Fig 2: Effects of TGF-ß1 on the motility of human osteoblasts are dependent upon PI3K/AKT signalling. (A) Migration of hFOB1.19 human osteoblasts treated for 24 h with 1 ng/ml TGF-ß1 and/or 10 µM LY, a PI3K/AKT inhibitor, as determined by a scratch-wound assay. The number of cells entering the wound indicated the migration of cells. (B) Migration of osteoblasts treated for 24 h with 1 ng/ml TGF-ß1 and/or 10 µM LY, as determined by a Transwell assay. Scale bar, 200 µm. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. AKT, protein kinase B; LY, LY294002; NC, negative control; PI3K, phosphatidylinositol 3-kinase; TGF-ß1, transforming growth factor ß1.

Fig 3: Effects of the inhibition of PI3K/AKT and mTOR/S6K1 signalling on TGF-ß1-mediated osteogenic induction. (A) Effects of treatment for 7 days with 1 ng/ml TGF-ß1 plus 10 µM LY, a PI3K/AKT inhibitor, or 100 nM Rapa, an mTOR/S6K1 inhibitor, on the activity of ALP in hFOB1.19 human osteoblasts. ALP-positive cells were stained with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium. (B) Ca2+ deposits following treatment of osteoblasts for 14 days with 1 ng/ml TGF-ß1 plus 10 µM LY or 100 nM Rapa, as identified by Alizarin red S staining. Scale bar, 200 µm. Data are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001 vs. TGF-ß1. AKT, protein kinase B; ALP, alkaline phosphatase; LY, LY294002; mTOR, mammalian target of rapamycin; NC, negative control; PI3K, phosphatidylinositol 3-kinase; Rapa, rapamycin; S6K1, S6 kinase 1; TGF-ß1, transforming growth factor ß1.

Fig 4: Microcystin-LR has inhibitory effect on IL-4 induced M2 polarization of macrophages.RAW264.7 cells were left alone or treated with IL-4 (5 ng/ml) for 48 h to induce polarization. In some of the assays, cells were also treated with 0.1 µM microcystin-LR for the same duration as indicated. a Percentages of CD206+/CD11b+ M2-like macrophages were analyzed by flow cytometry. b Relative expression of the M2-like macrophage markers CD206, TGF-ß1, arginase-1 (Arg1) and chitinase-like 3 (Ym1) was analyzed using qRT-PCR. *P < 0.05, **P < 0.01 determined by one-way ANOVA with S-N-K post hoc analysis (CD206: F3,8 = 38.234, P < 0.000; TGF-ß1: F3,8 = 6.900, P = 0.013; Arg1: F3,8 = 23.223, P < 0.000; Ym1: F3,8 = 15.990, P = 0.001; n = 3). c Protein levels of CD206, p-PI3K/PI3K, p-AKT/AKT and p-STAT6/STAT6 signaling pathway molecules were determined by western blot. *P < 0.05, **P < 0.01 determined by one-way ANOVA with S-N-K post hoc analysis (CD206: F3,8 = 6.475, P = 0.016; PI3K: F3,8 = 9.546, P = 0.005; AKT: F3,8 = 6.813, P = 0.014; STAT6: F3,8 = 67.900, P < 0.000; n = 3).

Fig 5: Role of Nhe1 in IgE-induced macrophage foam cell formation. Intracellular lipid quantification a and selected representative images b of macrophages from Apoe–/–Nhe1+/+ and Apoe–/–Nhe1+/– mice after treatment with or without IgE, ox-LDL, PI3 kinase inhibitor LY294002, AKT inhibitor triciribine, and mTOR inhibitor rapamycin as indicated. Bars: 200 µm. c Immunoblot analysis of (p)-AKT, (p)-PI3K, (p)-mTOR, and ß-actin in macrophages from Apoe–/–Nhe1+/+ and Apoe–/–Nhe1+/– mice after treatment with and without IgE. Gel density quantifications are shown to the right. Data are presented as mean ± SEM from three independent experiments. One-way ANOVA test followed by a post hoc Tukey’s test (p-AKT/total AKT in c) and Kruskal–Wallis test followed by Dunn's procedure (all other panels) were used to compare three or more groups. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file