Supplemental Figure Legends
Supplemental Figure 1. Inhibitory effect of IL-32 on colon cancer cell growth and apoptotic cell
death. A, Colon cancer cells were transfected with IL-32γ and cultured in the absence or presence of
paclitaxel (5 nM). At 24, 48, and 72 h, the cell numbers were calculated. All results are expressed as mean
± SD of three experiments, with triplicate tests in each experiment. B, Prostate (PC3), liver (HepG2), and
lung (NCIH4610) cells were transfected with IL-32 and cultured for 72h. Cell growth was measured by
direct counting after trypan blue staining.*p<0.05 compared with the cancer cells transfected vecor alone.
C, Apoptosis of colon cancer cells following co-transfection of IL-32γ and siIL-32γ. The numbers of
apoptotic cells (DAPI-stained, TUNEL-positive cells) were estimated by direct counting. D, SW620 cells
were transfected with IL-32 and treated with its antibody. The number of apoptotic cells (DAPI-stained,
TUNEL-positive cells) were estimated by direct counting. All results are expressed as mean ± SD of three
experiments, with triplicate tests in each experiment.
Supplemental Figure 2. Effect of IL-32 protein on DNA and transcriptional activation of NF-B in
colon cancer cells. A, Nuclear extracts of SW620 cells that were cultured for 24 h and transfected with
the vector or each IL-32 isoform (, , or ) were added to a 32P-end-labled oligonucleotide containing
the NF-κB sequence. The DNA binding activity of NF-κB was investigated using EMSA, as described in
Methods. Control: untransfected cells. B, Nuclear extracts from PC3, HepG2, and NCIH4610 cells that
were cultured for 24 h and transfected with the vector or with IL-32 were added to a 32P-end-labled
oligonucleotide containing the NF-κB sequence. The DNA binding activity of NF-κB was investigated
using EMSA. C, The DNA binding activity of NF-B was determined via EMSA in the nuclear extracts
of SW620 cells that were treated with recombinant IL-32 protein (25 or 50 ng/ml). D, For competition
assays, nuclear extracts from SW620 cells treated with IL-32 protein (50 ng/ml) were incubated for 1 h
before EMSA with unlabeled NF-B oligonucleotide. For supershift assay, nuclear extracts were
incubated for 1 h before EMSA with specific antibodies against p50 and p65. SS: supershifted. Each
band is representative of three independent experiments. E, Colon cancer cells were transfected with
pNF-B-Luc plasmid (5 x NF-B) and activated with TNF- (10 ng/ml) alone or with different
concentrations (10-50 ng/ml) of recombinant IL-32 protein, and the luciferase activity was determined.
The results are expressed as mean ± SD of three experiments, with triplicates in each experiment.
#
p<0.05, compared with the colon cancer cells untreated. *p<0.05, compared with the colon cancer cells
treated withTNF- alone. RLU: relative to luciferase activity of transfected, unstimulated cells.
Supplemental Figure 3. Effect of IL-32 on translocation of p65 and p50. Celluar localization of p65
(green) and p50 (red) was observed by confocal microscopy after immunofluorescence staining of colon
cancer cells transfected with IL-32 alone or with its siRNA. Each image is representative of three
independent experiments.
Supplemental Figure 4. Effect of IL-32 on the level of cytokines. The levels of IL-10, TNF-, IL-1,
and IL-6 in the tumor tissues of xenograft nude mouse (n=3) (A), and in colon cancer SW620 cells
transfected with IL-32 in the absence (B) or presence of IL-32 siRNA (C) were measured using
quantitative real time PCR, as described in Methods. *p<0.05 compared with tumor tissues inoculated
with colon cancer (SW620) cells expressing vector alone (A). *p<0.05 compared with colon cancer
(SW620) cells expressing vector alone (B). #p<0.05 compared with colon cancer (SW620) cells
expressing vector alone. *p<0.05 compared with tumor tissues inoculated with colon cancer (SW620)
cells expressing IL-32 (C).