Cosentino C , Grieco D , Costanzo V .

Cancer Research UK, 206281 European Research Council, ERC_206281 European Research Council

             double-strand break repair

	Figure 1. (A) Schematic representation of the pentose phosphate pathway. (B) Western blot with anti-Xenopus G6PD antibodies on Xenopus egg extract, which was not depleted (input), mock depleted (mock) or G6PD depleted (ΔG6PD). (C) Kinetic readings of NADPH fluorescence in mock (triangles) or G6PD depleted (squares) extracts. Fluorescent intensity is indicated in arbitrary units (a.u.). (D) Xenopus egg extract was treated with 20 ng/μl of DSBs in the presence or absence of 5 mM caffeine or left untreated. The samples were then loaded on 10% SDS–PAGE and analysed by western blot with anti-p-Ser1981 ATM. (E) Kinetic readings of NADPH fluorescence in untreated extract (ctr) or DSB-treated extract (DSBs). (F) Xenopus egg extract was treated with or without 20 ng/μl DSBs for 10 min and then incubated with 2′-5′-ADP-sepharose beads or protein G-sepharose as a control. An aliquot of the eluted protein was loaded on a 10% SDS–PAGE and the gel stained with Coumassie blue. (G) A second aliquot was instead used to measure G6PD activity. (H) Xenopus egg extract was untreated (ctr), treated with 20 ng/μl DSBs alone (DSBs) or in combination with 10 μM ATMi (ATMi) or 5 mM caffeine (Caffeine). The samples were then subjected to 2′-5′-ADP-sepharose pull down. G6PD activity was measured on the proteins eluted from beads after the pull down. Graphs shown in this figure indicate average values of three or more experiments. Error bars indicate s.d. A full-colour version of this figure is available at The EMBO Journal Online.
	Figure 2. HSP27 binding to G6PD. (A) Xenopus egg extract was left untreated (ctr) or treated for 15 min with 20 ng/μl DSBs (DSBs) in the presence or absence of 10 μM ATMi (ATMi) or supplemented with 3000 nuclei per microliter treated with 10 μM phleomycin (phl). Hundred microliters of extract were then diluted in PBS and incubated with 2′-5′-ADP-sepharose beads. The proteins bound to the beads were eluted in Laemmli buffer and loaded on a 4–12% SDS–PAGE gel, transferred onto nitrocellulose filter and analysed by western blot with anti-G6PD (upper panel) and anti-HSP27 (bottom panel) antibodies. (B) AG02603 fibroblast cells were left untreated or irradiated with 10 Gy. After 1 h, cells were collected and the proteins were extracted. The whole cell lysates were incubated with control (ctr) or anti-HSP27 antibodies. The samples were then analysed by western blot with anti-G6PD (upper panel) and anti-HSP27 (bottom panel) antibodies. (C) AG02603 cells were treated with 0.1% DMSO or 10 μM ATMi before being irradiated with 10 Gy or left untreated. G6PD was immunoprecipitated with specific anti-G6PD antibodies and the samples were analysed by western blot with anti-HSP27 antibodies. Non-immune serum was used as control (ctr). (D) In vitro G6PD activity assay: 300 ng of recombinant G6PD was incubated for 10 min at 30°C with the indicated amount of recombinant Hsp27. G6PD activity was then assessed. The histogram represents average enzymatic activities relative to untreated control (0). Experiment was repeated three times. Error bars represent s.d. (E) Human fibroblasts were exposed to 10 Gy of IR in the presence or absence of 10 μM ATMi. Total cell lysates were loaded on SDS–PAGE and then analysed by western blot with anti-phospho-Hsp27 (ser78), anti-tubulin and anti-γH2AX. (F) The histogram represents the average of three independent experiments in which Hsp27 phosphorylation was determined. Error bars represent s.d.
	Figure 3. G6PD activity in human cells. (A) AG02603 cells were treated with or without 10 μM ATMi or 5 mM caffeine before being exposed to 10 Gy of IR. Cells were then lysed and G6PD activity was determined over the time as indicated. (B) Normal fibroblasts (GM0024B), A-T fibroblasts (GM03395), normal lymphoblasts (GM0558) and A-T lymphoblasts (GM03382) were irradiated with 10 Gy or left untreated. These cells were then lysed and G6PD activity was determined on whole cell extracts. The histogram represents the average fold increase of G6PD activity over non-treated corresponding cells. Error bars indicate s.d.
	Figure 4. Silencing of Hsp27 in human fibroblasts. (A) Cell transfected with control or Hsp27 targeting siRNA were lysed. Total protein extract was loaded on SDS–PAGE and analysed for Hsp27 content (lower panel) and tubulin (upper panel). (B) G6PD activity assay in cells where Hsp27 was silenced. Graph indicates average values from independent experiments. Error bars indicate s.d.
	Figure 5. Incorporation of 14C into RNA. Human lymphoblasts were irradiated in the presence of D-glucose 6-phosphate-UL-14C. RNA was extracted and count per minutes (CPM) obtained for the irradiated cells were plotted as average fold increase compared to the correspondent non-irradiated cells. Graph represents average values derived from independent experiments. Error bars indicate s.d.
	Figure 6. DSB repair in human fibroblasts upon G6PD silencing. (A) Cells were transfected with a scrambled siRNA (ctr) and G6PD targeting siRNA. An aliquot of the cells was collected, lysed and analysed by western blot anti-G6PD (upper panel) and α-tubulin (bottom panel). (B) Another aliquot of the cells was either left untreated or irradiated with 10 Gy. Residual DSBs remaining after the indicated times were determined by neutral comet assay. The histogram represents the average comet tail moment. Experiment was repeated three times. Error bars represent s.d. (C) Cells transfected with control or G6PD-specific siRNA were exposed to 2 Gy of IR. γH2AX foci were visualized by immunofluorescence. (D) The histogram represents number of foci per cells. The data are the average of three independent experiments. Error bars indicate s.d.
	Figure 7. Schematic representation of G6PD regulation and downstream effect. DSBs activate ATM, which in turn promotes the interaction between Hsp27 and G6PD. This association leads to increased activity of G6PD and stimulation of the PPP. ATM also regulates glycolysis and PPP at transcriptional level (dashed arrows) through p53-mediated induction of TIGAR. As a consequence of these events, ROS levels are reduced and the dNTP pool is increased, allowing efficient DNA repair and promoting cell survival.

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