EMBO/SourceData huggingface dataset:
Figure 6SUMO conjugation assays performed under multiple turnover conditions as in Fig 4. Experiments were performed in technical triplicates (six times for WT). The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.
Figure 7SUMO conjugation assays performed under multiple turnover conditions as in Fig 4 and using different Siz2∆CT variants. The structural models highlight the position of the mutations using a homology model of the SAP domain of Siz2 obtained using the structure of Siz1 (PDB 2RNN) as a template. A dashed box indicates the relative position of residues that undergo chemical shift perturbation upon DNA binding according to (Suzuki et al, 2009). Bands for Mw marker are fully annotated in Figure 1B.
Figure 8Electrophoretic mobility shift assays (EMSA) to assess binding of RPA or SUMO-RPA to 25nM single-stranded 6-FAM-labeled 32dT DNA and displacement of SUMO-RPA from 25nM SUMO-RPA bound to single-stranded 6-FAM-labeled 32dT DNA by RPA and vice versa. All titrations were done in technical triplicates. One representative gel is shown.
Figure 9Electrophoretic mobility shift assays (EMSA) performed with 10 nM 6-FAM-ds20ss32, a 6-FAM-labeled 20-mer dsDNA with a 32 nucleotide 3' overhang and its complexes with RPA, RPA with K170-SUMO modified Rfa1 or RPA reconstituted with ∆WH-Rfa2 and serially diluted Siz2. All titrations were done in technical triplicates. One representative gel is shown.
Figure 2A Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%) or the VPS34 inhibitors, VPS34IN1 (3 µM) or SAR405 (20 µM). Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. VPS34 inhibition causes reduced excitatory synaptic transmission down to 46.0 ± 4.1 % and 55.0 ± 4.4 % for VPS34IN1 and SAR405 at the end of 60-min treatment. N = 6 slices per condition from ≥ 4 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001B Increased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses after VPS34 inhibition. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below, indicating an increased facilitation of the second response in VPS34IN1-treated acute hippocampal slices. PI(3)P depletion causes a significant facilitation of the second fEPSP, indicative of reduced initial release probability. N = 6 slices per condition from 6 mice; Two-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001C Synaptophysin-pHluorin responses normalized to baseline from neurons stimulated with 12 consecutive trains of 20 APs (20 Hz, 1 s) with 30 s gap between each stimulus. Neurons are acutely treated with DMSO (0.1 %) or VPS34 inhibitor1 (50 µM) starting from the onset of 2nd stimulation. The timing of the treatment and the stimulation trains are indicated. D Quantification of data from C. Net difference of Synaptophysin-pHluorin fluorescence 2 s before and 28 s after each stimulation (2 s before the next stimulation) to estimate relative surface retention of SVs. SV endocytosis is blocked within 5 minutes of treatment with VPS34 Inhibitor1, leading to progressive accumulation of Synaptophysin-pHluorin signal. N = 4 or 5 independent measurements per condition; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; **p < 0.01; ***p < 0.001
Figure 3A Electron micrographs of VPS34IN1- and DMSO-treated synapses following four consecutive trains of 200 AP (40 Hz, 5 s; 90 s gap between each train) stimulation. The postsynaptic compartment and the postsynaptic density are highlighted in green and purple, respectively. Note the reduced SV numbers and plasma membrane-derived endocytic intermediates in VPS34IN1-treated synapses. Scale bars, 500 nm (left) and 200 nm (right). B High magnification views of endocytic intermediates accumulated in stimulated hippocampal neurons treated with VPS34IN1. Scale bar, 200 nm. C 3D-reconstruction of a synaptic terminal treated with VPS34IN1 and stimulated as in A. The plasma membrane is colored in turquoise. Synaptic vesicles (yellow), plasma membrane-derived invaginations (blue), endosome-like vacuoles (green) and clathrin-coated vesicles (red) are highlighted. Scale bar, 500 nm (top) and 200 nm (bottom). Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001D-H Morphometric quantification of data shown in A. Synapses of VPS34IN1-treated stimulated neurons display reduced numbers of SVs/ bouton area in (D) and an accumulation of endocytic membrane invaginations (E), endosome like vacuoles (ELV) (F), clathrin-coated pits (CCP) (G), and clathrin-coated vesicles (CCV) (H). n = 100 (SV) and n = 200 (invagination, ELV, CCP, CCV) profiles per condition; Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; ***p < 0.001; ****p < 0.0001
Figure 4A-D Normalized Synaptophysin-pHluorin responses from neurons treated with VPS34IN1 (10 µM for 1h prior to imaging) and stimulated with four consecutive trains of 50 APs (10 Hz, 5 s) in A or 200 APs (40 Hz, 5 s) in C. PI(3)P depletion in VPS34IN1-treated neurons causes the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface in neurons stimulated with consecutive trains of 50 APs in B or 200 APs in D. Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive stimulation trains. N ≥ 4 independent experiments; Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001E Cultured hippocampal neurons pre-treated with DMSO (0.1%) or VPS34IN1 (10 µM, 1 h) were either field-stimulated with four trains of 200 APs (40 Hz, 5 s; 90 s gap between each train) (Stim) or left unstimulated (-), fixed and stained for surface-stranded Synaptotagmin-1, total Synaptotagmin-1, and vGlut1. Scale bar, 10 µm. F Fraction of endogenous Synaptotagmin-1 stranded on the surface of presynaptic nerve terminals from the images shown in E. N = 3 independent experiments (≥ 40 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001G Surface retention of Synaptophysin-pHluorin 90 s post-stimulus is plotted for each of the four successive 200 AP stimulation trains in neurons treated with SAR405 (20 µM). Data are similar to those shown in C and D. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001H Exogenous supply of membrane-permeant PI(3)P acutely rescues defective Synaptophysin-pHluorin endocytosis induced by pharmacological inhibition of VPS34-mediated PI(3)P synthesis in hippocampal neurons. Data are similar to those shown in C and D; where indicated exogenous PI(3)P-AM (20 µM) was supplied in parallel to VPS34IN1 (10 µM) for 1 h prior to imaging. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001I Kinetic block of Synaptophysin-pHluorin endocytosis at hippocampal synapses depleted of VPS34. Hippocampal neurons depleted of endogenous VPS34 by specific shRNA (shVPS34) or treated with non-targeting control shRNA (shCtrl) and stimulated as in C and analyzed as in D. N =3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Sidak's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001;
Figure 5A-C The kinetic block of SV endocytosis at excitatory synapses depleted of PI(3)P depends on prior neuronal activity and Ca2+. VPS34IN1-induced defects in SV endocytosis are reverted by parallel treatment with TTX (0.2 µM) in A and EGTA (2 mM) in B and AP5 (20 µM) in C, in Synaptophysin-pHluorin-expressing neurons N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001D Overexpression of dominant-negative Rab5 (Rab5DN), but not constitutively active Rab5 (Rab5CA), rescues the progressive accumulation of Synaptophysin-pHluorin on the surface of VPS34IN1-treated hippocampal neurons stimulated and analyzed as in A-C. N = 3 independent experiments (≥ 10 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001E Levels of various synaptic proteins in DMSO (0.1%) and VPS34IN1 (10 µM) treated cultured cerebellar granule neurons analyzed by western blot. VPS34 inhibition results in p35 processing to hyperactive p25, a reaction blocked by Calpain inhibitor ALLN (100 µM).F-H Calpain inhibition by ALLN (100 µM) in F or Calpain 2 knockdown via lentiviral shRNA (shCAPN2) in H rescue the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1. Calpain 1 knockdown via lentiviral shRNA (shCAPN1) in G had no impact on VPS34IN1-induced kinetic delay of SV endocytosis. N = 3 independent experiments (≥ 11 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM ; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Figure 6B Cdk5 inhibition by Roscovitine (Ros, 10 µM) rescues the progressive accumulation of Synaptophysin-pHluorin on the neuronal surface induced by VPS34IN1 (10 µM). N = 4 independent experiments (20 images per condition); Two-way ANOVA; Tukey's Multiple Comparisons Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001C Calcineurin inhibition by Cyclosporin A (10 µM) phenocopies VPS34IN1-induced loss of PI(3)P with respect to the accumulation of Synaptophysin-pHluorin on the neuronal surface after four successive stimulation trains (40 Hz, 5 s). Data were analyzed as in B. N = 3 independent experiments (15 images per condition); Two-way ANOVA; Tukey's Multiple Comparison Test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001D Basal excitatory synaptic transmission of CA3-CA1 connections in acute hippocampal slices treated with DMSO (0.03%), Roscovitine (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative fEPSP traces (above) and the relative slope of fEPSPs (below) are shown. Concomitant application of VPS34IN1 and Roscovitine facilitates excitatory synaptic transmission, akin to Roscovitine alone, and in contrast to VPS34IN1 alone N = 6 slices per condition from 6 mice; Student's t-test. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001E Decreased paired paired-pulse facilitation (PPF) at hippocampal CA3-CA1 synapses treated with Roscovitine alone (Ros, 10 µM) or VPS34IN1 (3 µM) plus Roscovitine. Representative traces of fEPSPs with a 20 ms interpulse interval is shown above. The ratio of the slope of the second to the first response over a range of interstimulus intervals (10-500 ms) is plotted below. The data show decreased facilitation of the second response in Roscovitine or VPS34IN1 plus Roscovitine-treated acute hippocampal slices, unlike VPS34IN1 alone . N = 6 slices per condition from 6 mice; Two-way RM ANOVA. Data information: Data presented as mean ± SEM n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001
Figure 7A Activity-dependent polyspiking of population spikes (PS) during 1 Hz stimulation is reduced by VPS34IN1 and facilitated by Roscovitine compared to DMSO-treated control slices. Top, representative traces of the 1st and 30th stimulus responses with polyspikes indicated by arrows. N = 12 slices per condition from 12 mice. B Quantification of polyspiking amplitudes in response to the 30th stimulus in the presence of DMSO, VPS34IN1, or Roscovitine. Polyspiking amplitudes are increased by Roscovitine and reduced by VPS34IN1. N = 12 slices per condition from 12 mice; One-way ANOVA. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.C The relative changes in the PS and polyspike areas in the presence of picrotoxin (50 µM) and a subsequent treatment with DMSO or VPS34IN1 (10 µM). VPS34IN1 significantly reduced the PS and polyspike area compared to DMSO. N = 6 slices per condition from 6 mice. D Mean area of PS and polyspikes in picrotoxin-treated slices 40 min post-application of DMSO or VPS34IN1. N ≥ 6 slices per condition from ≥ 6 mice; Student's t-test. Data information: Data presented as mean ± SEM ; *p < 0.05; ***p < 0.001.
Figure 1A Specific CD8+ T-cell (OT-I) proliferation in response to immature BMDCs incubated with fdOVA, fdOVA/sc-αDEC or OVA257-264 peptide chemically coupled to anti-DEC-205 antibody (NLDC:pOVA). Percentage of CFSE-labeled proliferating T cells was evaluated on CD8+-gated cells after 72 h of DC:T co-culture. The mean ±SD of three independent experiments is reported.B Proliferation of adoptively transferred OT-I T cells in C57BL/6mice (n = 3/group) immunized with vehicle (PBS), NLDC:pOVA, NLDC:pOVA plus CpG, fd, fd/sc-αDEC or LPS-free ovalbuminprotein. Percentage of CFSE-labeled proliferating OT-I T cells was evaluated after three days on Vα2+CD8+-gated cells. The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.C Percentage of IFN-γ-producing CD8+ splenocytes isolated from mice immunized as in (B). The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6 mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.D-F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.
Figure 3IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/−mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/−mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.Proliferation of OT-ICD8+T cells after 72 h of co-culture with 1 × 104DCs isolated from C57BL/6mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fd/sc-αDECbacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-ICD8+T cells. As a control, the proliferation of OT-ICD8+T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).
Figure 4BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDECbacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocalmicroscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.
Figure 5A-D BMDCs from C57BL/6mice were stably transduced with YFP-tagged TLR9. Cells grown on coverslips were then incubated with TRITC-conjugated wild-type (A, B) or fdsc-αDEC (C, D) bacteriophages for 6 h. Cells were then washed, fixed and analyzed by confocal microscopy. Nuclei were counterstained with Hoechst. (B, D) are enlargements of a single cell. Representative images are shown. Numbers at bottom of right panels indicate the percentage of TLR9bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm.
Figure 1(C-D) Diverse outcome for tumor cells following interactions with CTLs. OT-I CTLs and OVA+ Eµ-myc B cells were labeled with the calcium-sensitive dye Indo-1, cultured together in the presence of propidium iodide (PI) and subjected to live imaging. Representative time-lapse images (C) and corresponding fluorescence quantification (D) showing the three distinct outcomes for CTL-tumor cell contacts (left, calcium response in T cells, and right, calcium and PI signal in targets): no detectable damage (no calcium signal in the target cells), reversible damage (transient calcium elevation and low uptake of PI), and irreversible damage (prolonged calcium elevation and bright PI staining). CTLs are outlined by white dashed lines. White arrowheads indicate tumor cells. Red arrowheads indicate tumor cells with internalized PI. Scale bar, 5 µm.
Figure 2(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CTL (during the duration of the contacts). Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.
Figure 4(A) Representative time-lapse images showing tumors without CTLs (upper panels) or with OT-I CTLs (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CTLs appear in white. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm. Data information: Data were pooled of four independent experiments. (*p<0.05, ***p<0.001, unpaired t‑test).
Figure 5CTLs exhibit extensive differences in lytic activity in vivo. Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct-GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. (E) Representative two-photon time-lapse images showing a representative CTL with high lytic capacity (killing 4 targets) and a CTL with no lytic activity for up to 4 hours. Blue arrows mark apoptotic tumor cells. Live and apoptotic tumors appear in grey and blue, respectively. CTLs appear in green. Scale bar, 10 µm. Representative of 3 independent experiments.
Figure 6(A-B) Calcium responses were recorded over time in (A) unconjugated tumor cells or in (B) tumor cells forming a conjugate with a CAR T cell. Each line corresponds to an individual tumor cell and each square to a time point. Squares are colored in magenta upon calcium elevation or in blue otherwise. Apoptotic events (detected by caspase 3 activity) are indicated by an overlaid green dot. Data is representative of three independent experiment.
Figure 7(A) Representative time-lapse images showing tumors without CAR T cells (upper panels) or with CAR T Cells (lower panels). Tumor cells with low and high intracellular calcium appear in blue and red, respectively. CAR T cells appear in light green. Red arrows indicate tumor cells with elevated calcium. Scale bar, 5 µm.
Figure 1A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates).C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR.D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172).E, MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with indicated dose. 72 hours later, cells were harvested for immunoblotting with indicated antibodies.F, Same condition as E except the irradiation dose with 20 Gy, mRNA levels of inflammatory genes as indicated were assessed by real-time quantitative PCR, presented as mean + standard error of the mean of three biological replicates.
Figure 2A, MCF10A WT cells were pretreated with AZD6738(250 nM) and RO-3306 (9 μM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.B, Same condition except that RO-3306 is replaced by PD-0332991 (1µM).C, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 hours before harvesting for flow cytometry.D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting.E, MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A over-expressed cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cells were harvested for immunoblotting with indicated antibodies.F, Same condition except that cells were harvested 72 hours later.