A B Supplementary Figure 1: A, TRAIL-induced apoptosis in melanoma cells and melanocytes. Cells were treated with TRAIL (200ng/ml) for 24 hours before measurement of apoptosis by the propidium iodide method using flow cytometry. B, Inhibition of cystatin B does not sensitize melanoma cells to cisplatin-induced apoptosis. Mel-RM and Mel-FH cells with cystatin B stably inhibited by shRNA were treated with cisplatin (10  g/ml) for 48 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are the mean  SE of three individual experiments.

Supplementary Figure 2: The cathepsin L specific inhibitor z-FF-fmk does not block TRAIL-induced apoptosis of melanoma cells. Cells were treated with z-FF-fmk (10  M) for 3 hours before the addition of TRAIL (200ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are the mean  SE of three individual experiments.

Supplementary Figure 3: Inhibition of cystatin B enhances TRAIL-induced activation of Bax in melanoma cells. Mel-RM and Mel-FH cells with cystatin B stably knocked down by shRNA treated with TRAIL (200ng/ml) for 16 hours were subjected to measurement of activation of Bax by flow cytometry using an antibody that specifically recognize activated Bax. The filled histograms represent isotype controls. The thick open histograms were generated from cells transduced the control shRNA treated with TRAIL, and the open thin histograms from cells with cystatin B stably knocked down treated with TRAIL. The data shown are representative of three individual experiments.

Supplementary Figure 4: Inhibition of cystatin B does not significantly alter the mRNA levels of FLIP in melanoma cells. Total RNA from Mel-RM and Mel-FH cells was isolated and subjected to Real-time PCR analysis for FLIP mRNA expression. The relative abundance of mRNA expression in corresponding parental cells was arbitrarily designated as 1. The data shown are the mean  SE of three individual experiments.

A B Supplementary Figure 5: Over-expression of cystatin B prolongs the half- life time of FLIP L. A, Whole cell lysates from Mel-CV and MM200 stably transfected with cDNA encoding cystatin B was subjected to Western blot analysis. B, The half-life time of FLIP L was prolonged in melanoma cells over-expressing cystatin B. Mel-CV and MM200 cells over-expressing cystatin B as shown in A were treated with cycloheximide (10  g/ml) for indicated periods. Whole cell lysates were subjected to Western blot analysis. The data shown are representative of three individual experiments.

Supplementary Figure 6: The levels of cystatin B expression do not in general correlate with sensitivity of melanoma cells to TRAIL- induced apoptosis. The levels of cystatin B expression as shown in Figure 1 were semi-quantitated. The levels of apoptosis induced by TRAIL were as shown in Supplementary Figure 1. Regression analysis was carried out with the Excel software in an IBM computer.