Fig. 5

Reversibility of subtilisin. Panel (a) illustrates the experimental paradigm. MDA-MB-231 cells, as a mixture of flattened and rounded cell types attached to the dish together with some detached, floating cells (b(i),(iv)), were treated with a relatively high concentration of subtilisin (1 μM) for 24 h. This caused most cells to detach from the culture plate and appear as spherical cells floating in the culture medium either as individual cells (b(v)) or as aggregates. After 24 h, the cells were washed in fresh medium and transferred to new wells, where the detached cells re-attached to the well surface and grew to reform the apparently normal mixture of flat and rounded, adherent cultures (b(vi)). Control cells were treated similarly by washing (b(ii)) and transference to new wells (b(iii)) in parallel with the subtilisin-treated dishes. The ability to recover from subtilisin treatment was supported by protein expression (c). There was a substantial reduction in the expression of neogenin and unc-5C, with preservation of RhoA levels (c) in the presence of subtilisin. When the cells were washed and re-plated as above, expression of these proteins recovered to their orginal levels within 24 h, in parallel with the recovery of normal cell morphology. Scale bar: 100 μm