Supplementary Figure 1: Experimental manipulation of the NMD pathway
Five breast cancer cell lines were incubated with emetine for 8 hours, after which cells
were washed and actinomycin D was added for a total of 2 hours. Every 30 minutes,
cells were lysed and total RNA was extracted. Control cells were incubated with
actinomycin only. RNAs from the tested sample or the control served as templates for
the synthesis of cDNAs that were hybridized to a 44k oligonucleotide expression array
(Agilent).
Supplementary Figure 2: Schematic overview of the integrated genomic strategy.
The step-wise selection of the 53 candidate TSGs involves combining data obtained from
five breast cancer cell lines undergoing NMD manipulation (see Supplementary Figure
1), SNP array and gene expression profiling.
Supplementary Figure 3: Proliferation of cancer cells transfected with ARID1A
Bar graph represents proliferation at day 5 after plating, as measured by Alamar blue dye
reduction in ARID1A transfected and empty vector transfected T47D cells. Means and
standard errors were obtained from two independent experiments each performed in
quadruplicate. Difference was analyzed by the t-test. *p 0.001
Supplementary Figure 4: ARID1A gene silencing stimulates proliferation in MCF-7
cells. (A) ARID1A inhibition increases proliferation of MCF-7 cells. Cells were treated
with a control siRNA (CTL) or a siRNA targeted against ARID1A. 72 hours after the
treatment, cells were plated in a 96 well plate (2500 cells/well) and incubated for 5 days.
Proliferation was monitored at day 5 using the alamarBlue assay. Data are means ±
S.E.M from three independent experiments each performed in quadruplicate. *p=0.01
versus CTL. (B) Immunoblot analysis of ARID1A expression levels in MCF-7 cells
treated with the control or ARID1A siRNA. Immunoblots are from one experiment
representative of three that gave similar results. TCL, total cell lysates. (C) ARID1A
expression levels in MCF-7 and T47D cell lines. ARID1A expression levels in MCF-7
and T47D cells were detected by immunoblotting of nuclear extracts with the Baf250a
(PSG3) antibody. The nuclear marker p84 was used as loading control.
Supplementary Table 1: PCR primers for ARID1A mutation screening