H₂O₂ induces DNA DSB signaling in WT and uPAR−/− mouse VSMC.

<p>VSMC isolated from WT (A) and uPAR−/− (B) mice were treated with 100 µM H₂O₂ for indicated time. Phosphorylation of ATM, Chk-2 and H2AX was assessed by western blotting. C. H₂O₂-induced phosphorylation of ATM, Chk-2 and H2AX was quantified from 3 independent experiments. Data are shown as folds of increase relative to unstimulated control and normalized to the total level of corresponding protein.</p

H₂O₂ induces DNA damage foci formation in WT and uPAR−/− mouse VSMC.

<p>A. WT and uPAR−/− mouse VSMC were treated with H₂O₂ for 1 h, then fixed and stained for P-Chk-2 (Alexa 488) and P-ATM (Alexa 594). B. Cells treated as in C were stained for γH2AX (Alexa 488) and P-ATM (Alexa 594). Scale bar 10 µm. C. Quantification of H₂O₂-induced DNA damage foci number per cell nucleus was performed using Particle analysis tool of ImageJ. D. Average size of DNA damage foci was calculated using ImageJ.</p

H₂O₂-induced PSMD6 nuclear import is impaired in the absence of uPAR.

<p>A. SiCo and uPARsi -nucleofected human VSMC were treated with 100 µM H₂O₂ for 1 h at 37°C. Then cells were fixed and stained for PSMD6 (Alexa 488) DraQ5 was used as nuclear stain. B. Human VSMC were treated as in A and subcellular fractionation was performed. PSMD6 content in nuclear fraction was assessed by western blotting. Histon H3 was used as loading control. Scale bar 100 µm. C. H₂O₂ -induced nuclear import of PSMD6 was quantified from 3 independent experiments.</p

MMS-induced increase of proteasome activity and PSMD6 nuclear import are abrogated in uPARsi VSMC.

<p>A. SiCo and uPARsi human VSMC were treated with 1.2(B) and uPARsi (C) VSMC were treated with 1.2 mM MMS for 1 h, then fixed and stained for PSMD6 (Alexa 488) and 20Sα7 subunit (Alexa 594). DraQ 5 was used as nuclear stain. The right panels in B, C show colocalization of PSMD6 and 20Sα7 indicated by color coding. The colormap was created using colocalization colormap plugin of ImageJ software. Scale bar 100 µm.</p

uPAR is required for MMS-induced PSMD6 phosphorylation.

<p>A. SiCo and uPARsi human VSMC were treated with 1.2(A) and serine phosphorylation (B) was assessed using Duolink proximity ligation assay. The right panels show the number of Duolink PLA signals per cell quantified using ImageJ software. DraQ5 was used as nuclear stain. C. Serine phosphorylation of PSMD6 was assessed by immunoprecipitation. PSMD6 from lysates of control and MMS-stimulated cells was immunoprecipitated. P-Ser was detected by western blotting. The lower panel shows loading controls.</p

uPAR regulates PSMD6 recruitment to 26S proteasome and its activity.

<p>A. SiCo and uPARsi human VSMC were treated with 1.2</p

uPAR is essential for MMS-induced DNA SSB signaling and DNA repair.

<p>A. Growth arrested SiCo and uPARsi -nucleofected human VSMC were treated with 1.2 mM MMS for indicated time points. Phosphorylation of Chk1 and Chk2 kinases was detected by western blotting. B. HEK 293 cells were infected with control lentivirus or uPAR-FLAG-expressing virus, and stimulated with 1.2 mM MMS for indicated time points. Phosphorylation of Chk1 and Chk2 kinases was detected by western blotting. C. WT and uPAR−/− mouse VSMC were treated with MMS for 20 min on ice to induce DNA damage. After H₂O₂ removal VSMC were allowed to repair DNA for 4 hrs. Comet tails were quantified as described in the Materials and Methods. D. WT and uPAR−/− mouse VSMC were treated with different concentrations of MMS for 20 min to induce DNA damage. The number of viable cells was calculated 24 hrs after DNA damage using 5(6)CFDA as described in Material and methods.</p

DNA SSB signaling and DNA repair are impaired in uPARsi cells.

<p>A. SiCo and uPARsi -nucleofected human VSMC were treated with 100 µM H₂O₂ for indicated time points. Phosphorylation of ATR and Chk1 kinase was detected by western blotting. B. HEK 293 cells were infected with control lentivirus or uPAR-FLAG-expressing virus, and stimulated with 100 µM H₂O₂ for indicated time points. Phosphorylation of Chk1 and Chk2 kinases was detected by western blotting. C. WT and uPAR−/− mouse VSMC were treated with H₂O₂ for 20 min on ice to induce DNA damage. After H₂O₂ removal VSMC were allowed to repair DNA for 4 hrs. Comet tails were quantified as described in the Materials and Methods. D. WT and uPAR−/− mouse VSMC were treated with different concentrations of H₂O₂ for 20 min on ice to induce DNA damage. The number of viable cells was calculated 24 hrs after DNA damage using 5(6)CFDA as described in Material and methods.</p

Recruitment of 19S subunits to the proteasome complex calculated as the ratio of normalized intensity of peptide peak to the total input cell extract.

<p>Recruitment of 19S subunits to the proteasome complex calculated as the ratio of normalized intensity of peptide peak to the total input cell extract.</p

Detection of apoptosis induced by ASHD using flow cytometry and confocal microscopy.

<p>Reh (<b>A</b>) and K562 (<b>B</b>) cells were cultured with ASHD (50 and 250 μM) for 72 h and processed for Annexin V- FITC/PI double-staining. The cells were then quantitatively or qualitatively monitored. In panels <b>A</b> and <b>B</b>, lower left quadrant shows cells which are negative for both Annexin V-FITC and PI, lower right shows Annexin V positive cells which are in the early stage of apoptosis, upper left shows only PI positive cells which are dead, and upper right shows both Annexin V and PI positive, which are in the stage of late apoptosis or necrosis. The values mentioned in the quadrants show the percentage of cells positive for both the Annexin V and PI (Top) or Annexin V alone (Bottom). In both panels <b>A</b> and <b>B</b>, cells treated with DMSO (a), ASHD, 50 μM (b), and ASHD 250 μM (c) are shown. In both panels, bar diagram showing comparison of early and late apoptotic cells at different doses of ASHD treatment are presented (d). (<b>C</b>) and (<b>D</b>) shows confocal microscopy visualization of Reh or K562 cells, following treatment with ASHD. Cells incubated with DMSO alone (<b>a</b>), or ASHD 50 μM (<b>b, c</b>) and 250 μM (<b>d, e</b>) respectively are used for the study.</p