Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-6

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>mined by immunofluorescent microscopy in transfected 293 cells. Tax is expressed in nuclear foci in the absence of DNA damage (A) and is localized in the cytoplasm following UV irradiation (B). The addition of a UB tag at either the N- (C) or C- (E) termini caused Tax to be localized in the cytoplasm but treatment with LMB (D and F) sequestered UB-Tax and Tax-UB to nuclear foci. All images are shown at a magnification of 63×. The percentage of cells expressing nuclear foci (black bar) or cytoplasmic Tax (gray bar) was scored in five hundred transfected cells, in three independent experiments (G). Western blot showing expression of UB-Tax, Tax-UB, and native Tax proteins (H)

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-3

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p> HA-UB. Cells were either mock (-) or UV irradiated (+) and the ubiquitination status of Tax was examined by immunoblot analysis for Tax

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-1

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>or UV irradiated (30 J/m, 30 minute recovery). The immunoprecipitates were analyzed by immunoblot for Tax (left) or HA (right)

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-4

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>ed in 293 cells for 48 hrs and subjected to 30 J/mUV irradiation (B, D, F, H, J) or mock irradiated (A, C, E, G, I) and allowed to recover for 30 min. sc35 (left column) and Tax (left center column) were visualized by immunofluorescent microscopy separately, together (Merged, right center column), or together with DAPI (DAPI Merged, right column). All images are shown at a magnification of 63×

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export-2

<p><b>Copyright information:</b></p><p>Taken from "Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export"</p><p>http://www.retrovirology.com/content/4/1/95</p><p>Retrovirology 2007;4():95-95.</p><p>Published online 14 Dec 2007</p><p>PMCID:PMC2234431.</p><p></p>bsence (black bars) or presence of 30 J/mUV irradiation (gray bars) is shown. Error bars represent the standard error in three independent experiments. (B) Western blot showing expression of each Tax mutant analyzed

Tax expression accelerates S-phase entry following DNA damage.

<p>Synchronized CREF-neo and CREF-Tax cells were exposed to 30 J/m² of UV irradiation 12 hours after release from G0, which is shown as “0” h post irradiation. The percent of cells in G1 phase (A) and S phase (B) are displayed at the indicated times post-irradiation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055989#s2" target="_blank">Results</a> shown are the average of three independent experiments. (error bars represent standard error of the mean; * p-value≤0.1, ** p-value≤0.05).</p

Tax expressing cells fail to form UV-induced γH2AX and p-RPA foci.

<p>(<b>A</b>) UV irradiated CREF-neo and CREF-Tax cells were stained with either anti- p-RPA2(S33), anti- p-RPA2(S4/8), or anti-γH2AX (all stained in red). Cells were counterstained with Dapi (blue) to visualize nuclei. (<b>B</b>) CREF-Neo and CREF-Tax cells were either mock-irradiated or damaged with 30 J/m² UV. Cells were collected at the indicated timepoints and stained for γH2AX (red) and DAPI (blue). (<b>C</b>) Quantitation of γH2AX immunofluorescence intensity of 12 fields of at least 20 cells. Error bars represent standard error and ‡ represents a p-value≤0.001.</p

Tax interacts with WIP1.

<p>(<b>A</b>) 293 cells were transfected with either a Flag-WIP1 construct alone or Flag-WIP1 along with a Tax expression construct. Lysates were immunoprecipitated using either anti-WIP1 or anti-Tax antibodies followed by western blotting with anti-Flag or anti-Tax antibodies. (<b>B</b>) Coomassie stain of purified His-WIP1 and GST-Tax used in panel C. (<b>C</b>) His-WIP1 was incubated with either Glutathione sepharose beads or GST-Tax immobilized on Glutathione sepharose. Reactions were analyzed by western blot with anti-Tax and anti-His antibodies.</p

Tax expression alters the G₁ phase arrest following DNA damage.

<p>Asynchronous CREF-neo and CREF-Tax cells were exposed to 30 J/m² of UV irradiation. The percent of cells in G₁ phase (A) and S phase (B) are displayed at the indicated times post-irradiation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055989#s2" target="_blank">Results</a> shown are the average of three independent experiments (error bars represent standard error of the mean; * p-value≤0.1, ** p-value≤0.05).</p

Inhibition of WIP1 in Tax-expressing cells restores γH2AX levels following DNA damage.

<p>(<b>A</b>) CREF-Tax cells were transfected with a control siRNA or siRNA targeted to WIP1. 48 hours post-transfection cells were treated with 30 J/m² UV, allowed to recover for 4 hours, and analyzed by western blot for γH2AX and actin. (<b>B</b>) UV-damaged control siRNA transfected and WIP1 siRNA transfected CREF-Tax cells were analyzed by quantitative RT-PCR for WIP1 expression. (<b>C</b>) Uninfected (CEM) and HTLV-1 infected (MT4) cells (untreated or treated with the UV-mimetic drug 4-NQO) were harvested at the indicated times and analyzed by western blot. (<b>D</b>) CEM and MT4 cells were left untreated (−), treated with 4-NQO (+), or treated with the WIP1 inhibitor Compound M for 1 hour followed by 4-NQO (+M) and harvested after a 4 hour recovery followed by analysis by western blot for γH2AX.</p