conceived the research plan

conceived the research plan. SUMOylation and the ATR-ATRIP conversation, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling. = 3). Immunofluorescence and Laser Micro-irradiation HeLa and U2OS cells transfected with control or PIAS3 siRNA were seeded on coverslips in 6-well plates. To detect RPA, H2AX, and ATRIP at DNA damage sites, cells were treated with CPT or irradiate with UV through 5-m filter (Millipore). Subsequently cells were pre-extracted with 0.5% Ubenimex Triton X-100 in PBS buffer for 3 min on ice, fixed with 3% paraformaldehyde/2% sucrose for 15 min, and then extracted again with 0.5% Triton X-100 in PBS buffer for 3 min on ice. Cells were incubated with primary antibodies to RPA, H2AX, ATRIP diluted in 1 PBS made up of 3% BSA/0.05% Tween 20 for 2 h at room temperature. After 3 washes with 1 PBS made up of 0.05% Tween 20, cells were incubated with Cy3-conjugated anti-rabbit antibody and Alexa Fluor 488-conjugated anti-mouse antibody at room temperature for 1 h. Cells were then washed three times with 1 PBS made up of 0.05% Tween 20, and DNA was stained by DAPI (4,6-diamidino-2-phenylindole). To test if PIAS3 localizes to laser-induced DNA damage stripes, U2OS cells were micro-irradiated with UV laser as previously described (20). The pre-extraction step of immunostaining was skipped to avoid potential loss of PIAS3 from DNA damage stripes. In Vitro Kinase Assay HEK293T cells were treated with control, PIAS3, or PIAS1 siRNA for 24 h followed by transfection of Flag-ATR plasmids. Two days after plasmid transfection, Flag-ATR and Flag-ATRC were immunoprecipitated and tested with kinase assay as previously described (21). Analysis of the UV-induced Replication Inhibition HeLa and U2OS cells transfected with control or PIAS3 siRNA were either irradiated with UV (10 J/m2) or left untreated. At 1 h after UV or mock treatment, cells were labeled with 10 m EdU (5-ethynyl-2-deoxyuridine) for 30 min, trypsinized, washed with 1 PBS, and fixed in 75% ethanol at ?20 C. EdU-labeled cells were processed using a Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay kit according to the manufacturer’s instructions (Invitrogen). Data acquisition was performed on a FACS LSRII apparatus and analyzed with Kaluza software (Beckman Coulter). RT-Quantitative Ubenimex PCR (RT-qPCR) of PIAS2 mRNA Total RNA of HeLa cells transfected with control siRNA or siRNA targeting each of the PIAS family member was isolated using PureLink RNA mini kit (Invitrogen). cDNA was synthesized using the (dT)16 primer and TaqMan Reverse Transcriptase Reagents (Life Technologies). Two primer pairs that specifically amplify (#1 forward primer 5-GCTATTTCCTTTGCCTGGCTAT-3; #1 reverse primer 5-TTCTTCCCAATTTCTGATGCC-3; #2 forward primer 5-CCAAGTTCAGTTGAGACTTTGC-3; #2 reverse primer 5-GTGGTGCATAGCCAGGCAA-3) were used in qPCR, which was performed using PowerUp SYBR Green Grasp Mix (Applied Biosystems) according to the manufacturer’s protocol. Reactions were analyzed by LightCycler 480 (Roche) and the relative mRNA levels were normalized to GAPDH (forward Ubenimex primer 5-CGGATTTGGTCGTATTGGGC-3 and reverse primer 5-TGGAAGATGGTGATGGGATTTC-3). Results PIAS3 Is the Only PIAS SUMO Ligase Indispensable for ATR Activation While members of the PIAS family of SUMO ligases have been implicated in the DDR, whether and how they contribute to DNA damage signaling is still unclear. To address this question, we used siRNAs to knock down all 4 members of the PIAS family in HeLa cells and Rabbit polyclonal to CREB.This gene encodes a transcription factor that is a member of the leucine zipper family of DNA binding proteins.This protein binds as a homodimer to the cAMP-responsive element, an octameric palindrome. analyzed the effects around the CPT-induced, ATR-mediated Chk1 phosphorylation at Ser317. The knockdown of PIAS1, PIAS3, and PIAS4 was confirmed by Western blot (Fig. 1mRNA levels were determined by RT-qPCR. and and and and 0.05. 0.05. and 0.05. PIAS3 Is usually Dispensable for ATRIP SUMOylation Our previous studies showed that ATRIP is usually.