Supplementary Information
Supplementary Tables
Supplementary Table 1. Transcription profile comparison between shRNAi
clone, TC71 wild type and mock (pS) in the early and late stage: genes upregulated after EWS-FLI1 knockdown.
Changes in expression of well known and putative EWS-FLI1 target genes assessed
by GeneChip analysis of EWS-FLI1 RNAi-suppressed TC71 cells. Alterations in the
transcriptome profile were followed on Affymetrix HG-U133A microarrays after
suppression of EWS-FLI1 by shRNAi. Two independent experiments have been
performed comparing shRNAi TC71 cells with TC71 wt and mock cells. Summary of
the most relevant genes up-regulated after EWS-FLI1 knockdown according to
Genetrix analysis. shRNAi clone corresponds to TC71 shRNAi clone 6.
Supplementary Table 2. Transcription profile comparison between shRNAi
clone, TC71 wt and mock (pS) in the early and late stage: genes down-regulated
after EWS-FLI1 knockdown.
Changes in expression of well known and putative EWS-FLI1 target genes assessed
by GeneChip analysis of EWS-FLI1 RNAi-suppressed TC71 cells. Alterations in the
transcriptome profile were followed on Affymetrix HG-U133A microarrays after
suppression of EWS-FLI1 by shRNAi. Two independent experiments have been
performed comparing shRNAi TC71 cells with TC71 wt and mock cells. Summary of
the most relevant genes down-regulated after EWS-FLI1 knockdown according to
Genetrix analysis. shRNAi clone corresponds to TC71 shRNAi clone 6.
Supplementary Table 3. Transcription profile comparison between shRNAi
clone and mock (pS) in the early and late stage: EWS-FLI1 well known target
genes down-regulated after EWS-FLI1 knockdown.
Changes in expression of well known EWS-FLI1 target genes assessed by Genedata
Expressionist Refiner (Genedata, Basel, Switzerland) analysis of EWS-FLI1 RNAisuppressed TC71 cells. Alterations in the transcriptome profile were followed on
Affymetrix HG-U133A microarrays after suppression of EWS-FLI1 by shRNAi. Two
independent experiments have been performed comparing shRNAi TC71 cells with
TC71 wt and mock cells. shRNAi clone corresponds to TC71 shRNAi clone 6.
Genedata Expressionist Refiner is based on MAS5 method. Genedata normalization
onto median of 105 was performed. To filter genes with unreliable signals, all probe
sets characterized by an “Absent Detection Call” in all experiments are left out.
Clustering was done using the Euclidean distance measure and the Single Linkage /
Complete Linkage methods. To identify differentially expressed genes we computed
two-sample, two-sided t-tests between groups “shRNAi” / “TC71wt”, “mock” /
“TC71wt” and for each condition in early and late stage. Genes with a p value less
than 0.05 were considered candidate genes.
Supplementary Table 4. Primers used for qRT-PCR.
Supplementary Table 5. Primers used for ChIP.
Supplementary Figures
Supplementary Figure 1. EWS-FLI1 shRNAi slightly reduced in vitro growth but
did not alter cell cycle pattern.
A) Proliferation index measured by MTT assay in the early and late stage. Cell
growth decrease in the early stage was almost lost through cellular passages. shRNAi
clone corresponds to TC71 shRNAi clone 6. Columns, mean of replicates of three
different replicates; bars, SD.
B) Cell cycle pattern assessed through FACS in the early and late stage. Cells were
seeded on 24 well plates and after 24h were stained with IP. No change in the cell
cycle distribution was detected after EWS-FLI1 inhibition. shRNAi clone corresponds
to TC71 shRNAi clone 6. The means ± standard deviations (error bars) of 4
independent experiments are shown.
Supplementary Figure 2. Validation of microarray data of several known EWSFLI1 targets.
A) EWS-FLI1 interference up-regulated CDKN1C/p57/Kip2 protein and mRNA level
in early and late stage. Histogram representation of CDKN1C/p57/Kip2 mRNA
expression level in the shRNAi clone as assessed by real-time RT-PCR normalized to
GAPDH. SYBR probes were used. Experiments were done in triplicate in an early
and late stage. Columns, mean of triplicates of three different experiments; bars, SD.
Two-sided t-tests were performed between groups for each condition (* p<0.05).
shRNAi clone showed CDKN1C/p57/Kip2 protein level up-regulation in the early
and late stage as assessed through Western Blot. Actin was used as loading control.
shRNAi
clone
corresponds
to
TC71
shRNAi
clone
6.
B) EWS-FLI1 inhibition downregulated NR0B1 (DAX1) and NKX2.2 protein
expression in both stages, early and late, as confirmed by Western Blot. Actin was
used as loading control. shRNAi clone corresponds to TC71 shRNAi clone 6.
C) Reduction in the expression of NR0B1 (DAX1) signal in the early stage of
shRNAi clone assessed through immunohistochemistry on cellular pellets. shRNAi
clone corresponds to TC71 shRNAi clone 6.