Supplementary Figures Legends
Figure S1. Structural organisation of DAPK1 and DAPK2. The kinase domain of
DAPK2 is 80% homologous to that of DAPK1 and both kinases are regulated by calcium
(Ca2+) and calmodulin (CaM). DAPK1 also has a series of ankyrin repeats (pink) important
for protein-protein interactions and appropriate localisation of the protein, P loops (purple
lines), a cytoskeletal binding motif (orange), a death domain (purple) and a serine-rich tail
(green), the latter two characteristic of pro-apoptotic proteins. Interestingly, DAPK2 has a
dimerization motif (turquoise) on the C-terminus.
Figure S2. Validation of siRNA oligonucleotide molecules that target different
sequences of DAPK2 mRNA, designed using different algorithms. U2OS (a, c-f and
k-n) or A549 (b, g-j and o-v) cells were transfected with non-targeting siNS, or different
DAPK2 siRNA oligonucleotides, as indicated in the figure. Forty-eight h after transfection
knockdown efficiency was determined by qPCR (a, b), cell survival rates in response to
TRAIL measured using crystal violet viability assays (c-j), and DR5 (k-r) and DR4 (s-v)
expression assessed by flow cytometry. For cell survival assays, 24 h after transfection,
cells were re-plated into 96-well plates at a density of 2x104 cells per well. The following
day, cells were treated with the indicated concentrations of TRAIL for 24 h. Cells were
then fixed using methanol and stained with crystal violet. Crystals were dissolved in 10%
(v/v) acetic acid and quantified by measuring the absorbance at 595 nm. Values were
normalised to the untreated samples. Data represents mean ± SEM of at least three
independent experiments, performed in triplicate. Statistical analyses were done using
two-way ANOVA test (** p<0.01, *** p< 0.005). Flow cytometry was used to assess the
levels of DR5 expression in U2OS cells (k-n) and A549 cells (o-r), DR4 levels were
assessed in A549 cells only (s-v). Data represents mean ± SEM of three independent
experiments. Cell surface expression quantification was done using geometric means of
those three independent experiments and plotted as fold change of surface expression in
Schlegel et al. 2014
Cell Death and Differentiation (re-re-submission)
relation to siNS. Statistical analysis was done using Student’s t-test (paired, one tailed) (*
p< 0.05, ** p<0.01, *** p< 0.005).
Figure S3. Silencing DAPK2 sensitises resistant cancer cell lines to TRAIL-induced
cell death. U2OS and A549 cells were transfected with either siNS, or siDAPK2. Fortyeight h after transfection, U2OS (a-e) and A549 (f-j) cells were treated with TRAIL (100
ng/ml) for 6 h. Cells were then fixed with ethanol, incubated with RNase A and PI. Cell
sub-populations were analysed using FlowJo. Cell cycle profiles representative of three
independent experiments are shown in histograms (1-d and f-i) and summarised
graphically in bar charts (e and j).
Figure S4. Silencing DAPK2 does not affect the levels of FLIP. U2OS (a, c) and A549
(b, d) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight h after
transfection the expression levels of FLIP were evaluated by quantitative western blotting
(a, b) and the induction of FLIP mRNA by qPCR (c, d). Blots shown are representative of
three independent experiments yielding identical data. qPCR data represents mean ±
SEM of three independent experiments with duplicate samples. Statistical analysis was
done using Student’s t-test (paired, one tailed).
Figure S5. Overexpression of BCL-XL does not rescue siDAPK2-mediated
sensitisation of U2OS cells to TRAIL. (a) U2OS cells were transfected with either siNS,
or siDAPK2 in combination with an empty vector pcDNA3 control plasmid (EV), or with a
pcDNA3 BCL-XL expression plasmid, and the knockdown and over-expression efficiencies
determined by quantitative western blotting. Cells transfected with the empty vector (b), or
with the BCL-XL-expressing vector (c) were re-plated into 96-well plates at a density of
2x104 cells per well 24 h after siRNA transfection. The following day cells were treated
with the indicated concentrations of TRAIL for 24 h and cell death assessed using crystal
violet assays, as described before. Values were normalised to the untreated samples.
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Schlegel et al. 2014
Cell Death and Differentiation (re-re-submission)
Data represents mean ± SEM of two independent experiments performed in triplicate.
Statistical analyses were done using two-way ANOVA test (* p< 0.05, ** p<0.01, *** p<
0.005).
Figure S6. siDAPK2-mediated sensitisation to TRAIL is p53-independent. Prostate
cancer PC3 (a, c) and colon cancer T24 (b, d) cells were transfected with either nonspecific siRNA (siNS) or DAPK2 siRNA. Forty-eight h after transfection knockdown
efficiency was determined by western blotting (c, d) and cell survival using crystal violet
viability assays (e, f). Staining was quantified by measuring the absorbance at 595 nm, as
described before. Values were normalised to the untreated samples. Data represent
mean±SEM of three independent experiments performed in triplicates. Statistical analysis
was done using two-way ANOVA test (* p< 0.05, ** p<0.01, *** p< 0.005).
Figure S7. Involvement of RELB in sensitising several cancer cell lines to TRAIL
following DAPK2 depletion. U2OS (a) and A549 cells (b) were transfected with either
siNS or with the following siRNA 1:1 mixes: siDAPK2+siNS, siRELB+siNS or
siRELB+siDAPK2. Twenty-four h after transfection cells were re-plated into 96 well plates
at a density of 2x104 cells per well. The following day cells were treated with TRAIL for 24
h at the indicated final concentrations. Cells were then methanol-fixed and stained with
crystal violet. Staining was quantified by measuring the absorbance at 595 nm, as
described before. Values were normalised to the untreated samples. Data represent
mean±SEM of three independent experiments performed in triplicates. Statistical analysis
was done using two-way ANOVA test (* p< 0.05, ** p<0.01, *** p< 0.005).
Figure S8. Efficiency of siRNA-induced silencing using two different siRNA
oligonucleotides concomitantly. U2OS (a) and A549 (b) cells were transfected with
either non-specific siRNA (siNS) or siRNA 1:1 mixes, as indicated in the figure to a final
siRNA concentration of 40 nM. Forty-eight h after transfection, knockdown efficiency of
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Cell Death and Differentiation (re-re-submission)
each target was determined by qPCR. Values were normalised to the untreated samples.
Data represents mean±SEM of three independent experiments performed in triplicate.
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Cell Death and Differentiation (re-re-submission)
Supplementary Tables
TABLE SI. siRNA oligonucleotide sequences used throughout this study
Target gene
Target sequence
Dharmacon ID
Non targeting (All Stars)
Luciferase GL3 Duplex
BID_3
BID_18
BID_19
BID_20
DAPK2_1#
DAPK2_3
DAPK2_4
DAPK2_5#
DAPK2_3'UTR
DAPK2_Qiagen 11
DR4_6
DR4_15
DR4_16
DR4_17
DR5_1
DR5_2
DR5_3
DR5_4
NFKB1_1
NFKB1_2
NFKB1_3
NFKB1_5
NFKB2_1
NFKB2_2
NFKB2_3
NFKB2_18
RELA_3
RELA_4
RELA_5
RELA_18
RELB_1
RELB_4
RELB_5
RELB_18
Not disclosed by Qiagen
CUUACGCUGAGUACUUCGA
GCACCUACGUGAGGAGCUU
GAGUAAGGGCACUGACGGA
GCCAGAAGCUACUGCGAUG
UGCAAUACAUACCACGCUA
GAGGAGAGCUCUUCGAUUU
GGAAACGGCUCACAAUCCA
GGAAUUUGUUGCUCCAGAA
GAGAUGGGCCCAAGGAAUU
GAGUGUGGACUUAGGAAAA
CAGCAUUCCCAAAGCTCUU
UGACAAUUCUGCUGAGAUG
CAACAAAACUGGACGGAAC
GAACAUAGCCCUUUGGGAG
CGGCAGAUUUGACAGGUGU
GGACAGAAGCUCACAACGA
UCAUGUAUCUAGAAGGUAA
ACACGAUGCUGAUAAAGUG
CAAGGUCGGUGAUUGUACA
GCAGGUAUUUGACAUAUUA
GCAAUAGCCUGCCAUGUUU
GAACCACGCCUCUAGAUAU
GGGCUACACCGAAGCAAUU
CCAAACAGUUCACCUAUUA
GGACGUGUCUGAUUCCAAA
GGUGAUGGAUCUGAGUAUA
GUAGACACGUACCGACAGA
GGAUUGAGGAGAAACGUAA
CUCAAGAUCUGCCGAGUGA
GGCUAUAACUCGCCUAGUG
GAUUGAGGAGAAACGUAAA
CAUCAGAGCUGCGGAUUUG
GCCCGUCUAUGACAAGAAA
GCACAGAUGAAUUGGAGAU
GUACCUGCCUCGCGACCAU
Qiagen-SI03650318
Dharmacon-001400-01-20
Dharmacon-004387-03
Dharmacon-004387-18
Dharmacon-004387-19
Dharmacon-004387-20
Dharmacon-004418-01
Dharmacon-004418-03
Dharmacon-004418-04
Dharmacon-004418-05
Dharmacon-custom
Qiagen-SI04988298
Dharmacon-008090-06
Dharmacon-008090-15
Dharmacon-008090-16
Dharmacon-008090-17
Dharmacon-004448-01
Dharmacon-004448-02
Dharmacon-004448-03
Dharmacon-004448-04
Dharmacon-003520-01
Dharmacon-003520-02
Dharmacon-003520-03
Dharmacon-003520-05
Dharmacon-003918-01
Dharmacon-003918-02
Dharmacon-003918-03
Dharmacon-003918-18
Dharmacon-003533-03
Dharmacon-003533-04
Dharmacon-003533-05
Dharmacon-003533-18
Dharmacon-004767-01
Dharmacon-004767-04
Dharmacon-004767-05
Dharmacon-004767-18
# Excluded from the siRNA pool following deconvolution analysis due to off-target effects
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TABLE SII. Oligonucleotide qPCR primer pair sequences used throughout this
study
Oligo name
Oligo sequence (5' to 3')
DAPK2 fwd
DAPK2 rev
DR4 fwd
DR4 rev
DR5 fwd
DR5 rev
FLIP fwd
FLIP rev
BID fwd
BID rev
NFKB1 fwd
NFKB1 rev
NFKB2 fwd
NFKB2 rev
RELA fwd
RELA rev
RELB fwd
RELB rev
cREL fwd
cREL rev
C-JUN fwd
C-JUN rev
TNFA fwd
TNFA rev
TCCTGGATGGGGTGAACTAC
CAGCTTGATGTGTGGAATGG
CATCGGCTCAGGTTGTGGA
TGCCGGTCCCAGCAGACA
GCACTCACTGGAATGACCTC
GCCTTCTTCGCACTGACAC
TCTCACAGCTCACCATCCCTG
CAGGAGTGGGCGTTTTCTTTC
TCCACACATGAATCTGCACATC
GGGAACCTGCACAGTGGAA
GCAGCACTACTTCTTGACCACC
TCTGCTCCTGAGCATTGACGTC
GGCAGACCAGTGTCATTGAGCA
CAGCAGAAAGCTCACCACACTC
CCAGGTTCTGGAAACTGTGGAT
CCCCACGAGCTTGTAGGAAAG
TGTGGTGAGGATCTGCTTCCAG
TCGGCAAATCCGCAGCTCTGAT
AGTTGCGGAGACCTTCTGACCA
CGTGATCCTGGCACAGTTTCTG
TCCAAGTGCCGAAAAAGGAAG
CGAGTTCTGAGCTTTCAAGGT
TCTTCTCGAACCCCGAGTGA
CCTCTGATGGCACCACCAG
GAPDH fwd
GAPDH rev
HPRT fwd
HPRT rev
AGCCACATCGCTCAGACAC
GCCCAATACGACCAAATCC
TGACCTTGATTTATTTTGCATACC
CGAGCAAGACGTTCAGTCCT
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