(A) Scheme illustrating the experimental layout for molecular restoration of KLF4 in myofibroblasts to evaluate their differentiation status and functional response to Fas-mediated apoptosis. (B–G) MRC5 cells stably transduced with DOX-KLF4 or DOX-GFP were treated for 48 hours with TGF-β to promote a myofibroblast phenotype. Cells were then treated with/without doxycycline (1 μg/mL) for 16 hours, and the expression of α-SMA (B) and COL1α2 (C) was analyzed by qPCR at 24 hours; or cells were further stimulated with Fas Ab and assessed for relative annexin V binding at 24 hours (D), caspase-3/7 activity at 16 hours (E), and expression of APAF1 (F) and BIRC5 (G) measured by qPCR at 24 hours. (H) Scheme illustrating the experimental layout for treatment of elicited myofibroblasts or IPF fibroblasts with the pharmacologic KLF4 inducer APTO-253 and evaluation of differentiation status and functional response to Fas-mediated apoptosis. (I–L) MRC5 cells were treated for 48 hours with TGF-β to elicit myofibroblast differentiation. They were then treated with/without APTO-253 (250 nM) for 36 hours and assessed for the induction of KLF4 by qPCR (I) and α-SMA protein by densitometric analysis of Western blot (J) or further incubated with Fas Ab and assessed for relative annexin V binding at 24 hours (K), with expression of APAF1 and BIRC5 measured by qPCR at 24 hours (L). (M–O) IPF fibroblasts were treated with/without APTO-253 (250 nM) for 36 hours and assessed for the induction of KLF4 by qPCR (M) or analyzed for the expression of α-SMA by qPCR (N) and protein densitometry (O). GAPDH mRNA and protein were used to normalize α-SMA and COL1α2 expression by qPCR and Western blot, respectively. (M–O) Each symbol represents a single patient lung-derived fibroblast line. mRNA values are expressed relative to control. (B and C) The dashed line represents the value of fibroblasts not treated with TGF-β. All data represent mean values (± SE) from 3–4 independent experiments. *P < 0.05, 2-way ANOVA.