Fig 1: IL-1β induced gene expression of gelatinases and TIMPs in MAPK-specific inhibitor-pretreated mono-cultured and co-cultured chondrocytes. (a) IL-1β at different concentrations induces gene expression of gelatinases and TIMPs in mono-cultured and co-cultured chondrocytes.1, 5, 10 and 20 = 1, 5, 10 and 20 ng·mL−1. GAPDH and β-actin were used as the reference genes. The product sizes are indicated in the left lane. The gels shown are representative of three independent experiments (n = 3). (b) High ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1 induced by different concentrations of IL-1β in inhibitor-pretreated mono-culture and co-culture chondrocytes. The data were the mean of three different experiments (n = 3). *Significant difference with respect to the normal ratios (P < 0.05). (c) The gene profiles of the gelatinases and TIMPs induced by IL-1β and/or specific inhibitors in mono-cultured and co-cultured chondrocytes. GAPDH and β-actin were used as internal controls. The product sizes are indicated in the left lane. The gels shown are representative of three independent experiments (n = 3). (d) Different ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1 inhibitor-pretreated mono-cultured and media co-cultured chondrocytes. The data are the mean of three different experiments (n = 3). *Significant difference with respect to control ratios (P < 0.05). (e) qPCR was done to confirm the gene expressions of gelatinases induced by IL-1β in mono-culture and co-culture group. *Significant difference with respect to monolayer chondrocytes (P < 0.05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; MMP, metalloproteinase; TIMP, tissue inhibitors of metalloproteinase.
Fig 2: IL-1β increases the activity of gelatinases in a dose-dependent manner in both mono-cultured and co-cultured chondrocytes. (a) Zymography showed different dose-dependent increases of the gelatinases in mono-cultured and co-cultured chondrocytes. Pro-MMP-9 (87 kDa), active-MMP-9 (83 kDa), pro-MMP-2 (68 kDa) and active-MMP-2 (65 kDa) are in the right lane. The gels shown are representative of three different experiments (n = 3). (b) The quantification was performed with Quantity One 4.6.3 software. The optical densities of the pro- and active-MMP-2 and -9 bands were added as the total value of activity for MMP-2 and -9. The data are the mean of three different experiments (n = 3). *Significant difference with respect to control (P < 0.05). (c) ELISA Kit confirmed the IL-1β-induced gelatinases secreted by chondrocytes in mono-culture and co-culture groups (mean ± standard deviation) (n = 4). *P < 0.05, **P < 0.025, compared with controls. ELISA, enzyme linked immunosorbent assay; IL, interleukin; MMP, metalloproteinase.
Fig 3: Osteoclast-conditioned media attenuate the activity of gelatinases secreted by chondrocytes. (a) Zymography demonstrating gelatinases in 0.5%, 1% and 2% fresh FBS culture media and collected osteoclast culture media. Osteoclast culture media (50%) shows the samples of this group loaded for electrophoresis were 50% dilution compared with other groups. (b) Zymography demonstrating the activity of gelatinases secreted by mono-cultured and co-cultured chondrocytes. 0.5%, 1% and 2% FBS show the active gelatinase contents in chondrocytes after culture with 0.5%, 1% and 2% FBS; monolayer chondrocytes and media co-cultured chondrocytes are shown in a gel to make a comparison. The gels are the representative of three different experiments (n = 3). (c) Quantification demonstrated time-dependent increases of MMP-2 in both mono-culture and co-culture chondrocytes. Quantification was performed with Quantity One 4.6.3 software. The optical densities of the pro- and active-MMP-2 bands were added as the total value of activity for MMP-2. The values at 24, 48 and 72 h were compared to the values at 12 h. The quantitative data about total activity refer to 72 h time points of the mono-cultured and co-cultured chondrocytes. The data are the mean of three different experiments (n = 3). *Significant difference with respect to monolayer chondrocytes (P < 0.05). (d) ELISA Kit confirmed the gelatinases secreted by chondrocytes in mono-culture and co-culture groups (mean ± standard deviation) (n = 4). *Significant difference with respect to monolayer chondrocytes (P < 0.05). DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme linked immunosorbent assay; FBS, foetal bovine serum; Mono, mono-culture.
Fig 4: IL-1β restores the high activities of gelatinases in MAPK-specific inhibitor-pretreated mono-cultured and co-cultured chondrocytes. (a) Zymography demonstrating that IL-1β (10 ng·mL−1) can restore the activities of gelatinases through ERK (in the presence of PD58059), p38 (in the presence of SB203580) and especially JNK (in the presence of SP600125). The gels shown are representative of three different experiments (n = 3). (b) The activities of pro- and active-MMP-2/9 in mono-cultured and co-cultured chondrocytes were quantified by the optical densities methods (Quantity One 4.6.3 software). The indicated quantitative data refer to 72 h time-points. The data are the mean of three different experiments (n = 3). *Significant difference with respect to IL-1β-induced control (P < 0.05); #Significant difference with respect to other two inhibitor-treated groups (P < 0.05). (c) IL-1β restored the total activities of gelatinases in MAPK inhibitor-pretreated chondrocytes (mean ± standard deviation) (n = 3). Scale bars, standard deviation. *P < 0.05, compared with control. (d) Due to high restored gelatinases in JNK inhibitor group, ELISA was done to explore the gelatinase secretion in both mono-culture and co-culture groups (mean ± standard deviation) (n = 4). *P < 0.05, **P < 0.025, compared with controls. ELISA, enzyme linked immunosorbent assay; ERK, extracellular signal-regulated kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; MMP, metalloproteinase.
Supplier Page from Abcam for Mouse MMP2 ELISA Kit