Figure 5. Perforin degradation by cathepsin and melanoma cell extracts.

(a,b) Measurement of perforin lytic activity on Jurkat cells. Jurkat cells were incubated with purified human perforin either in the absence or in the presence of CatB or of CatB plus CA074 for 1 h. PI entry in cells was measured by flow cytometry. (a) Plots show results from one representative experiment; (b) data are expressed as mean±s.e.m. of four independent experiments. (c,d) Degradation of perforin in isolated CTL lytic granules. (c) Western blot analysis of lytic granule perforin either untreated or incubated with CatB or with CatB plus CA074. (d) Western blot analysis of lytic granule perforin either untreated or incubated with increasing concentrations of melanoma cell lysates (+ 0.5 μg ml⁻¹; ++ 1 μg ml⁻¹; +++ 3 μg ml⁻¹). Results are from one representative experiment out of three. Numbers indicate band intensity fold increase. (e,f) Jurkat cells were incubated for 1 h with purified human lytic granules lysate either in the absence or in the presence of melanoma cell vesicular fraction lysates or of melanoma cell vesicular fraction lysates plus CA074 for 1 h. PI entry in cells was measured by flow cytometry. (e) Plots show result from one representative experiment; (f) data are expressed as mean±s.e.m. of three independent experiments. Unpaired Student's t-test using the GraphPad Prism software was used to determine the statistical significance of differences between the groups. ^***P<0.001, ^**P<0.01. LG, lytic granules; VF, vesicular fraction.