β1, 500 nM PGF_2α, or media control. (B) Gene expression analysis via qPCR in untreated adventitial and alveolar fibroblasts quantifying the expression of population-specific marker genes confirms the identity of target fibroblasts. (C) Quantification of transitional cluster maker genes after 48-hour challenge demonstrates the potential for FPr to induce the transitional state in adventitial fibroblasts. This is not observed in adventitial fibroblasts lacking the FPr. (D) Quantification of fibrotic cluster marker genes after 48-hour challenge confirms that TGF-β promotes entry adventitial fibroblasts into the fibrotic state independent of FPr status. Ordinary 1-way ANOVA testing was performed. *P < 0.05, **P < 0.005, ***P < 0.0005. Statistical significance between treatment and media control denoted by ^&P < 0.05.