Figure 1.
Effect of CTS on gene expression in AF cells derived from non-degenerate and degenerate IVDs.
Cells derived from non-degenerate (n = 3) or degenerate (n = 3) IVDs were mechanically stimulated with 10% CTS, 1.0 Hz frequency for 20 minutes and then incubated for up to 24 hours prior to analysis. QRT-PCR was used to analyse the gene expression of A) ADAMTS -4 or B) type I collagen, relative to the housekeeping gene GAPDH and normalised to the corresponding unloaded baseline control. Black and grey represent mechanically stimulated non-degenerate and degenerate AF cells, respectively. Values are mean of 3 donors+/− standard error mean. * denote a significant change (p≤0.05) in gene expression between mechanically stimulated and unstimulated baseline control.
Figure 2.
Effect of CTS on the gene expression of AF cells from non-degenerate and degenerate IVDs+/− peptides.
Cells derived from non-degenerate and degenerate IVDs were treated+/− RAD (50 µg/ml) or RGD (50 µg/ml) -peptides 30 minutes prior to mechanical stimulation with CTS at 10% strain, 1.0 Hz, for 20 minutes, then incubated for up to 24 hours prior to analysis. QRT-PCR was used to analyse the gene expression of A) ADAMTS -4 or B) type I collagen, relative to the housekeeping gene GAPDH and normalised to the corresponding unloaded baseline control in non-degenerate (n = 3) and degenerate (n = 3) AF cells, respectively. Black represents AF cells cyclically strained without peptide treatment, while speckles and stripes represent cells cyclically strained after treatment with RAD or RGD –peptides, respectively. Values are mean of 3 donors+/− standard error mean. *denote a significant change (p≤0.05) in gene expression between mechanically stimulated and unstimulated baseline control.
Figure 3.
Phosphorylation of FAK following treatment of AF cells derived from non-degenerate and degenerate IVDs with 1.0 Hz CTS.
AF cells derived from non-degenerate (n = 4) and degenerate (n = 3) IVDs were treated+/−1.0 Hz CTS in serum-free media and total protein extracted at timepoints of up to 20 minutes. Mechanically stimulated and unstimulated A) non-degenerate and B) degenerate protein samples (5 µg/well) were separated using 10% SDS-PAGE and probed using primary antibodies against phosphorylated FAK. Blots were then stripped using a stripping buffer, re-blocked and probed using an antibody against total FAK protein. C) The density of bands was quantified using a Syngene imaging system and the ratio of phosphorylated: total FAK protein normalised to timepoint controls and plotted as % change. *denotes a significant change (p≤0.05) between treatment groups.
Figure 4.
Phosphorylation of FAK following treatment of AF cells derived from non-degenerate IVDs with 1.0 Hz CTS, with and without pre-treatment with RGD or RAD peptides.
AF cells derived from non-degenerate IVDs (n = 4) were treated+/− RGD (50 µg/ml) or RAD (50 µg/ml) peptides, and mechanically stimulated (10% CTS, 1.0 Hz frequency) in serum-free media and total protein extracted at timepoints of 5 and 20 minutes. Mechanically stimulated and unstimulated,+/− RGD or RAD peptides, non-degenerate protein samples (5 µg/well) exposed to A) 5 minutes and B) 20 minutes of CTS, were separated using 10% SDS-PAGE and probed using primary antibodies against phosphorylated FAK. Blots were then stripped using a stripping buffer, re-blocked and probed using an antibody against total FAK protein. C) The density of bands were quantified using a Syngene imaging system and the ratio of phosphorylated: total FAK protein normalised to timepoint controls and plotted as % change. *denotes a significant change (p≤0.05) between treatment groups.