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Suppl. Fig. 1 Both Wip1 isoforms are bound to chromatin A) U2OS cells were transfected with three independent shRNAi plasmids targetting Wip1 or with control plasmids, selected with puromycine for 48h. Total cell lysates were probed with indicated antibodies. Note that both bands recognized by anti-Wip1 are reduced in knock-down samples. B) U2OS cells were transfected with pRS or with pRS-Wip1 and selected for 48h with puromycine. Cell lysates were probed with anti-Wip1 antibody preabsorbed either with His-Wip1 (preadsorbed) or with equivalent amount of BSA (control). Note reduced reactivity of antibody preadsorbed on recombinant His-Wip1. Arrowhead indicates the position of His-Wip1 (right panel). C) U2OS cell were fractionated as described in Methods in presence of indicated amounts of NaCl (mM) and samples were probed with indicated antibodies. Note that both bands recognized by antiWip1 are present in chromatin fraction. D) HCT116 p53+/+ and HCT116 p53-/- cells were treated with adriamycine (1M for 12h) and normalized cell lysates were probed with indicated antibodies. Note DNA damage-induced expression of both Wip1 bands in p53 wild type cells but not in p53-/- cells. E) U2TR-Wip1 cells were induced or not to express Wip1FLAG and were subjected to immunoprecipitation. Indicated amounts of NaCl were present throughout the immunoprecipitation. Suppl. Fig. 2. Wip1 dephosphorylates H2AX in vitro and in vivo. A) Indicated amounts of purified His-Wip1 wild-type or phosphatase dead were mixed with 100M synthetic peptides in the presence or absence of 60mM MgCl2 and incubated for 30min at 30C. Free phosphate was measured by colorimetric assay and determined from a standard curve. Error bars indicate SD from four independent experiments. B) U2OS cells were transfected with Wip-WT or Wip-D314A together with transfection markers CFP-Golgi and mCherry-NLS, respectively. Cells were synchronized by 1 thymidine and 5h postrelease exposed to doxorubicine (0.5M) for 1h. Cells were fixed and stained for H2AX. Suppl. Fig. 3. Wip1 activity on H2AX is independent of ATM. A) U2TR cells stably transfected with inducible Wip1 were synchronized by thymidine and 5h postrelease challenged with DNA damage (0.5M doxorubicin, 1h). Wip1 expression was induced or not with tetracycline and caffeine was added where indicated. All samples were treated with nocodazole to arrest the recovering cells in mitosis. Samples were collected at 2h intervals and probed with indicated antibodies. B) U2OS and GM05849 cells were exposed to 10M etoposide for 1h, fixed and probed with antiH2AX and anti-ATM antibodies (left panel) or analysed on westrn blot (right panel). C) GM05849 cells were transfected with EGFP-Wip1 or with EGFP-TPX2. DNA damage was induced 48h posttransfection by 10M etoposide for 1h. Cells were fixed and stained with anti-H2AX antibody. Suppl. Fig. 4. Depletion of Wip1 interferes with H2AX removal after doxorubicin treatment. U2OS cells were transfected with pRS-Wip1 or empty pRS and selected with puromycine. Cells were synchronized in G2, treated with 0.5M doxorubicin for 1h and extensively washed with fresh medium. Samples were collected at indicated times (h) and probed with indicated antibodies. Suppl. Fig. 5. Wip1 depletion prevents removal of H2AX in ATM deficient cells. AT cells were transfected with siRNA targeting GAPDH or Wip1 and 36h postransfection challenged with 10M etoposide for 2h. Cells were released in fresh 2 media, fixed at indicated times and probed with anti-Wip1 and anti-H2AX antibodies. Note that only a fraction of control depleted cells express detectable levels of Wip1 at 10h postrelease. Suppl. Fig. 6. Premature expression of Wip1 prevents formation of DNA damage foci. U2TR-Wip1 cells were transfected with MDC1-EGFP, synchronized in G2 and induced or not to express Wip1 by addition of tetracycline. Cells were fixed 1h after exposure to 0.2 M doxorubicin and probed with indicated antibodies. 3