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. 2019 Jul 11;116(5):956–969. doi: 10.1093/cvr/cvz187

Figure 4.

Figure 4

ADAMTS16 induces the activation of TGF-β. (A) Western blot analysis showed that overexpression of ADAMTS16 decreased the level of LAP-TGF-β in mouse cardiac fibroblasts transfected with LAP-TGF-β. The experiment was independently repeated for three times (n = 6). (B) Schematic diagram showing wild-type (WT) ADAMTS16 and mutant ADAMTS16 with the RRFR motif mutated into IIFI. (C) Western blot analysis showed that overexpression of mutant ADAMTS16 did not affect the level of LAP-TGF-β in mouse cardiac fibroblasts transfected with LAP-TGF-β (n = 6). (D) Co-IP analysis showed that the interaction between LAP-TGF-β and ADAMTS16 was reduced in mouse cardiac fibroblasts by mutation IIFI (n = 6). (E) ELISA for measurements of active TGF-β (pg/mL) in the supernatant of mouse cardiac fibroblasts co-transfected with LAP-TGF-β and WT-ADAMTS16 or Mut-ADAMTS16. Data are shown as mean ± SD. **P < 0.01 (n = 4). Statistical analysis was carried out by a one-way ANOVA test. (F) ELISA for measurements of total TGF-β (acid treated) in the supernatant of mouse cardiac fibroblasts treated as in (E). (G) Luciferase assays for TGF-β-mediated transcription activation in HeLa cells. Data are shown as mean ± SD. **P < 0.01 (n = 4/group). Statistical analysis was carried out by a one-way ANOVA test. (H) Effects of TGF-β NAb on TGF-β-mediated transcription activation in HeLa cells. Data are shown as mean ± SD. **P < 0.01, NS, not significant (n = 4/group). Statistical analysis was carried out by a one-way ANOVA test. (I) Western blot analysis showed that knockdown of Adamts16 expression by siRNA significantly increased the level of LAP-TGF-β in mouse cardiac fibroblast (n = 6/group). (J) ELISA for measurements of the level of active TGF-β in the supernatant of mouse cardiac fibroblasts transfected with Adamts16 siRNA or siNC. Data are shown as mean ± SD. **P < 0.01 (n = 4). Statistical analysis was carried out by a Student’s t-test. (K) ELISA for measurements of the level of total TGF-β (acid treated) treated as in (J). (L) Luciferase assays showing reduced TGF-β-mediated transcriptional activation by Adamts16 siRNA. Data are shown as mean ± SD. **P < 0.01 (n = 4). Statistical analysis was carried out by a Student’s t-test.