TRF1 a telomere-binding protein is important for telomere protection and homeostasis. TRF1 stability was not observed in hTERT-negative immortal cells but was pronounced when hTERT was ectopically indicated in the cells suggesting that hTERT may be needed in the PinX1-mediated TRF1 stability pathway. Interestingly the knockdown of both PinX1 and hTERT in HeLa cells stabilized TRF1 suppressed DNA damage response activation and restored chromosome stability. In summary our findings suggested that PinX1 may maintain telomere integrity by regulating TRF1 stability and that hTERT may act as both a positive and a negative regulator of TRF1 homeostasis inside a PinX1-dependent manner. gene using the primers 5′-CCGAATTCAAATGCAGATCTTCGTGAAG-3′ and 5′-AAGCGGCGCCTACCACCCTGAGACGGAG-3′ and the EcoRI and NotI sites are underlined. Amplified DNAs were gel-purified digested with EcoRI and NotI and ligated into the pCMV-HA. Site-directed mutagenesis was performed to produce the PinX1L291A and TRF1T122A mutants according to the manufacturer’s instructions (Stratagene). The primers utilized for the mutagenesis were as follows: TRF1T122A-F 5 TRF1T122A-R 5 PinX1L291A-F 5 PinX1L291A-R 5 Positive INCB018424 clones were confirmed by DNA sequencing (Cosmogenetech Seoul Korea). Transfection siRNA and Plasmids Cells at 50-60% confluence were transfected with 1 μg of plasmid or 50 nm siRNA using JetPRIMETM transfection reagent (Polyplus Illkirch France). StealthTM siRNAs Rabbit Polyclonal to OR2AG1/2. purchased from Invitrogen were as follows: PinX1 (HSS123667 HSS123668 HSS182773) hTERT (HSS144248 HSS144247 HSS144249) and control (catalog no. 2935-300). A mixture of three siRNAs was utilized for PinX1 and hTERT silencing. Some of the work was done with control siRNA purchased from Genolution (5′-ACGUGACACGUUCGGAGAAUU-3′; Genolution Seoul Korea). Plasmids encoding myc-PinX1 HA-PinX193-328 HA-PinX1149-268 HA-PinX1205-328 and GFP-PinX1 were described inside a earlier report (21). pcDNA3-hTERT-myc was generously provided by Dr. Ishikawa’s group. Immunoblotting and Antibodies Cell lysates were prepared from passive lysis buffer (Promega) comprising a mixture of protease inhibitors (Roche Applied Technology) and incubated with the following main antibodies: TRF1 (1:1 0 ab10579; Abcam Cambridge UK); PinX1 (1:3 0 H00054981-A01; Abnova Taipei City Taiwan); γ-H2AX (1:3 0 NB100-2280; Novus Biologicals Littleton CO); hTERT (1:5 0 1531 Epitomics Burlingame CA); GFP (1:5000 632381 Clontech Sparks MD); pT68-CHK2 (Thr-68) (1:3 0 2197 β-actin (1:5 0 4967 and GAPDH (1:5 0 2118 (Cell Signaling Technology); and c-myc (9E10) (1:5 0 sc-40) and HA-probe (Y-11) (1:500 sc-805; Santa Cruz Biotechnology). The secondary INCB018424 antibodies included horseradish peroxidase-conjugated anti-mouse (1:5 0 and anti-rabbit (1:5 0 from Cell Signaling Technology. Protein INCB018424 Stability Assay Cells were transfected with plasmids or siRNAs and cycloheximide (CHX) (Sigma) was added 24-48 h later on at 500 μg/ml for the changing times indicated in the numbers. Cell lysates prepared from INCB018424 cells collected at different time points were subjected to immunoblotting. Signal intensity of the bands was semiquantified using Amount One (Bio-Rad). In Vivo Ubiquitination Assay Cells were transfected with 50 nm siRNA against PinX1 or control. After 24 h cells were then transfected with 1 μg of plasmids encoding myc-TRF1 and HA-ubiquitin (for 36 h and then treated with 1 μm MG132 (Calbiochem) for 8 h to inhibit proteasome function. Cells were lysed with passive lysis buffer. With mild agitation 500 μg of INCB018424 clarified cell lysates was incubated with protein G-agarose (Amersham Biosciences) for 1 h at 4 °C. The supernatant was added to 0.5 μg of anti-myc antibody. After 1 h of incubation at 4 °C protein G-agarose was added and the combination was incubated for 1 h at 4 °C. The agarose beads were resuspended in SDS sample buffer (Cell Signaling Technology) and INCB018424 boiled for 5 min. The immunoprecipitates were then analyzed by Western blot analysis. Immunoprecipitation Immunoprecipitation (IP) assay was performed as explained in the protocol of the IP assay kit (Sigma). HeLa cells transfected with plasmids were lysed in passive lysis buffer comprising a 1× protease inhibitor combination (Roche Applied Technology). Then 800 μg of clarified cell lysates was incubated with 2 μg of anti-TRF1 antibody.