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Supplemenatry figure 1: Effect of HIF-1 silencing in myeloma cells
The HMCLs JJN3 and RPMI-8226 were transfected by electroporation with 2 nmol of smart pool
double stranded RNA oligonucletides (siRNA) against HIF-1 or with a non-specific control
siRNA (Cy). After 24 hours cells were incubated in the presence or absence of hypoxic condition
(1%O2, 5%CO2 atmosphere or CoCl2 treatment) for 12 hours. HIF-1 mRNA expression was
evaluated by RT-PCR whereas HIF-1 protein level and activity was detected by western blot and
ELISA, respectively, as described in the Methods. Graphs represent the mean ± SD of two
independent experiments measured in triplicate (OD= optical density). Nuclear extracts of COS-7
treated with CoCl2 in the presence or absence of wild-type (wt) or mutated (mt) competitor
oligonucleotides were tested as control (Con); *= p<0.05 (a).
VEGF, IL-8, OPN, TGF, PGF and GAPDH mRNA expression was evaluated by RT-PCR in nonspecific control siRNA (Cy) in normoxic and hypoxic condition (Norm.= normoxia; Hypox.:
hypoxia) and in JJN3 transfected with siRNA against HIF-1 in hypoxic condition (b).
HIF-1, VEGF, IL-8, OPN mRNA levels were quantified by real time PCR in JJN3 transfected
with siRNA against HIF-1 or with a non-specific control siRNA (Cy) and incubated in normoxic
or hypoxic condition. Data are expressed as mean - Ct ± SD, as described in the Methods (c).
Aliquots of conditioned media of both JJN3 and RPMI-8228 transfected cells in normoxic and
hypoxic condition were tested by ELISA assay to detect VEGF or IL-8 levels. Graphs represent the
mean ± SD of VEGF or IL-8 levels in three independent experiments measured in triplicate;
*=p<0.05, ** p<0.01 (d).
Conditioned media obtained from JJN3 transfected with siRNA against HIF-1 or with a nonspecific control siRNA (Cy) either in normoxic or hypoxic condition were tested by angiogenesis
antibody array and the protein expression was quantified as described in the Methods. Bars
represent the median fold change in the expression of selected pro-angiogenic factors of three
independent experiments (e).
Supplementary Table 1: Functional annotations of the 714 differentially expressed genes
identified by a SAM supervised analysis in purified CD138+ PC samples from 11 MM patients
compared in hypoxic versus normoxic condition. Genes are ordered according to their SAM
score(d) and the probe set with the highest SAM score is shown, whenever more than one
recognizes the same gene.
Supplementary Table 2: Functional annotations of the 13 differentially expressed genes identified
by a dChip supervised analysis in JJN3 cell line triplicates compared after electroporation with
siRNA anti-HIF-1 or siRNA control (Cy), performed in normoxic condition. Genes are ordered
according to their fold change (FC) and the probe set with the highest FC value is shown, whenever
more than one recognizes the same gene.
Supplementary Table 3: Functional annotations of the 671 differentially expressed genes
identified by a dChip supervised analysis of JJN3 cell line triplicates compared after electroporation
with siRNA anti-HIF-1 or siRNA control (Cy), performed in hypoxic condition. Genes are
ordered according to their fold change (FC) and the probe set with the highest FC value is shown,
whenever more than one recognizes the same gene. * symbol and red color indicate the 463 genes
also modulated in hypoxic versus normoxic condition analysis, after probe set and gene symbol
matches of the two identified lists.
Supplementary Table 4: Functional annotations of the 1196 differentially expressed genes
identified by a dChip supervised analysis in JJN3 cell line triplicates compared in hypoxic versus
normoxic condition. Genes are ordered according to their fold change (FC) value and the probe set
with the highest FC value was selected whenever more than one recognized the same gene. Red
color indicates the 733 genes not comparing among the differentially expressed genes in siRNA
anti-HIF-1 versus siRNA control (Cy) in hypoxic condition analysis, after probe set and gene
symbol matches of the 1196 and 671 gene lists.