Figure 5

PE inhibited the activation of RUNX-2 in primary human OA chondrocytes. (a) Primary human OA chondrocytes were pretreated with PE for 2 h and stimulated with IL-1β for 30 minutes and the presence of activated RUNX-2 in the nucleus was determined by Immunoblotting. Protein loading was verified by using an antibody against the nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1). Band images were digitally captured and the band intensities (pixels/band) were obtained as described under Figure 1. Average pixel values presented as mean ± SD. (b) PE inhibited the DNA binding activity of RUNX-2 in IL-1β-stimulated human OA chondrocytes. DNA binding activity of activated RUNX-2 present in the nucleus was determined by using a Transcription Factor ELISA kit specific for RUNX-2 (Active Motif). The Saos-2 nuclear extract supplied with the kit was used as a positive control. Data is representative of two experiments and is presented as Mean ± SEM. Values differ without a common letter P < 0.05. (c) PE does not block the interaction of IL-1β with IL-1 receptor. Human T cells HSB.2 (70 to 80% confluent) were stimulated with IL-1β and PHA in the presence or absence of PE and the production of IL-2 was quantified by a sandwich ELISA (R&D Systems) and the values were calculated from a standard curve. Data presented as Mean ± SEM; data without a common letter differ, P < 0.05.