Journal: Oncology Reports

Article Title: POSTN + fibroblast-secreted small extracellular vesicles drive macrophage M2 polarization through BMP4/BMPR2/Smad signaling

doi: 10.3892/or.2026.9067

Figure Lengend Snippet: BMP4 activates BMPR2/Smad signaling to induce macrophage M2 polarization. (A) PCA analysis of macrophages were treated with sEV-derived CAF-S6 transfected with shNC and shPOSTN in three replicate times. (B) Venn-diagram of DEGs between shPOSTN sEV vs. CTRL and shPOSTN sEVs vs. shNC sEVs. (C) Heatmap showing the top DEGs in each group. (D) Volcano plot showing BMP4 downregulation in macrophages treated with shPOSTN sEVs vs. shNC sEVs. (E) GSEA analysis of hallmark pathways in macrophages treated with sEV-derived CAF-S6 transfected with either shPOSTN or shNC. (F) GO enrichment analysis was performed to identify pathways associated with the representative DEGs. (G) The bubble plot displays the top 20 markedly enriched KEGG pathways for the DEGs. (H) The mRNA expression of BMP4 in macrophages induced sEVs derived from CAF-S5/-S6 transfected with shNC and shPOSTN were analyzed by RT-qPCR. (I) The mRNA expression of TNF-α, IL-6, TGF-β and IL-10 in macrophages stimulated with BMP4 at concentrations of 0, 50 and 100 ng/ml. (J) The expression of CD163 and CD206 in macrophages treated with BMP4 at concentrations of 0, 100 ng/ml examined by flow cytometry. (K) The level of BMPR2, pSmad1/5/9, Smad 5 and Smad 9 in macrophages treated with BMP4 (100 ng/ml) for 48 h were examined by western blotting (n=3 per group). (L) The level of pSmad1/5/9, Smad5, Smad9, CD163 and CD206 in macrophages treatment with or without LDN193189 inhibitor (n=3 per group). Statistical significance was determined using a one-way ANOVA test, ns, not significant *P<0.05; **P<0.01; ****P<0.0001. Error bars represent the mean ± SEM. Source data for blotting assays, see . PCA, principal component analysis; sEV, small extracellular vesicles; sh, short hairpin; NC, negative control; DEGs, differentially expressed genes; POSTN, perostin; sEV, small extracellular vesicles; GSEA, gene set enrichment analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR; pSmad, phosphorylated Smad.

Article Snippet: The primary antibodies included POSTN antibody (cat. no. ab14041; 1:500; Abcam), CD9 (cat. no. ab236630; 1:1,000; Abcam), CD81 (cat. no. ab79559; 1:1,000; Abcam), Calnexin (10427-2-AP; 1:1,000, Proteintech, Wuhan, China), CD80 (cat. no. ab134120; 1:1,000; Abcam), CD86 (cat. no. abs115477; 1:1,000; Absin Bioscience), CD163 (sc-33715; 1:1,000, Santa Cruz Biotechnology), CD206 (cat. no. ab64693; 1:1,000; Abcam), BMPR2 (cat. no. abs147034; 1:1,000; Absin Bioscience), Phospho-Smad 1 (Ser463/465)/Smad5(Ser463/465)/Smad9(Ser465/467) (cat. no. 13820; 1:1,000; Cell Signaling Technology, Inc.), Smad5 (12534; 1:1,000; Cell Signaling Technology, Inc.), Smad9 (cat. no. abs131190; 1:1,000; Absin Bioscience) and GAPDH (cat. no. 10494-1-AP; 1:5,000; Proteintech Group, Inc.), followed by incubation with a HRP-conjugated Goat anti-Rabbit IgG (H+L) as the secondary antibody (cat. no. SA00001-2; 1:3,000; Proteintech Group, Inc.) for 2 h. After washing the membrane three times with 1X TBST, the protein bands were visualized using an ECL detection system.

Techniques: Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction