Supplementary Figure 2. Effect of IL-32 and siRNA of IL-32 on melanoma, colon and prostate cancer cell growth and apoptosis as well as IL-32 expression.

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Supplementary Figure 2. Effect of IL-32 and siRNA of IL-32 on melanoma, colon and prostate cancer cell growth and apoptosis as well as IL-32 expression. (A), To determine effects of the IL-32 expression level on melanoma, colon and prostate cancer cell growth, the cells were inoculated into 24-well plates (5 × 104 cells/well), and transfected with the IL-32 plasmid (0.1-0.4 g/well) in the absence or presence of siRNA of IL-32b. Cell growth was measured by direct counting after trypan blue staining. Control, untransfected cells. *, p<0.05 compared with the colon and prostate cancer cells transfected with vector. (B), Effect of IL-32 on apoptotic cell death in colon and prostate cancer cell growth. Cells were transfected with the vector or the IL-32 plasmid for 24 h. The apoptotic index was determined as the number of DAPI-stained, TUNEL-positive cells counted. The results are expressed as mean ± SD of three experiments with each experiment performed in triplicate. *, p<0.05 compared with the colon and prostate cancer cells transfected with vector. (C), Expression of IL-32 was determined with specific antibody of IL-32b as described in material and Methods. The expression band representative of three experiments with each experiment performed in triplicate.

A B C Supp. Fig. 2 Melanoma SW620 PC3 IL-32b IL-32b IL-32b b-actin 20 40 60 80 100 120 Cell viability (% of control) * # PC3 siCON (nM) - - 50 100 - - siIL-32 (nM) - - - - 50 100 IL-32 (0.4 g/well) - - 50 100 - - - - - - 50 100 Melanoma A B * 10 20 30 40 50 # # * 10 20 30 40 50 # # Apoptotic rate (%) Apoptotic rate (%) Apoptotic rate (%) * siCON (nM) - - 100 - siIL-32 (nM) - - - 100 IL-32 (0.4 g/well) siCON (nM) - - 100 - siIL-32 (nM) - - - 100 IL-32 (0.4 g/well) siCON (nM) - - 100 - siIL-32 (nM) - - - 100 IL-32 (0.4 g/well) C IL-32b IL-32b IL-32b b-actin b-actin b-actin Fold increase * 1 2 3 4 5 # 8 # 5 # 6 # 4 # Fold increase 4 Fold increase 3 * 2 2 * 1 1 siCON (nM) - - 100 - siIL-32 (nM) - - - 100 IL-32 (0.4 g/well) siCON (nM) - - 100 - siIL-32 (nM) - - - 100 IL-32 (0.4 g/well) siCON (nM) - - 100 - siIL-32 (nM) - - - 100 IL-32 (0.4 g/well)