Fig 2: The impact of activin-A on TGF-β and Hippo signaling pathway in BMDMs. (A) Activin-A promoted SMAD2 phosphorylation. Left panel: the expression of p-SMAD2 and SMAD2 after 2-h activin-A stimulation. (B) Activin-A promoted p-YAP and p-TAZ dephosphorylation, as well as the nuclear accumulation of SMAD2, YAP, and TAZ. Left panel: the expression and location of p-SMAD2, p-YAP, p-TAZ, SMAD2, YAP, and TAZ after 2-h activin-A stimulation. (C) IF staining detected the nuclear translocation of SMAD2, YAP, and TAZ in activin-A-treated BMDMs; the representative images were taken at 630× magnification. (D) The expression of p-SMAD2 after 2-h activin-A stimulation. Silencing of TAZ blunted activin-A-induced p-SMAD2 expression. (E) The expression of p-SMAD2 after 2-h activin-A stimulation. Silencing of YAP decreased activin-A-induced SMAD2 phosphorylation. (F) Overexpression of TAZ promoted the activin-A-induced p-SMAD2 expression. (G) Overexpression of YAP increased the levels of p-SMAD2 under activin-A treatment. (H) Overexpression of TAZ and YAP promoted the expression of Fn1. (I) Yap or Taz deficiency reduced Inhba mRNA quantity. (J) The overexpression of Yap or Taz increased the mRNA levels of Inhba. Quantitative results are presented as mean ± standard error of the mean. *, P<0.05; **, P<0.01; ***, P<0.001. +/−: with/without the stimulation of activin-A. ns, no significance; α-SMA, alpha smooth muscle actin; BMDM, bone marrow-derived macrophage; FST, follistatin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; PBS, phosphate-buffered saline; p-SMAD2, phosphorylated SMAD2; SMAD, suppressor of mothers against decapentaplegic; TAZ, transcriptional coactivated with PDZ-binding motif; TGF-β, transforming growth factor-β; YAP, yes-associated protein.

Fig 3: Transcriptome analysis of activin-A treatment of BMDMs. (A) Representative imagines of coimmunostaining (IF staining, 400×) for ALK4 (green) and CD206 (red) in BALF cells from ALI mice and control subjects. White arrows: macrophages co-expressing ALK4 and CD206. (B) Heatmap of the top DEGs in the control and activin-A-treated BMDMs. Red arrow: increased expression of Inhba gene in activin-A treated BMDMs. (C) Heatmap of significant DEGs related to inflammation cytokines and chemokines. (D) Heatmap of significant DEGs related to ECM organization. (E) KEGG analysis indicating that the top DEGs were enriched in TGF-β and Hippo signaling pathway. AGE, advanced glycation end products; RAGE, receptor for advanced glycation end products; ALI, acute lung injury; ALK4, activin receptor-like kinase 4; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid; BMDM, bone marrow-derived macrophage; CD206, mannose receptor; DEG, differentially expressed gene; ECM, extracellular matrix; KEGG, Kyoto Encyclopedia of Genes and Genomes; TGF-β, transforming growth factor-β; IF, immunofluorescence.

Fig 4: Analysis of activin-A expression in LPS-treated mice and BMDMs. (A) Depletion of macrophages displayed a protective effect against LPS-induced ALI [representative results of H&E staining (100×)]. (B) The semiquantitative lung injury scores, indicating the severity of ALI. (C,D) Levels of TNF-α and activin-A detected by ELISA. (E) Representative images of coimmunostaining (400×) for CD206 (red) and Inhba (green) in BALF from ALI mice and control subjects. (F) qPCR analysis of LPS-induced activin-A expression in BMDMs at different concentrations. (G) Levels of activin-A in LPS-induced BMDMs. Quantitative data are presented as mean ± standard error of the mean. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ALI, acute lung injury; BALF, bronchoalveolar lavage fluids; CD206, mannose receptor; BMDM, bone marrow-derived macrophage; ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; LPS, lipopolysaccharide; qPCR, quantitative real-time polymerase chain reaction; TNF-α, tumor necrosis factor alpha; HPF, high power field.