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J. 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 in 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-CCAGTCTAACAGCTTGCCAGTTGAGAGCTATATACATATGTC-3; TRF1T122A-R, 5-GACATATGTATATAGCTCTCAACTGGCAAGCTGTTAGACTGG-3; PinX1L291A-F, 5-CCGGGACTTCACCGCGAAGCCCAAAAAGA-3; PinX1L291A-R, 5-TCTTTTTGGGCTTCGCGGTGAAGTCCCGG-3. Positive clones were confirmed by DNA sequencing (Cosmogenetech, Seoul, Korea). Transfection, siRNA, and Plasmids Cells at 50C60% confluence were transfected with 1 g of plasmid or 50 nm siRNA using JetPRIMETM Basmisanil transfection reagent (Polyplus, Illkirch, France). StealthTM siRNAs purchased from Invitrogen were as follows: PinX1 (HSS123667, HSS123668, HSS182773), hTERT (HSS144248, HSS144247, HSS144249), and control (catalog no. 2935C300). A mixture of three siRNAs was utilized for PinX1 and hTERT Aviptadil Acetate silencing. Some of the work was done with control siRNA purchased from Genolution (5-ACGUGACACGUUCGGAGAAUU-3; Genolution, Seoul, Korea). Plasmids encoding myc-PinX1, HA-PinX193C328, HA-PinX1149C268, HA-PinX1205C328, and GFP-PinX1 were described in a previous 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) made up of a mixture of protease inhibitors (Roche Applied Science) and incubated with the following main antibodies: TRF1 (1:1,000, ab10579; Abcam, Cambridge, UK); PinX1 (1:3,000, H00054981-A01; Abnova, Taipei City, Taiwan); -H2AX (1:3,000, NB100-2280; Novus Biologicals, Littleton, CO); hTERT (1:5,000, 1531-1; Epitomics, Burlingame, CA); GFP (1:5000, 632381; Clontech, Sparks, MD); pT68-CHK2 (Thr-68) (1:3,000, 2197P), -actin (1:5,000, 4967), and GAPDH (1:5,000, 2118) (Cell Signaling Technology); and c-myc (9E10) (1:5,000, sc-40) and HA-probe (Y-11) (1:500, sc-805; Santa Cruz Biotechnology). The secondary antibodies included horseradish peroxidase-conjugated anti-mouse (1:5,000) and anti-rabbit (1:5,000) from Cell Signaling Technology. Protein Stability Assay Cells were transfected with plasmids or Basmisanil siRNAs, and cycloheximide (CHX) (Sigma) was added 24C48 h later at 500 g/ml for the times indicated in the figures. Cell lysates prepared from cells collected at different time points were subjected to immunoblotting. Signal intensity of the bands was semiquantified using Quantity 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 g of plasmids encoding myc-TRF1 and HA-ubiquitin (for 36 h and then treated with 1 m MG132 (Calbiochem) for 8 h to inhibit proteasome function. Cells were lysed with passive lysis buffer. With gentle agitation, 500 g of Basmisanil clarified cell lysates was incubated with protein G-agarose (Amersham Biosciences) for 1 h at 4 C. The supernatant was added to 0.5 g 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 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 made up of a 1 protease inhibitor combination (Roche Applied Science). Then, 800 g of clarified cell lysates was incubated with 2 g of anti-TRF1 antibody at 4 C overnight. For precipitation of myc-TRF1, 1 g of anti-myc antibody (9E10) was added to 500 g of lysates for 1 h. Protein G-agarose suspension was then added to each reaction and incubated for 4 h at 4 C. After sequentially washing the beads, the proteins were subjected to immunoblot analysis. Chromatin Immunoprecipitation (ChIP) Assay and Telomere Slot Blotting ChIP assay was performed with an EZ ChIPTM assay kit (Upstate, Charlottesville, VA). In brief, cells were fixed in formaldehyde and lysed, followed by.