1. Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression 
H. Pratsinis, A. Papadopoulou, C. Neidlinger-Wilke, M. Brayda-Bruno, H.-J. Wilke, D. Kletsas 
Osteoarthritis and Cartilage 
Volume 24, Issue 4, Pages (April 2016)
DOI: /j.joca
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
MAPK activation by CTS. AF cell cultures were subjected to CTS and MAPK activation was assessed by western analysis. In (A) the various magnitudes were applied under constant frequency (1 Hz) and lysates were collected at 30 min. In (B) the various frequencies were assessed at a magnitude of 4% and lysates were collected at 30 min. In (C) CTS (4%; 1 Hz) was applied for the indicated time intervals. One representative experiment is depicted out of three ones performed in lysates from different donors.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
CTS effect on DNA and collagen synthesis. (A) AF cell cultures arrested in DMEM 0.2% FBS were labeled with tritiated thymidine and subjected to the indicated CTS magnitudes (frequency 1 Hz) for 12 h. After further 12 h DNA synthesis was estimated. Horizontal lines indicate mean (±95% confidence interval) of three experiments performed in cultures from different donors. (B) AF cell cultures arrested in DMEM 0.2% FBS were treated with PDGF or subjected to CTS (magnitude 4%; frequency 1 Hz) for 12 h; then the cells were fixed and subjected to flow cytometric cell cycle analysis. A representative experiment out of three ones is depicted (mean ± 95% confidence interval of two technical replicates). (C) AF cell cultures arrested in DMEM 0.2% FBS were labeled with tritiated proline and subjected to the indicated CTS magnitudes (frequency 1 Hz) for 12 h. After further 12 h, culture supernatants were used for determination of newly synthesized collagen. Horizontal lines indicate mean (±95% confidence interval) of three experiments performed in cultures from different donors.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Proinflammatory gene expression following CTS effect of various magnitudes. AF cell cultures were subjected to CTS of the indicated magnitudes, frequencies and durations and mRNA was collected and used for qRT-PCR. Horizontal lines indicate mean (±95% confidence interval) of three experiments performed in cultures from different donors (*P < 0.05; **P < 0.01; compared to unstressed).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Proinflammatory gene expression following CTS in the presence of MAPK inhibitors. AF cell cultures were preincubated with the indicated kinase inhibitors (25 μM PD98059; 10 μM SB203580; 20 μM SP600125), subjected to CTS (8%; 1 Hz) for 12 h, and mRNA was collected and used for qRT-PCR. Mean values from three independent experiments are presented (*P < 0.05; **P < 0.01; CTS in the absence of inhibitors is compared to unstressed; CTS in the presence of inhibitors is compared to CTS in the absence of inhibitors).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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6. Supplemental Fig. 1
Validation of CTS assay system. AF cells were cultured on fibronectin-coated silicone dishes under low serum conditions and stimulated with 10 ng/ml human recombinant platelet-derived growth factor (PDGF; R&D Systems, Minneapolis, MN, USA) or human platelet-rich plasma (PRP). In (A) MAPK activation was assessed 30 min after stimulation by western analysis (one representative experiment out of two is presented). In (B) DNA synthesis was assessed 24 h after stimulation based on tritiated thymidine incorporation (mean from three experiments performed with cultures from different donors; **P < 0.01).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

7. Supplemental Fig. 2
MAPK activation by CTS. Images presented in Fig. 1 were analyzed using the Advanced Image Data Analyzer (AIDA , Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany). The signal intensities of phosphorylated proteins were normalized to those of the corresponding unphosphorylated ones.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

8. Supplemental Fig. 3
MMP gene expression and activity after CTS. AF cell cultures were subjected to CTS under the indicated conditions of magnitude, frequency and duration. In (A) and (B) mRNA was collected and used for qRT-PCR. In (B) frequency was 1 Hz and duration 12 h. Horizontal lines indicate mean (±95% confidence interval) of three experiments performed in cultures from different donors. In (C) after CTS (frequency 1 Hz) for 12 h, cells were incubated for further 12 h and then, culture supernatants were assayed for MMP activity using the substrate TNO211, as described in Skevaki et al. (2012) Clin. Transl. Allergy 2: 14. A representative experiment out of two ones is depicted (mean ± 95% confidence interval of three technical replicates).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

9. Supplemental Fig. 4
COX-2 protein expression after CTS. AF cell cultures were subjected to CTS of the indicated magnitudes (1 Hz) for 12 h, incubated for further 12 h and then cell lysates were subjected to 10% SDS-PAGE and immunoblotting using anti-COX-2 (D5H5; Cell Signaling Technology) and anti-actin (ACTN05; Neomarkers/Thermo Fisher Scientific). In the lower panel, densitometric analysis of COX-2 expression after normalization to that of actin (see above Supplemental Fig. 2) is presented.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions