Figure 1.
The increased expression of MMP-3 mRNA and MMP-3 protein in odontoblast-like cells.
(A) Increased expression of MMP-3 and GAPDH (a housekeeping gene) transcripts measured using RT-PCR. (B) Western blot analysis of the levels of MMP-3 and β-tubulin. The β-tubulin protein served as the internal control to confirm that equal amounts of the total protein extract had been loaded into each well of the gel. Each experiment was repeated three times and the results shown are representative of these three independent experiments.
Figure 2.
IL-1β induces the expression of TIMP mRNA and TIMP in odontoblast-like cells.
(A) Expression of TIMP-1, TIMP-2 and GAPDH (housekeeping gene) transcripts measured using RT-PCR. (B) Western blot analysis of TIMP-1 and TIMP-2 protein expression in each cell line. Data are representative of at least three independent experiments.
Figure 3.
Effect of exogenous IL-1β on MMP-3 activity in odontoblast-like cells.
Effect of exogenous IL-1β on MMP-3 activity in odontoblast-like cells derived from iPS cells, ES cells and KN-3, as evaluated by the immunoprecipitation-MMP-3 assay. The cells were incubated in serum-free medium in the absence or presence of IL-1βfor the times indicated (6 h = white bars, or 12 h = black bars). Data are presented as the mean ± SD of six independent experiments; (vs. control, *P<0.05).
Figure 4.
Effects of exocytosis inhibitor Exo1 treatment with IL-1β on MMP-3 activity in odontoblast-like cells.
The cells were incubated in serum-free medium in the absence or presence of Exo1 (0, 1, or 3 µM) and IL-1βfor the times indicated (6 h = grey bars, or 12 h = black bars). Data are presented as the mean ± SD of four independent experiments;. (**P<0.01 vs. control; #P<0.05 vs. Exo1 (1 µM); †P<0.01, as indicated by the bracket).
Figure 5.
Effect of exogenous IL-1β on DNA fragmentation and cell proliferation in odontoblast-like cells.
(A) Effect of exogenous IL-1β on DNA fragmentation in odontoblast-like cells derived from iPS cells, ES cells, and KN-3, as evaluated by the detection of BrdU-labeled DNA fragments. Data are presented as the mean ± SD of six independent experiments; (vs. control, *P<0.05, **P<0.01). (B) Effect of exogenous IL-1β on cell proliferation, as evaluated using the BrdU-cell proliferation ELISA. The cells were incubated in the absence or presence of IL-1β (0, 0.25, 2.5, and 25 ng/mL) for 24 h. Data are presented as the mean ± SD of at four independent experiments; (vs. control, *P<0.05, **P<0.01).
Figure 6.
The effect of transfection of odontoblast-like cells with siRNA on the levels of MMP-3.
(A) RT-PCR analysis of the MMP-3 gene expression in cells 24 h after transfection with siRNA. (B) Western blot analysis of the increased expression of MMP-3 in cells 24 h after transfection with siRNA. Each experiment was repeated three times and the results shown are representative of these three independent experiments.
Figure 7.
The effect of transfection of odontoblast-like cells with siRNA on the levels of MMP-3 activity.
Effects of the combination of IL-1β treatment (0, 0.25, 2.5 and 25 ng/mL) on MMP-3 activity released from cultured cells 24 h after transfection with siRNA. The cells were incubated in serum-free medium in the absence or presence of exogenous IL-1β at the concentrations indicated for either 6 h (white bars) or 12 h (black bars). Data are presented as the mean ± SD of four independent experiments. *, # and † denote significant differences; (*P<0.05 vs. control; #P<0.05 vs. control siRNA; †P<0.01, as indicated by the bracket).
Figure 8.
Effect of MMP-3 siRNA on DNA fragmentation in odontoblast-like cells.
DNA fragmentation of the cells 24-3 evaluated by the detection of BrdU-labeled DNA fragments; (**P<0.01 vs. control; ##P<0.01 vs. control siRNA; †P<0.01, as indicated by the bracket.) Data are presented as the mean ± SD of six independent experiments.
Figure 9.
Effect of MMP-3 siRNA on cell proliferation in odontoblast-like cells.
Cell proliferation of odontoblast-like cells derived from iPS cells, ES cells and KN-3 24 h after transfection with siRNA designed to target MMP-3 evaluated using BrdU-cell proliferation ELISA; (**P<0.01 vs. control; ##P<0.01 vs. control siRNA; †P<0.01 as indicated by the bracket). Data are presented as the mean ± SD of six independent experiments.
Figure 10.
Effect of exogenous MMP-3 on cell proliferation in odontoblast-like cells.
Effects of exogenous MMP-3 on odontoblast-like cells proliferation derived from iPS cells, ES cells and KN-3. Cells were incubated in serum-free medium in the absence or presence of various concentrations of MMP-3 (0, 10, 30, 50 and 100 ng/mL) for 24 h prior to cell proliferation evaluation using the BrdU-cell proliferation ELISA. Data are the mean ± SD of six independent experiments; (vs. control, *P<0.05, **P<0.01).
Figure 11.
Effects of exogenous MMP-3 on odontoblast-like cells apoptosis induced by IL-1βandMMP-3 siRNA treatment.
Cells were incubated in serum-free medium in the absence or presence of MMP-3 (5, 10, 20 and 30 ng/mL), and MMP-3 siRNA combined with IL-1β (0 and 0.25 ng/mL) for 24 h, and then DNA fragmentation (i.e., apoptosis index) was evaluated by the BrdU-labeled DNA fragmentation ELISA. Data are the mean ± SD of six independent experiments; (vs. as control with the IL-1βaddition (0.25 ng/mL), **P<0.01, †P<0.05 as indicated by the bracket).