FIG 2.

Induced cystatin E/M expression leads to cell growth inhibition. (A) Induced expression of cystatin E/M with doxycycline results in growth inhibition of CST6 cells. Slope calculations indicate P = 0.0047 for 1.0 μg/ml dox versus no dox and P = 0.0004 for 2.0 μg/ml doxycycline versus no doxycycline. (B) CST6 cells show expression of cystatin E/M that is accompanied with decreased expression of cathepsin L (arrow above 28 kDa) and the formation of multimeric Bax protein (arrow above 38 kDa). tet-ind, tetracycline induction. (C) Immunoprecipitation with mouse anti-cystatin E/M antibody followed by Western blotting with biotinylated anti-cystatin E/M antibody shows cystatin E/M expression in the cell-free supernatants, indicating secretion of the protein into the medium. Bacterial recombinant cystatin E/M proteins (R & D Systems, Inc., Minneapolis, MN) (15 and 21 kDa) were used as positive hybridization controls.