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Fig. 6 | Journal of Biomedical Science

Fig. 6

From: Crosstalk between transforming growth factor β-2 and Autotaxin in trabecular meshwork and different subtypes of glaucoma

Fig. 6

Effects of ATX on the expression levels of TGF-βs. AC n = 4) Relative mRNA expression levels of TGF-β1–3 in hTM cells were evaluated following ATX stimulation. The expression of TGF-β1 was significantly upregulated with ATX stimulation, and this change was significantly attenuated by treatment with 10 μM SB431542 or 10 μM HA130 (A). The expression of TGF-β2 was also significantly upregulated with ATX stimulation, and this change was attenuated by treatment with 5 or 50 μM SB431542, or 1 μM HA130 (B). However, the expression of TGF-β3 did not significantly differ following ATX stimulation (C). *P < 0.05, **P < 0.01, ***P < 0.001. DE Western blotting analysis of TGF-β2 when hTM cells were stimulated with ATX (D) or LPA (E). Figures on the right side present representative WB bands when hTM cells were stimulated with 40 μM ATX or 10 μM LPA with or without corresponding inhibitors (10 μM HA130 or 10 μM Ki16425). The expression of TGF-β2 was upregulated upon stimulation with ATX or LPA, and this change was attenuated by treatment with the corresponding inhibitors (D and E). Quantitative analysis revealed significant differences (n = 3, E and G). *P < 0.05, **P < 0.01. ATX induced expression of CAGA-12 promoter activity, TGF-β-induced protein and CTGF and fibrotic markers in hTM cells (F-K, n = 4). F Luminescence in hTM cells expressing a luciferase reporter gene fused with 12 repeats of the Smad-binding element (CAGA-12) was measured using luciferase assay. Relative activity of the promoter was measured under treatment with 10 ng/ml TGF-β2, 40 µM ATX with or without its inhibitor (10 µM HA130), and 10 µM LPA with or without its inhibitor (10 µM Ki16425). The relative levels of CAGA-12 promoter activity were significantly upregulated after treatment with TGF-β2, ATX, and LPA, and those changes were significantly downregulated when either ATX inhibitor (HA130) or LPA inhibitor (Ki16425) were applied (F). *P < 0.05. G The relative mRNA expression level of TGF-β-induced protein was evaluated following ATX stimulation in hTM cells. The expression of TGF-β-induced protein was significantly upregulated with ATX stimulation, and this change was inhibited by treatment with 50 μM SB431542 but not ATX inhibitor (G). *P < 0.05. (H) Measurement of ATX-induced CTGF mRNA expression. CTGF was significantly upregulated by ATX treatment, and the changes were attenuated by inhibition of TGF-β or ATX. *P < 0.05, **P < 0.01, ***P < 0.001. (I-K): Effects of ATX on the mRNA expression levels of fibrotic markers. Relative mRNA expression levels of COL1A1, fibronectin, and α-SMA were analyzed under ATX stimulation with or without 5 μM or 50 μM SB431542, or 1 μM or 10 μM HA130. Expression levels of COL1A1, fibronectin, and α-SMA were enhanced upon ATX stimulation, and these changes were significantly suppressed by treatment with either TGF-β inhibitor or ATX inhibitor (D, E, and F)

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