Figure 6. Effect of Rottlerin on biosynthesis and molecular size of CSPG and the CS-chains.

A, B: Cells in the absence (control) or presence of 10⁻⁷ M PMA were incubated with increasing concentrations of Rottlerin for 8 h and 23 h in serum free medium containing either [³⁵S]sulphate (A) or [³H]glucosamine (B). A typical experiment is shown, where the results (total cpm not adjusted to the amount of living cells) are presented as mean ± s.d. (n = 2). C–F: Cells were incubated for 24 h in serum free medium containing either [³⁵S]sulphate or [³H]glucosamine. C: [³⁵S]sulphate and [³H]glucosamine labelled macromolecules were applied to Q-Sepharose chromatography and the bound CSPG were eluted with a 0.15–1.5 M NaCl gradient (----) as shown in the upper graph. ○, absence of Rottlerin; Δ, presence of 1 µM Rottlerin. D, E: Eluted CSPG from the Q-Sepharose column was either untreated (solid line) or treated with cABC (---) or 0.5 M NaOH (^…….) prior to application on a Superose 6 gel chromatography column as described in Material and Methods. Cont., control without Rottlerin; Rotl., presence of 1 µM Rottlerin and Fr.No., fraction number. F: [³⁵S]CSPG (•) and free [³⁵S]CS-chains (○) from control and Rottlerin-treated cells were subjected to Q-Sepharose chromatography as in (C). Arrow shows the elution position of shark cartilage CS. G: [³⁵S]Sulphate labelled CSPG (isolated from control, PMA (10⁻⁷ M) and Rottlerin treated cells) was subjected to SDS-PAGE (upper panel: 4% stacking gel and 7.5% separating gel; lower panel: 4–12% gradient gel) followed by autoradiography (see Materials and Methods). An equal amount of radioactivity (based on scintillation counting) was loaded to each well in order to be able to compare the bands. Arrowhead shows the border between the separating and stacking gel and the position of molecular size markers are shown. Small arrow shows the bottom of the application well.