1A, right panel)

1A, right panel). to UV and have a much greater risk of developing sun-induced skin malignancy than that of normal population [4C7]. Due to the role of PolH in repairing UV-induced DNA damage, current studies have been focused on identifying various pathways that regulate PolH expression and activity. Recently, we exhibited that Pirh2 E3 ubiquitin ligase promotes PolH degradation in a ubiquitin-independent manner [8]. In addition, Kim ubiquitination assay was performed as described [21]. RKO cells were transfected with indicated plasmids and treated with 5 M MG132 for 6 h prior to harvest. Cleared cell lysates were immunoprecipitated with anti-HA antibody followed by Western blot with anti-HA and anti-Ubiquitin antibodies to detect PolH ubiquitination. ubiquitination assay using immunopurified proteins was performed as described [21]. 35S-labeled PolH proteins were mixed with immunopurified FLAG-Mdm2 or Mdm2 (1-441) and then incubated on ice for 1 h to form Mdm2:PolH complexes. The complexes were added to ubiquitination buffer made up of E1, E2, and ubiquitin, and Piperidolate incubated at 30C for 2 h. Finally, the reaction mixtures were separated on SDS-PAGE and analyzed by autoradiography. E1, E2, and ubiquitin were purchased from Boston Biochem. 35S-labeled PolH was produced by the TNT T7-coupled reticulocyte lysates system (Promega). To purify Mdm2 from RKO cells, whole cell lysates from RKO cells transfected with FLAG-Mdm2 or FLAG-Mdm2(1C441) expression vector were then immunoprecipitated by anti-FLAG (Sigma) antibody. ubiquitination assay using bacterial purified proteins was carried out as described above except that recombinant GST-Mdm2 and Piperidolate GST-Mdm2(1C441) were used as E3 ligase. 2.7. GST fusion protein preparation DNA sequences corresponding to Piperidolate the ORFs of human Mdm2 and Mdm2(1C441) were subcloned into the bacterial expression vector pGEX-4T-3 (Amersham Pharmacia Biotech) to produce GST-tagged fusion protein. The GST fusions of Mdm2 and Mdm2 (1C441) were expressed in BL21 (DE3) (Novagene) upon induction with 0.5 mM IPTG for 4 h at 37C. Bacterial cells were harvested, sonicated, and clarified by centrifugation. The recombinant GST-tagged proteins were purified by glutathione-Sepharose beads (Amersham Pharmacia Biotech) as described previously [22]. 2.8. DNA histogram analysis DNA histogram analysis was performed as previously described [20]. RKO, H1299 and GM00024 cells were transfected with scramble or Mdm2 siRNA for 2 days. Both floating cells in the medium and live cells around the plates were collected 24 h post 15 J/m2 UV irradiation and fixed in 70% prechilled ethanol and followed by PI staining. Stained samples were analyzed by BD FACS Calibur? flow cytometer. 2.9. Cell proliferation assay H1299 cells were transfected with scramble or Mdm2 siRNA for 48 h and then an appropriate number of cells were inoculated in 6 well plates in triplicate. 24 h after seeding, cells were washed with PBS and exposed to 15 J/m2 UV irradiation and cultured over a 9-day period. Cells were harvested and counted at the indicated occasions. 2.10. Immunofluorescence assay Immunofluorescence assay was performed as previously described [22]. Briefly, H1299 cells were transfected with scramble or Mdm2 siRNA for 72 h and then an appropriate number of cells were seeded on cover slide in triplicate. 6 CREB3L3 h after UV irradiation (15 J/m2), cells were fixed with formaldehyde and then incubated with a mixture of anti-PolH and anti-PNCA antibodies. Nuclei were visualized using 4-6-Diamidino-2-phenylindole (DAPI). 3. Results 3.1. Mdm2 regulates the steady-state level of PolH In our previous study, we showed that PolH protein stability can be regulated by Pirh2 [8]. However, Pirh2-mediated PolH degradation is usually accomplished in a ubiquitin-independent manner. To investigate whether PolH stability is regulated by other E3 ubiquitin ligases, we showed that upon transient knockdown of Mdm2, the level of PolH was increased (Fig. 1A, left panel). To confirm this, Mdm2 was inducibly knocked down in MCF7 cells (Mdm2-KD). Similarly, PolH was increased upon knockdown of Mdm2 (Fig. 1A, middle panel). To confirm the effect of Mdm2 on PolH expression in a p53-impartial manner, we showed that PolH was increased upon Mdm2-KD in p53?/? HCT116 cells (Fig. 1A, right panel). To further validate this, Mdm2 was knocked down in MCF7 cells over a 3-day period. We showed that as the level of Mdm2 was progressively decreased (Fig. 1B, Mdm2 panel), Piperidolate the level of endogenous PolH protein was gradually increased with concomitant induction of.