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. 2022 Jun 28;206(5):596–607. doi: 10.1164/rccm.202106-1503OC

Figure 4.

Figure 4.

F2-isoprostane (F2-IsoP) induction of the TGF-β pathway requires TBXA2R. (A) Luciferase assay was performed to quantify Smad2/3 transcriptional activity after transfection with Smad2/3-binding element–driven luciferase reporter construct using wild-type (WT) and Tbxa2riKO (Rosa26-CreER+TBXA2Rf/f) lung fibroblasts at 8 hours after F2-IsoP or vehicle treatment. n = 3 for each group. *P < 0.05. (B) Western blot for T-Smad2, P-Smad2, and Timp1 (tissue inhibitor of metalloproteinase-1) using WT and Tbxa2riKO lung fibroblasts at 24 hours after F2-IsoP or vehicle treatment. (C) Western blot for T-Akt, P-Akt, T-p44/42, and P-p44/42 using WT and Tbxa2riKO lung fibroblasts at 24 hours after F2-IsoP or vehicle treatment. Western blots were performed in triplicate samples and repeated at least twice. (D) WT mouse lung fibroblasts were treated with or without F2-IsoPs (100 nM) and LY2109761, an inhibitor of TGF-β receptor type 1/type II kinases, for 24 hours. Quantitative PCR analysis for Serpine1 and Timp1 gene expression was performed. n = 3 for each group. *P < 0.05. Significance was determined using the Wilcoxon rank sum test. Error bars denote SEM. Ctrl = control; Hsp70 = heat shock protein 70; N.S. = not significant; P-Akt = phospho-Akt; P-p44/42 = phospho-p44/42; P-Smad2 = phospho-Smad2; SERPINE1 = serpin family E member 1; T-Akt = total Akt; TBXA2R = thromboxane–prostanoid receptor; TGF-β = transforming growth factor-β; T-p44/42 = total p44/42; T-Smad2 = total Smad2.