Figure 3. Synthesis of proMMP-9 and CSPG in the absence and presence of PMA.
A–C: Typical gelatin zymographies of 20 times diluted conditioned medium from THP-1 cells. A: Cells were incubated for 72 h in the presence of various concentrations of PMA as indicated. B: Cells were incubated for various time periods (as indicated) in the absence or presence of 10−7 M and 10−5 M PMA. C: Cells were either incubated for 72 h in the absence (−) or presence (+) of 10−7 M PMA at serum free conditions, or pre-incubated for 3 h in the absence (−) or presence (+) of 10−7 M PMA and/or 10% fetal calf serum. After the pre-incubation, cells were washed three times in PBS and thereafter incubated in serum free medium for 72 h. A–C: The position of proMMP-9 homodimer (225 kDa), monomer (92 kDa) and proMMP-2 (72 kDa) standards is indicated. D and E: Conditioned medium from THP-1 cells incubated with [35S]sulphate was passed over a G-50 Sepharose column in order to separate labelled macromolecules from free [35S]sulphate. The amount of labelled macromolecules was determined by counting the entire pass through fraction in a liquid scintillation spectrometer. D: Cells were incubated in the absence or presence of various concentrations of PMA for 72 h as indicated and in (E) the cells were incubated for various time periods in the absence or presence of 10−7 M PMA. (D: upper and lower panel) Results (mean ± s.d) were normalized against the control (without PMA). Lower panel, results were in addition normalized against the number of viable cells. E: Results (mean ± s.d) are presented as cpm (upper panel) and cpm/viable cells (lower panel). The results in (D) and (E) are from a typical experiment with four parallels in (D) and three parallels in (E), where *p<0.05 compared to control without PMA.