Fibrillin-1 regulates periostin expression during maintenance of periodontal homeostasis

Figures

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  Figure 1. Cytotoxicity under mechanical force. Cytotoxicity was significantly increased by the application of 135 g for 12 and 24 hours compared with 40 g. However, it was not significantly increased by the application of 90 g for 12 and 24 hours compared with 40 g (n = 3). For subsequent experiments, 90 g mechanical force was applied for 24 hours. ∗p < 0.05, 40 g versus 90 g versus 135 g.

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  Figure 2. Changes in the expression levels of FBN1 and POSTN under mechanical force in HPDLFs, evaluated by quantitative reverse transcription-polymerase chain reaction (n = 3). The relative expression levels are shown with the 0 g group regarded as 1. There were no significant differences in the expression of FBN1 (A) and POSTN (B) upon exposure to 90 g for 24 hours. FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: human periodontal ligament fibroblasts, POSTN: periostin.

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  Figure 3. Changes in the expression levels of FBN1 and POSTN under mechanical force in HPDLFs upon knockdown of FBN1, evaluated by quantitative reverse transcription-polymerase chain reaction. (A) Expression of FBN1 in HPDLFs. The relative expression levels are shown with Scr regarded as 1. Expression of FBN1 was decreased compared with the Scr group (p < 0.01) (n = 3). This result confirmed reduced expression of FBN1. (B) Expression of POSTN under mechanical force of 90 g in HPDLFs upon knockdown of FBN1. The relative expression levels are shown with Scr-0 g regarded as 1. Expression of POSTN in Si-0 g was decreased compared with Scr-0 g (p < 0.01) (n = 3). Similarly, the expression of POSTN in Si-90 g was significantly decreased compared with Si-0 g (p < 0.05) (n = 3). (A) ∗p < 0.05, Scr versus Si. (B) ∗p < 0.05 or ∗∗p < 0.01, Scr-0 g versus Scr-90 g versus Si-0 g versus Si-90 g FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: human periodontal ligament fibroblasts, POSTN: periostin, Scr: scrambled siRNA, Si: siRNA against FBN1.

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  Figure 4. Changes in the expression levels of BMP2 and CTGF under mechanical force in HPDLFs upon knockdown of FBN1, evaluated by quantitative reverse transcription-polymerase chain reaction. The relative expression levels are shown with Scr-0 g regarded as 1. (A) Expression of BMP2 was significantly increased under mechanical force of 90 g, regardless of FBN1 knockdown (p < 0.01) (n = 3). (B) There was no change in the expression of CTGF under mechanical force of 90 g, regardless of FBN1 knockdown (n = 3). ∗∗p < 0.01 versus Scr-0 g. BMP2: bone morphogenetic protein 2, CTGF: connective tissue growth factor, FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: human periodontal ligament fibroblasts, POSTN: periostin, Scr: scrambled siRNA, Si: siRNA against FBN1.

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  Figure 5. Changes in the phosphorylation of Smad2 under mechanical force in HPDLFs upon knockdown of FBN1, evaluated by western blotting. β-actin was used as the internal loading control. (A) FBN1 knockdown caused a decrease in the phosphorylation of Smad2. Furthermore, centrifugation caused a decrease in the phosphorylation of Smad2. (B) The phosphorylation rate of Smad2 shown in (A) was quantified by ImageJ software. The relative expression levels are shown with Scr-0 g regarded as 1. The phosphorylation of Smad2 was significantly decreased under mechanical force of 90 g, regardless of FBN1 knockdown (p < 0.05) (n = 3). ∗p < 0.05 ∗∗p < 0.01, Scr-0 g versus Scr-90 g versus Si-0 g versus Si-90 g FBN1: fibrillin-1, HPDLFs: human periodontal ligament fibroblasts, Scr: scrambled siRNA, Si: siRNA against FBN1.

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  Figure 6. Changes in the expression of POSTN upon treatment with TGF-β1, evaluated by quantitative reverse transcription-polymerase chain reaction. (A) Expression of POSTN was increased by supplementation with 0.1, 1.0, or 10 ng/ml TGF-β1 (p < 0.01) (n = 3). (B) Expression of POSTN in Si-90 g was rescued by supplementation with 0.1 ng/ml TGF-β1 (p < 0.01) (n = 3). (A) ∗∗p < 0.01, versus control. (B) ∗∗p < 0.01, versus Scr-0 g and Si-0 g (without TGF-β1 supplementation). FBN1: fibrillin-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, HPDLFs: human periodontal ligament fibroblasts, POSTN: periostin, Scr: scrambled siRNA, Si: siRNA against FBN1.