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Suplementary Tables
Table S1. Regulation of the genes that were not mapped within the deleted or
amplified chromosomal regions in the entire population of DU145 parental (P) cells versus
DU145 radioresistant (RR) cells.
Table S2. Gene set enrichment analysis (GSEA) for DU145 radioresistant (RR) cells
versus DU145 parental (P) cells, NOM p value < 0.05
Table S3. Regulation of the genes involved in WNT signaling pathway in the entire
population of DU145 parental (P) cells versus DU145 radioresistant (RR) cells.
Table S4. Regulation of the genes involved in WNT signaling pathway in the ALDH+
population of DU145 parental (P) cells versus ALDH- population of DU145 P cells.
Table S5. Regulation of the genes involved in WNT signaling pathway in the ALDH+
population of DU145 radioresistant (RR) cells versus ALDH- population of DU145 RR cells.
Supplementary Figures
Figure S1. Irradiation-induced changes in the expression of stem cell markers. A,
The parental prostate cancer cell lines DU145 and LNCaP and their radioresistant
counterparts DU145-RR and LNCaP-RR were treated with 4 Gy of X-ray and protein
expression was analyzed by western blotting or flow cytometry during 6 weeks after
irradiation. B, Flow cytometry analysis of the ABCG2 expression in DU145 and DU145-RR
cells within 6 weeks after irradiation with 4Gy of X-ray. Error bars represent S.E.M, n≥3. C,
Protein expression in sham radiated (SR) DU145 cells. D, The DU145-RR and LNCaP-RR
sublines showed enhanced ALDH activity as compared to the parental cells. E, ALDH+
DU145 cell population did not show an increased proliferation after irradiation as compared
to ALDH- DU145 cells. Cells were sorted at day 1 or day 7 after 4Gy irradiation.
Figure S2. Genetic alterations in the irradiated prostate cancer cells. A,
Microsatellite polymorphism analysis for DU145 and DU145-RR cells revealed loss of
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heterozygosity
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for vWA locus 17 (chromosome 12). B, Array CGH profiles showed
marked differences between DU145 RR and paternal DU145 cells. Besides common
(identical) alterations, there were additional changes only present in DU145-RR (e. g. gains
on chromosome 15), while also several of the complex alterations found in the paternal
DU145 were not present anymore in the DU145 RR. An example is illustrated in (C) for
chromosome 4, showing complex alterations with different breakpoints in the paternal DU145
(-4p15.33p15.1,+4p15.1p11,-4q12q35.2,+4q31.21q31.22,-4q31.22q35.2), while in DU145
RR only two of the alteration, +4q31.21q31.22 and -4q31.22q35.2, are still present.
Figure S3. Regulation of histone methylation in response to cell irradiation. A,
Densitometric analysis of the protein immunoblots shown in Figure 2C and Figure S3B. B,
Irradiation induces long-lasting changes in the expression of EZH2, SETD2 and ASH1
histone methyltransferases in prostate cancer cells DU145 and LNCaP. C, Ingenuity pathway
analysis (IPA) revealed EZH2 as one of the most significantly upregulated transcriptional
regulators in DU145-RR cells (p value < 10-8). The diagram shows potential EZH2 targets
and their regulation in DU145-RR cells as compared to DU145 cells. D, Analysis of H3K4
and H3K36 methylation in LNCaP P and LNCaP-RR cells. Error bars represent S.E.M; n=4;
*, p < 0.05.
Figure S4. Differential expression of stem cell and EMT markers in response to
irradiation in the parental and radioresistant prostate cancer cells. A, The delayed effect
of X-ray irradiation on the expression of the stem cell and EMT markers in parental and RR
cells. After irradiation with 4 Gy of X-ray, the lysates of DU145, DU145-RR, LNCaP and
LNCaP-RR cells were prepared at the indicated time points within 6 weeks after irradiation
and analyzed by Western blotting; n.d. – signal was not detected. B, Induction of the ALDH+
cell population after irradiation of ALDH+ and ALDH- cell subsets. FACS sorted ALDH+ and
ALDH- cells were plated in a 6 well plate and irradiated with 4Gy 18 hours later. Irradiation
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with 4Gy was repeated at day 7 after first irradiation. The cells were analyzed at day 14 by
flow cytometry. Error bars represent S.E.M; n=3; *, p < 0.05.
Figure S5. The role of ALDH1A1 expression in regulation of spherogenicity,
tumorigenicity and radioresistance of the parental and radioresistant prostate cancer
cells. A, Luciferase reporter assay in PC3 cells that were sham-irradiated or irradiated with
4Gy of X-ray. An assay was performed using the ALDH1A1-promoter luciferase reporter,
TOPFlash and FOPFlash (TOPFlash mutant) reporters. Error bars represent S.E.M; n=5; *, p
< 0.05. Knockdown of ALDH1A1 gene expression mediated by siRNA resulted in a
significant decrease in prostate cancer cell radioresistance (B), ALDH activity (C) and
spherogenicity (D). Error bars represent S.E.M; n=3; *, p < 0.05. E, DU145-RR ALDH+ cells
showed an increase in spherogenic potential as compared with DU145-RR ALDH- cells. The
automated analysis of the sphere forming properties of ALDH+ and ALDH- cell population
was performed using the Celigo cytometer. Error bars represent S.E.M. F, DU145-RR
ALDH+ cells possess higher tumorigenic properties compared to DU145-RR ALDH- cells.
Analysis of the limiting dilution assay was performed using the ELDA software. G,
Spherogenic cell survival assay has shown no significant differences in radiobiological
response of DU145-RR ALDH+ and DU145-RR ALDH- cells. Error bars represent S.E.M. H,
Immunofluorescence analysis of the residual γ - H2A. X foci in the DU145-RR ALDH+ and
DU145 ALDH- cells 24 h after irradiation revealed no differences between these two cell
populations. Error bars represent S.E.M.
Figure S6.
Analysis and validation of the global gene expression data. A,
Expression of ALDH1A1 and ALDH1A3 genes in radioresistant and radiosensitive DU145 and
LNCaP cells revealed from the global gene expression profiling. B, RT-PCR analysis of the
ALDH1A3 expression in radioresistant and radiosensitive DU145 and LNCaP cells; n=3
(DU145 P and DU145 RR cells) or n=2 (LNCaP P and LNCaP RR cells). *, p < 0.05. C,
Expression of ALDH1A3 genes in ALDH+ and ALDH- DU145 cells. The data were extracted
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from the global gene expression profiling of the FACS purified DU145 ALDH+ and ALDHcell populations. D, FACS-isolated ALDH+ and ALDH- cell populations were analyzed by
MTT viability assay. Error bars represent S.E.M; n≥2. E, Migratory potential of the FACSisolated ALDH+ and ALDH- cell populations were analyzed was analyzed by transwell
migration assay. Error bars represent S.E.M; n=4 (DU145 P and DU145 RR cells) or n=2
(LNCaP P and LNCaP RR cells); *, p < 0.05. F, Plating efficacy of the parental and
radioresistant DU145 and LNCaP cell lines pre-treated with XAV939 inhibitor. The cells
were serum starved in DMEM with 1% FBS for 24 h followed by treatment with XAV939
antagonist at concentration 1μM for 3 days. Error bars represent S.E.M; n=4; **, p < 0.01; *,
p < 0.05. G, LEF1 expression in DU145 parental and DU145 RR cells. LEF1 expression data
was obtained from the global gene expression dataset. Error bars represent S.E.M; n=3; *, p <
0.05.
Figure S7. Analysis of cell viability, plating efficacy and post-irradiation
clonogenic survival in response to inhibition of the histone methylation activity. A, MTT
assay revealed that BIX01294 showed similar toxicity for radioresistant and parental DU145
and LNCaP cells. IC50 values were determined after 24 h and 72 h of treatment with the
drugs; n≥3 (24h) or n≥2 (72h). B, IC50 values based on plating efficiency of the cells treated
with DZNep for 72 h; n≥2. C, LNCaP parental and LNCaP RR cells were pre-treated with
BIX-01294 at the different concentrations for 72h and their susceptibility to X-ray irradiation
was evaluated by clonogenic cell survival assay; Error bars represent S.E.M..
Figure S8. Effect of DZNep treatment on apoptosis induction and γH2A.X level
in prostate cancer cell lines. A, Analysis of apoptosis induction after treatment of cells with
DZNep at a concentration of 1 µM for 72h alone or in combination with 6Gy of X-ray
irradiation. Cells treated with DMSO were used as control. B, Western blot analysis of the
induction of PARP cleavage after treatment of the LNCaP and LNCaP-RR cells with DZNep
or BIX1294 at concentration 1 µM for 72h. C, Fluorescent microscopy analysis of the
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induction of PARP cleavage after treatment of the LNCaP-RR and DU145-RR cells with
DZNep at concentration 1 µM for 72h. Scale bars, 100 μm. Error bars represent S.E.M; n=2.
Figure S9. Analysis of apoptosis induction and γH2A.X increase by DZNep
treatment in prostate tumor tissues and primary cell cultures. A, Analysis of the cleaved
PARP and γH2A.X increase in the specimens of radical prostatectomy (PT2 and PT3), which
were treated ex vivo with 4 Gy of X-ray alone or in combination with 2 µM of DZNep given
for 48 h. The tissues were fixed 24h after irradiation. Hematoxylin and eosin staining of the
tissue specimens showed an increase in apoptosis in response to combination treatment. B,
Human prostate tumor primary cells 312/13 and 311/13 were treated with 4 Gy of X-ray alone
or in combination with 2 µM of DZNep given for 48 h. The tissues were fixed 24h after
irradiation. Error bars represent S.E.M. C, Treatment of the immortalized, non-tumorigenic
cell line RWPE-1 with DZNep does not lead to a significant increase in residual DNA damage
as compared to the treatment by irradiation alone. Cells were treated with 4 Gy of X-ray alone
or in combination with 2 µM of DZNep given for 48 h. The tissues were fixed 24h after
irradiation. Error bars represent S.E.M. D, Western blot analysis of LNCaP RR cells treated
with DZNep or BIX1294 at concentration 5 µM or 10 µM for 72h. Control cells were treated
with DMSO. E, Western blot analysis of ALDH1A1 and H3K36me3 in DU145 cells treated
with 4 Gy of X-ray alone or in combination with 1 µM of DZNep given for 72h. F,
Fluorescent microscopy analysis showed that DU145 cells treated with DZNep and
subsequently irradiated with 4Gy of X-ray show an inhibited ALDH1A1 expression as
compared to the cells treated only with 4Gy of X-ray. Scale bars, 25 μm. Error bars represent
S.E.M; n=3; *, p < 0.05.
Figure S10. Co-regulation of EZH2, ALDH1A1 and CTNNB1 in cell lines and
prostate tumors. A, siRNA-mediated inhibition of β-catenin expression decreases expression
of EZH2 and ALDH1A1 in PC3 prostate cancer cells. Error bars represent S.E.M; n=2. B,
Expression of ALDH1A1 and EZH2 genes co-occur with expression of the CTNNB1 (β5
catenin) in a prostate cancer dataset from the Taylor study55. Data was analyzed using
cBioPortal for Cancer Genomics. C, Expression of ALDH1A1 and EZH2 correlated with
reduced disease-free survival in patients with prostate cancer. The analysis is based on the
provisional TCGA data set. Data were extracted from http://www.cbioportal.org/. The cutoff
for expression was set as z-score +/-2.
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