MiaPaCa2 Control Gem 100nm AZD1775 200nM Gem AZD1775 ID: 659502
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Slide1
Condition MiaPaCa-2ControlGem (100nm) AZD1775 (200nM) Gem + AZD1775 Panc-1ControlGem (50nm) AZD1775 (200nM) Gem + AZD1775 Capan-1 ControlGem (50nm) AZD1775 (50nM) Gem + AZD1775
Supplementary Table 1. Summary of sensitization to gemcitabine-radiation by AZD1775
Supplementary Table 1. Summary of sensitization to gemcitabine-radiation by AZD1775. Cells were treated as described in Fig. 1. Data are the mean of mean ± standard error of 4-5 independent experiments with statistical significance indicated vs GemRT* (P<0.05). The mean inactivation dose (MID) used to calculate RER is shown.
MID2.7 ± 0.12.2 ± 0.42.4 ± 0.11.3 ± 0.13.7 ± 0.13.2 ± 0.13.4 ± 0.12.4 ± 0.22.3 ± 0.21.4 ± 0.2 2.2 ± 0.21.2 ± 0.1
Cytotoxicity1.00.9 ± 0.11.1 ± 0.10.7 ± 0.31.00.9± 0.040.9 ± 0.040.5 ± 0.051.0 0.9 ± 0.1 1.0 ± 0.1 0.8 ± 0.2
RER
1.0
1.3
± 0.2
1.1 ± 0.1
2.1 ± 0.2
*
1.0
1.1
± 0.04
1.1 ± 0.03
1.5 ± 0.2
*
1.0
1.8
±
0.2
1.0 ±
0.1
1.9 ± 0.2
Slide2
Suppl. Fig. 1
Supplementary Figure
1. The effects of AZD1775 and gemcitabine-radiation on DNA damage response signaling. A,
Panc-1 cells were treated with gemcitabine, AZD1775 and radiation (6Gy) as described in Fig. 1, except that cells were harvested for immunoblotting at 6 hours post-RT. B, pCDK1 (Y15) and pCHK1 (S345) protein levels from gemcitabine-radiation and AZD1775 treatment conditions (as described in Fig. 2) were quantitated from 3 independent experiments. Data are the mean fold change ± standard error in pCDK1 or pCHK1 protein levels in response to gemcitabine-radiation and increasing concentrations of AZD1775 relative to gemcitabine-radiation with no AZD1775 treatment. A. Panc1pCHK1 (S345)pCDK1 (Y15)0
10050200 50 200GemRT1000CDK1
CHK1
GAPDH
AZD1775 (
nM
):
B.Slide3
ControlpHistone H3 MiaPaCa-2DNA contentGemAZD1775GemAZD1775RTDNA contentGemRTDNA content
AZD1775RTDNA content
GemAZD1775RTt0t
2t48t24 AZD1775RTGemFACS
M:1.4%
M:1.2%
M:2.7%
M:2.3%
M:0.5%
M:0.3%
M:1.4%
M:2.8%
pHistone
H3
Suppl. Fig. 2
Supplementary Figure 2.
Abrogation of the G2 checkpoint
by
AZD1775.
MiaPaCa-2 cells were treated as described in Fig. 2C. Data are from a single representative experiment. Slide4
Suppl. Fig. 3M:1.6%M:1.5%M:2.4%
M:2.0%M:0.5%
M:0.7%M:1.0%M:2.6%
pHistone H3pHistone H3 AZD1775RTGemFACS
t0t2t48t24ControlDNA contentGemAZD1775GemAZD1775
RT
DNA content
GemRT
DNA content
AZD1775RT
DNA content
GemAZD1775RT
Capan-1
Supplementary Figure 3.
Abrogation of the G2 checkpoint
by
AZD1775.
Capan-1 cells were treated as described in Fig. 2D. Data are from a single representative experiment. Slide5
ConAZD1775Gem
GemAZD1775
RT
GemRTAZD1775RTGemAZD1775RTSuppl. Fig. 4Supplementary Figure 4. Induction of DNA damage by AZD1775. MiaPaCa-2 cells were treated as described in Fig. 3C with gemcitabine (50nM), AZD1775 (200nM) and radiation (6Gy). Cells were fixed for immunofluorescence at 16 hours post-RT . Cells were stained for γH2AX (red) and with DAPI (blue). Data are from a single representative experiment. Slide6
BRCA2GAPDHDLD1 parentalDLD1 BRCA2-nullSuppl. Fig. 5Supplementary Figure 5. Characterization of BRCA2 isogenic DLD1 cells. DLD1 parental of BRCA2 null cells were subjected to immunoblotting for the indicated proteins. The absence of BRCA2 protein in BRCA2 null cells confirms genomic deletion of BRCA2. Slide7
Supplementary Table 2. Summary of sensitization to gemcitabine-radiation by AZD1775 Supplementary Table 2. Summary of sensitization to gemcitabine-radiation by AZD1775. Cells were treated as described in Fig. 4. Data are the mean ± standard error of 3 independent experiments with statistical significance indicated vs Control* (P<0.05).ConditionDLD1 BRCA2 wild-typeGem (100nm) AZD1775 (200nM) Gem + AZD1775 DLD1 BRCA2
nullGem (50nm)
AZD1775 (200nM) Gem + AZD1775
Cytotoxicity 0.9 ± 0.021.1 ± 0.030.7 ± 0.070.7± 0.070.9 ± 0.140.8 ± 0.12RER1.3 ± 0.07 1.3 ± 0.03 1.5 ± 0.09* 0.9 ± 0.07 1.1 ± 0.08
1.0 ± 0.04 Slide8
Suppl. Fig. 6Supplementary Figure 6. RAD51 depletion sensitizes pancreatic cancer cells to gemcitabine-radiation. MiaPaCa-2 cells were plated at clonal densities and treated with non-specific (siNS) or RAD51 siRNA, followed 24 hours later by treatment with gemcitabine (100nM), AZD1775 (200nM), and radiation, according to the schedule illustrated in Fig. 1A. Data are from a single experiment that is representative of three independent experiments.Slide9
Con
AZD1775DLD1 BRCA2 wild-type
GemAZD1775RTGemRTAZD1775RT
GemAZD1775RTGemDLD1 BRCA2 nullConGemRTSuppl. Fig. 7Supplementary Figure 7. Inhibition of RAD51 focus formation in DLD1 cells treated with AZD1775. DLD1 cells were treated as described in Fig. 4E. Cells were stained for RAD51 (red) and with DAPI (blue). Slide10
Suppl. Fig. 8Supplementary Figure 8. The effects of AZD1775 and gemcitabine-radiation on DNA damage response signaling. Patient-derived xenografts were treated as described in Fig. 5A. Data are from 2-3 independent sets of tumors. Lines indicate where images were cropped for consistent arrangement of sample order. ConAZD1775GemGem+AZD1775Con
AZD1775Gem
Gem+AZD1775RTGem+AZD1775
Con
AZD1775GemGem+AZD1775ConAZD1775Gem
Gem+
AZD1775
RT
GAPDH
RAD51
pCHK1(S345)
CHK1
CDK1
pCDK1(Y15)Slide11
Suppl. Fig. 9ConAZD1775GemGemAZD1775RTGemRTAZD1775RTGemAZD1775RT
Supplementary Figure 9.
Sensitization of patient-derived tumor xenografts to gemcitabine-radiation by AZD1775 involves inhibition of HR. Patient-derived xenografts (#08-444T) were treated as described in Fig. 5A. Xenografts were stained for RAD51 (green) and with DAPI (blue).