Supplemental Figure Legends
Figure S1. Influence of p53 status on cell cycle and apoptosis gene ontologies.
(A and B) A subtractive comparison of significant gene changes observed following 72 hr of
treatment with AZD1152 (200 nM) without doxycycline vs. AZD1152 with doxycycline (50 ng/ml)
was performed to identify all significant gene changes observed only in the presence of functional
p53 (i.e., without doxycycline). Those genes that were unique to AZD1152 without doxycycline
were defined as p53-dependent and analyzed independently using gene ontology and pathway
analyses.
A comprehensive cell cycle network was constructed by conjoining individual
subnetworks generated by unbiased pathway analyses. The parental datasets [AZD1152 without
doxycycline (A) and AZD1152 with doxycycline (B)] were then applied to the network.
Red
indicates significant upregulation, blue indicates significant dowregulation, and gray indicates upor down-regulation that was less than 1.5 fold. Solid lines indicate direct regulation, and dashed
lines indicate regulation that is indirect. (C and D) Genes regulated independently of p53 were
defined as those regulated in common between D54MG shp53 treated for 72 hr with 200 nM
AZD1152 in the presence and absence of functional p53 (AZD1152-dox vs. AZD1152+dox). That
common gene set was then subjected to gene ontology and pathway analyses to empirically
identify individual relational subnetworks of apoptotic genes.
A comprehensive network of
apoptotic genes was then constructed by connecting these discrete subnetworks. The parental
datasets [AZD1152 without doxycycline (A) and AZD1152 with doxycycline (B)] were then applied
to the network. Red indicates significant upregulation, blue indicates significant dowregulation,
and gray indicates up- or down-regulation that was less than 1.5 fold. Solid lines indicate direct
regulation, and dashed lines indicate regulation that is indirect. The position of TNFRSF10B,
which encodes TRAIL-R2, is indicated.
Figure S2. Cell surface expression of TRAIL-R1, TRAIL-R2, and OPG.
D54MGshp53 cells were treated with 200 nM AZD1152 for 72 hr then stained with a PE-labelled
secondary alone or with PE-conjugated antibodies raised against CD44, TRAIL-R1, TRAIL-R2, or
OPG. Relative cell surface expression was then determined by flow cytometry. There is a 3.8-
fold increase in surface staining of PE alone or CD44 after 72 hr of polyploidization due to the
increase in cell size, and thus surface area, that occurs during polyploidization. To correct for
this, a gate was set at 3.8-fold relative to the mean peak intensity for a given stain in DMSOtreated cells (green). An increase in surface staining >3.8-fold (orange), therefore, represents
increased surface expression in addition to that resulting from increased surface area.
The
expression of TRAIL-R2 (7.5-fold) and OPG (5-fold) on the cell surface increases with AZD1152
treatment, whereas CD44 (3.8-fold) and TRAIL-R1 (3.8-fold) does not.
Figure S3. Sensitivity of glioma cell lines to Aurora kinase/TRAIL combinations.
LN18 (A), U87MG (B), and U138MG (C) glioma cells were treated simultaneously with Aurora
kinase inhibitors and TRAIL and cell viability was determined after 72 hr of drug addition. (D)
Immunoblot analysis of cell lysates prepared from human glioma lines either treated with DMSO
or 200 nM AZD1152 for 72 hr.
Figure S4. Identification of potent TRAIL-R1 and TRAIL-R2 siRNAs.
(A) D54MGshp53 cells were treated with siNontarget, siLuciferase, or with individual TRAIL-R1
siRNAs (designated siTRAIL-R1-1-4) and cultured in the presence or absence of 200 nM
AZD1152 for a further 48 hr.
Cell extracts were prepared and TRAIL-R1 knockdown was
assessed by immunoblotting. Asterisk indicates non-specific band. (B) D54MGshp53 cells were
transfected with siNontarget or siTRAIL-R1-2 (designated siTRAIL-R1) oligos for 48 hr prior to the
simultaneous addition of 200 nM AZD1152 and TRAIL in 2-fold dose response. Cells were
cultured for a further 72 hr, and cell viability was determined. (C) D54MGshp53 cells were either
untreated or treated with transfection reagent alone (Mock), siNontarget, siLuciferase, or with
individual TRAIL-R2 siRNAs (designated siTRAIL-R2-1-4) and cultured in the presence or
absence of 200 nM AZD1152 for a further 48 hr. Cell extracts were prepared and TRAIL-R2
knockdown was assessed by immunoblotting.