Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: Increased ionizing radiation-dependent phosphorylation of H2AX at DNA double strand breaks upon depletion of TPX2. A, transient transfection of TPX2 siRNA or doxycycline-induced expression of an exogenous miRNA specific for TPX2 significantly increases phosphorylation of H2AX in HeLa cells 1 h after 10 Gy as indicated by Western blot analysis: control siRNA +IR (100.0 ± 13.2) versus TPX2 siRNA +IR (279.0 ± 17.6), p < 0.001, n = 4; −doxycycline +IR (100.0 ± 20.9) versus +doxycycline +IR (871.5 ± 23.4), p < 0.001, n = 4; group (mean ± S.E.), unpaired t test. B, GFP-TPX2 expression reduces the increased levels of γ-H2AX caused by depletion of TPX2 with an siRNA targeting the 3′-untranslated region of TPX2 mRNA. C, time course analysis of H2AX phosphorylation in HeLa cells depleted of TPX2 by siRNA (n = 3–5): 15 min, control siRNA (78.6 ± 31.7) versus TPX2 siRNA (221.3 ± 25.9), p < 0.05; 1 h, control siRNA (90.4 ± 18.5) versus TPX2 siRNA (354.1 ± 54.8), p < 0.05; 2 h, control siRNA (100.0 ± 0.0) versus TPX2 siRNA (206.9 ± 38.4), p < 0.05; group (mean ± S.E.), unpaired t test. Note the similar ATM activation in irradiated control and TPX2-depleted cells as indicated by the levels of p-ATM (Ser-1981). D, increase in the number of U2OS cells with more than five high intensity γ-H2AX ionizing radiation-induced foci (i.e. intensity two standard deviations above the mean focus intensity) following TPX2 depletion by siRNA, 1 and 2 h after 4 Gy: 1 h, control siRNA (1.7 ± 0.4%) versus TPX2 siRNA (6.7 ± 1.1%), p < 0.05, n = 3; 2 h, control siRNA (1.9 ± 0.3%) versus TPX2 siRNA (5.2 ± 0.9%), p < 0.05, n = 3; group (mean ± S.E.), unpaired t test. The number of cells with these high intensity ionizing radiation-induced foci declines 3 h post-IR. Representative pictures of γ-H2AX ionizing radiation-induced foci in control RNAi- and TPX2 RNAi-treated cells 2 h after 4 Gy are shown. E, TPX2 depletion in U2OS cells by siRNA does not cause a significant change in the mean number of γ-H2AX ionizing radiation-induced foci after 4 Gy (n = 3). NS, non-significant, unpaired t test, S.E. F, increased phosphorylation of H2AX 1 h after 10 Gy in caspase-3-deficient MCF-7 cells depleted of TPX2 for 24 or 48 h by siRNA (n = 3) as detected by Western blots. MCF-7 cells do not undergo ionizing radiation-induced apoptosis associated with DNA fragmentation (54, 55). G, increased phosphorylation of H2AX in TPX2-depleted HeLa cells 2 h after a non-lethal dose of 2 Gy (n = 4). H, increased γ-H2AX levels in TPX2-depleted cells 1 h after ionizing radiation are unaffected by the broad spectrum caspase inhibitor Z-VAD-fmk. Z-VAD-fmk was applied at a known effective dose (51) and inhibited PARP1 cleavage (a marker for apoptosis) in the presence of the DNA-damaging drug camptothecin (top blots). Relative quantifications of γ-H2AX signals from independent experiments are shown in bar charts (A, C, F, and G). n = number of independent experiments; NS, non-significant; *, p < 0.05, **, p < 0.01; ***, p < 0.001. Error bars represent S.E. Bar in D, 10 μm. A.U., arbitrary units; Con, control.

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Phospho-proteomics, Transfection, Expressing, Western Blot, Control, Activation Assay, Irradiation, Marker

Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: TPX2 regulates the levels of ionizing radiation-dependent γ-H2AX and forms ionizing radiation-induced foci in G1 phase cells. A, enhanced levels of γ-H2AX 1 h after 10 Gy in G1 phase HeLa cells depleted of TPX2 by doxycycline-induced TPX2 miRNA expression. Note that the γ-H2AX augmentation in these TPX2-depleted cells is ionizing radiation-dependent. Cells were synchronized using a double thymidine block, released into fresh medium, and then used at specified time points for ionizing radiation treatment as indicated. Unirradiated cells were used for flow cytometry-based cell cycle profiling (top bar charts). Relative quantifications of γ-H2AX signals from independent experiments are shown in bar charts on the right: 11 h, −doxycycline +IR (229.0 ± 21.0) versus +doxycycline +IR (748.6 ± 39.4), p < 0.001; 12 h, −doxycycline +IR (135.1 ± 34.2) versus +doxycycline +IR (466.1 ± 98.6), p < 0.05; group (mean ± S.E.), unpaired t test; n = 3 (independent experiments); *, p < 0.05; ***, p < 0.001. Error bars represent S.E. Note that 11 h after release from the thymidine block TPX2-depleted cultures contain slightly more G2/M phase cells (8.66%) than control cultures (3.25%). Thus, on the Western blot, a 12 h-11 h loading hierarchy is chosen to facilitate comparison between control 11 h versus +doxycycline 12 h (3.96% G2/M cells). Although the cell cycle profile between these two samples is highly similar, TPX2-depleted cells exhibit approximately twice the levels of γ-H2AX than control cells. B, U2OS cell cultures synchronized with a double thymidine block as in A (see flow cytometry-based cell cycle profiles in top histograms; NS, non-synchronized control) form TPX2 ionizing radiation-induced foci 1 h after irradiation that partially co-localize with γ-H2AX during G1 phase. All images were taken under identical experimental and microscopic conditions. See text for details. C, TPX2 maintains its association with the mitotic spindle in the presence of DNA double strand breaks marked by γ-H2AX. Early and late mitotic figures as identified via DAPI and TPX2 staining with and without DNA damage are shown. Commercially available TPX2 antibody 184 was used in all immunofluorescence images. See supplemental Fig. 5 and Fig. 5 for specificity of TPX2 184 antibody. A.U., arbitrary units.

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Expressing, Blocking Assay, Flow Cytometry, Control, Western Blot, Comparison, Irradiation, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: Regulation of γ-H2AX levels by TPX2 is distinct from its mitotic functions and occurs in postmitotic G0 primary neurons. A, depletion of Ndel1 does not increase γ-H2AX levels after ionizing radiation treatment. B, depletion of NUF2 does not increase γ-H2AX levels after ionizing radiation treatment. The mitotic arrest upon NUF2 depletion was confirmed with the increased levels of the mitotic marker H3S10p (see text for details). C, significant decrease in TPX2 levels in mouse primary cortical neurons co-transfected with a TPX2 shRNA-encoding construct and GFP (ratio, 5:1) as detected by immunofluorescence and confocal microscopy using commercially available TPX2 antibody 184. See supplemental Fig. 5 for the expression pattern of TPX2 in brain tissues, cellular distribution of TPX2 in primary neurons, and specificity of the TPX2 shRNA and antibody 184. D, enhanced levels of γ-H2AX in G0 postmitotic primary neurons co-transfected with vectors encoding TPX2 shRNA and GFP (ratio, 10:1) 1 h after 10 Gy compared with surrounding untransfected cells or cells co-transfected with vectors encoding a control shRNA and GFP (ratio, 10:1) as determined by immunofluorescence and confocal microscopy. E, decreased levels of γ-H2AX in G0 postmitotic primary neurons transfected with GFP-TPX2 compared with surrounding untransfected cells or cells transfected with GFP 1 h after 10 Gy as determined by immunofluorescence and confocal microscopy. Note that the γ-H2AX signals in control shRNA- and GFP-expressing neurons are of an intensity similar to that of untransfected surrounding cells. F and G, quantification of the relative changes in γ-H2AX signals in neurons in D and E expressed by the average ratio of total nuclear γ-H2AX signals of transfected neurons/average total nuclear γ-H2AX signals of non-transfected surrounding cells. F, ratio control shRNA (n = 10)/non-transfected (n = 196) = 1.7 ± 0.4 (S.E.) versus ratio TPX2 shRNA (n = 16)/non-transfected (n = 459) = 6.4 ± 0.9 (S.E.); p < 0.001, unpaired t test. G, ratio GFP (n = 9)/non-transfected (n = 37) = 1.0 ± 0.1 (S.E.) versus ratio GFP-TPX2 (n = 8)/non-transfected (n = 33) = 0.5 ± 0.1 (S.E.); p < 0.001, unpaired t test. H and I, quantification of the relative changes in γ-H2AX immunofluorescence signals in neurons co-transfected with vectors encoding a control or TPX2 shRNA and GFP (ratio, 10:1) (H) or a GFP or GFP-TPX2 construct (I) 1 h after 3 Gy. H, ratio control shRNA (n = 58)/non-transfected (n = 300) = 1.1 ± 0.1 (S.E.) versus ratio TPX2 shRNA (n = 75)/non-transfected (n = 330) = 1.3 ± 0.1 (S.E.); p < 0.001, unpaired t test. I, ratio GFP (n = 50)/non-transfected (n = 297) = 1.2 ± 0.1 (S.E.) versus ratio GFP-TPX2 (n = 47)/non-transfected (n = 420) = 0.9 ± 0.1 (S.E.); p < 0.01, unpaired t test. Values were calculated as in F and G. NS, non-significant; **, p < 0.01; ***, p < 0.001. Error bars represent S.E. Bars, 20 μm. Ctrl., control. White arrows indicate transfected cells (C–E).

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Marker, Transfection, shRNA, Construct, Immunofluorescence, Confocal Microscopy, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: Effects of TPX2 overexpression on the levels of γ-H2AX and MDC1 ionizing radiation-induced foci. A and B, overexpression of His-TPX2 or GFP-TPX2 significantly decreases phosphorylation of H2AX in HeLa (A) and MCF-7 cells (B) 1 h after ionizing radiation treatment as indicated by Western blot analysis (10 Gy) (A) and immunofluorescence microscopy (B) (left panel, 2Gy; right panel, 4Gy). A, relative quantifications of γ-H2AX signals from independent experiments are shown in bar charts: control +IR (100.0 ± 6.9) versus His-TPX2 +IR (16.0 ± 2.9), p < 0.001, n = 4 (independent experiments); group (mean ± S.E.), unpaired t test. Levels of actin and H2A were used as loading controls. NS, non-significant; ***, p < 0.001. Error bars represent S.E. C, dose-dependent effects of GFP-TPX2 or His-TPX2 on the levels of ionizing radiation-induced γ-H2AX. The amount of plasmid transiently transfected per 6-cm cell culture dish is indicated. Proteins on Western blots were visualized with the indicated antibodies. TPX2 antibodies recognize endogenous and exogenous TPX2 on the same Western blot, thereby allowing comparison of absolute protein levels (see text for details). D, overexpression of His-TPX2 or GFP-TPX2 inhibits MDC1 ionizing radiation-induced focus formation in MCF-7 cells after 2 or 4 Gy (15-min recovery), respectively. Bar in B and D, 10 μm. Merged images include DAPI staining (B and D). A.U., arbitrary units. Transfected cells are indicated by white frame and asterisks (B and D).

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Over Expression, Phospho-proteomics, Western Blot, Immunofluorescence, Microscopy, Control, Plasmid Preparation, Transfection, Cell Culture, Comparison, Staining

Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: TPX2 localizes to DNA double strand breaks. A and B, TPX2 partially co-localizes with γ-H2AX-positive ionizing radiation-induced foci after 5 Gy in LAN1 (A) and U2OS cells (B), respectively (see also supplemental Fig. 5). TPX2 is found in the nucleus and cytosol in neuroblastoma LAN1 cells and neurons (see text and supplemental Fig. 5 for details). C, TPX2 co-localizes with γ-H2AX at 4-hydroxytamoxifen (4-OHT)/AsiSI-induced DNA double strand breaks. U2OS cells stably expressing AsiSI-estrogen receptor were left untreated or treated with 300 nm 4-hydroxytamoxifen for 4 h and subsequently immunostained for TPX2 and γ-H2AX. D, TPX2 accumulates in DNA double strand break-containing laser tracks (indicated by white arrows) marked by γ-H2AX. U2OS cells were either mock-treated (−MP) or microirradiated with a multiphoton laser (+MP). A representative image shows TPX2 accumulation at 10 min after irradiation. The intensity profiles of γ-H2AX and TPX2 immunofluorescence signals were measured in the yellow bars perpendicular to the laser tracks. Commercially available TPX2 antibody 184 was used in all immunofluorescence images. See supplemental Fig. 5 and Fig. 5 for specificity of TPX2 184 antibody. Bars, 10 μm. AU, arbitrary units.

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Stable Transfection, Expressing, Irradiation, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: TPX2 associates with MDC1, p-ATM, NBS1, and γ-H2AX. A, Y2H experiment using bait-TPX2 (aa 8–747) and prey-MDC1 (aa 1683–2089 or 1809–2089). Plasmids were co-transformed as indicated. −Leu/−Trp selection agar plates are controls for transformation efficiency. Colonies on −Ade/−His/−Leu/−Trp selection agar plates reveal an interaction between bait-TPX2 and prey-MDC1. Plates were incubated for 6 days. B, an ectopic C-terminal fragment of MDC1 (C-MDC1-FLAG; aa 1807–2089) associates with endogenous TPX2 in HeLa cells as indicated by co-immunoprecipitations with TPX2 antibody 184 from total cell lysate (left panel). Ectopic GFP-TPX2 also co-immunoprecipitates with endogenous MDC1 (right panel). The input lane for the C-MDC1-FLAG (left panel) is from a shorter exposure of the same Western blot. The input lane for the endogenous MDC1 (right panel) is from a stronger exposure of the same Western blot. C, GST-TPX2 pulls down MDC1 and the positive control Aurora A from total HeLa cell lysate. For MDC1, the input is a shorter exposure of the same blot. D, co-immunoprecipitations from U2OS cell lysates with the specified antibodies (see text for details on antibodies). TPX2 was co-immunoprecipitated with MDC1 from these cells using MDC1 antibody (left panel). MDC1 also co-immunoprecipitated with TPX2 antibody 184 (left panel). TPX2 was also found in complex with NBS1 and MDC1 species that migrate slower on SDS-PAGE gels when the co-immunoprecipitations were performed with the TPX2 KiS2 antibody (right panel; see text for further details). E, TPX2 and MDC1 associate in HeLa and LAN1 cells as detected by co-immunoprecipitations with the specified antibodies from total (left and middle panels) or nuclear (right panel) lysates. TPX2 from neuroblastoma LAN1 cells (and primary neurons) migrates as a doublet on gels (see supplemental Fig. 5). TPX2 is also found in complex with p-ATM and γ-H2AX after ionizing radiation treatment. Beads alone or antibodies against c-Myc were used as negative controls as indicated. IP, immunoprecipitation.

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Transformation Assay, Selection, Incubation, Western Blot, Positive Control, Immunoprecipitation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates ?-Histone 2AX (?-H2AX) Levels upon Ionizing Radiation *

doi: 10.1074/jbc.M112.385674

Figure Lengend Snippet: The ionizing radiation-dependent increase in γ-H2AX caused by TPX2 depletion is antagonized by inhibition or knockdown of MDC1 or ATM. A, an siRNA-mediated knockdown of MDC1 antagonizes the ionizing radiation-triggered γ-H2AX hyperamplification in HeLa cells caused by TPX2 depletion (lane 3 versus lane 4). Note that during the DNA damage response TPX2 depletion and MDC1 depletion have an opposite effect on γ-H2AX levels (lane 1 versus lane 3). B, inhibition of ATM with KU55933 antagonizes the ionizing radiation-dependent increase in γ-H2AX caused by depletion of TPX2. Inhibition of DNA-PK with NU7441 does not rescue this γ-H2AX hyperamplification phenotype. C, siRNA-mediated loss of ATM abrogates the ionizing radiation-dependent increase in γ-H2AX caused by depletion of TPX2. siRNA-mediated loss of DNA-PKcs partially decreases ATM levels (as previously described in Ref. 71) but does not rescue the γ-H2AX hyperamplification caused by TPX2 depletion. D, quantification of γ-H2AX signals in C. γ-H2AX signals of control siRNA-treated cells were considered as 100% and compared with the respective TPX2 siRNA-treated cells (n = 3 (independent experiments); N.S., non-significant; *, p < 0.05, unpaired t test, S.E.). Error bars represent S.E. All cells were treated with 10 Gy and harvested after 1-h recovery. Ctrl., control.

Article Snippet: Antibodies used for co-immunoprecipitations were MDC1 (Abcam) and TPX2 (184 from Novus Biologicals, TPX2 serum, and KiS2).

Techniques: Inhibition, Knockdown, Control