Supplementary Information s71

Supplementary Information

Materials and Methods

Chemicals, reagents and antibodies

Okadaic acid sodium salt, Phosphatase Inhibitor Cocktail 2, wortmannin, Phosphatase

Inhibitor Cocktail 1 and 4-amino-1, 8-naphthalimide (4ANI) and Fostriecin sodium salt were from Sigma-Aldrich (St-Louis, MO). Tautomycetin was from Calbiochem. Calf

Intestinal Alkaline Phosphatase (CIP) was from New England Biolabs Inc. The anti-

PARP-1 (H-250) antibody, the anti-Ku-86 (B-1) antibody and the anti-WRN (H-300) antibody, purchased from Santa Cruz Biotechnology (Santa Cruz, CA ) were used for western blot analysis. For immunoprecipitation an anti-PARP-1 polyclonal antibody from

Cell Signaling Technology (Danvers, MA) was employed. The anti-Ku (p70/p80) Ab-3

(clone 162) antibody, used for immunoprecipitation, was purchased from Lab Vision

Corporation (Fremont, CA). The anti-β-actin antibody (clone AC-15) was obtained from

Sigma-Aldrich (St-Louis, MO). The anti-phospho-histone H2AX (ser139) and the anti- nucleolin antibodies were from Upstate Cell Signaling Solutions (Lake Placid, NY). The anti-Poly(ADP-ribose) (10H) antibody was obtained from Alexis Biochemicals (San

Diego, CA). The anti-PP5/PPT monoclonal antibody was from BD Biosciences

(Mississauga, ON). PP5 cDNA, amplified from a HeLa cDNA library, was in pFLAG-

CMV-2 (Sigma-Aldrich) with an N-terminal FLAG tag. Cell culture and treatments

HeLa, V15B, V15B + Ku80 (Schild-Poulter et al., 2003), V79 cells and M059J derivatives were maintained in DMEM containing 10% serum. HCT116 cells were grown as monolayers in McCoy's 5A medium containing 10% serum. γ-ray irradiation was performed by exposing cells to 137Cs γ-rays from Gammacell 3000 Elan (MDS Nordion,

Canada) at a dose rate of 5 Gy/min. UV irradiation (25 J/m2) was performed by exposing cells to germicidal UV lamps for 30 sec. Cells were allowed to recover for 0-2 h prior to lysis.

For siRNA suppression, HeLa cells were transfected with 200 pmol of annealed control double-stranded RNA (acuaccguuguuauaggug) or 100 pmol of ON-TATGETplus

SMARTpool siRNA targeting PP5C (Dharmacon, Chicago, IL) using Oligofectamine

Reagent (Invitrogen, Carlsbad, CA ). Cell extracts were prepared 3 days after the transfection. Overexpression of PP5 was carried out in HCT116 cells transfected with pFLAG-CMV-2 or pFLAG-CMV-2 PP5 using FuGene 6 transfection Reagent (Roche) for 3 days prior to treatment and protein extraction.

For in vivo [32P]-orthophosphate labeling, cells were incubated in phosphate- and serum- free DMEM medium for 2 hours and then labeled for 4 hours with [32P]-orthophosphate

(200 µCi/mL).

Immunofluorescence analysis of poly (ADP-ribose) (PAR)

Hela cells and murine embryonic fibroblasts were fixed in 4% paraformaldehyde in PBS for 10 min, rinsed twice in PBS, permeabilized in PBS with 0.4% Triton X-100 for 15 min, rinsed twice in PBS and blocked in PBS with 1% BSA, 0.4% Triton X-100 for 10 min. The coverslips were incubated in PBS with anti-PAR antibody (H-10) (1:200), 1%

BSA, 0.4% Triton X-100 and 10 M ADP-HPD at 37°C for 1 h, rinsed twice in PBS, and incubated with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:200) at 37°C for 30 min. Cells were rinsed twice in PBS and mounted after adding mounting medium with 4, 6 diamidino-2-phenylindole.

Preparation of cell extracts

Cells were harvested and lysed on ice for 10 min in Lysis buffer (25 mM Hepes, pH 7.5,

0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5% Triton X-100, 0.5 mM DTT and 1mM phenylmethylsulfonyl fluoride). PARP-1 was released into the supernatant (15800×g,

4°C, 10 min). The pellet was resuspended in 1×SDS loading buffer (50 mM Tris-HCl, pH

6.8, 100 mM DTT, 2% SDS, 10% glycerol) and sonicated prior to SDS-PAGE analysis of histones.

Immunoblotting and immunopreciptation

Fifty µg of cell extracts were resolved by 6% SDS-PAGE and transferred onto PVDF.

Following antibody incubation, membranes were developed by chemiluminescence using a peroxidase coupled secondary antibody. For the immunoblotting of  H2AX , 10 µg of proteins from cell pellet were resolved by 15% SDS-PAGE. For immunoprecipitation,

500 µg of proteins in 500 µl of IP buffer (12.5 mM Hepes, pH 7.5, 150 mM NaCl, 0.75 mM MgCl2, 0.1 mM EDTA, 0.25% Triton X-100, 0.25 mM DTT and 1mM phenylmethylsulfonyl fluoride) were incubated with 1.5 µg of anti-Ku70/80 monoclonal antibody (Ku Ab-3) or 10 µl of anti-PARP-1 antibody at 4 °C for 2 hr. Immunoprecipitates were recovered with protein G or protein A sepharose beads, washed five times with IP buffer, and analyzed by SDS-PAGE. Input lanes correspond to 10% of immunoprecipitated material.

ADP-ribosylation

Fifty µg of cell extract in 50 µl of IP buffer with 10 µM ADP-HPD (Calbiochem, La

Jolla, CA) was incubated with 1 µCi of [32P] adenylate NAD+ (~1000 Ci/mmol)

(Amersham Biosciences, ) at 30 °C for 10 min. Reactions were terminated with SDS loading buffer, resolved by SDS-PAGE, PVDF transfer and autoradiography. For experiments with protein kinase and protein phophatase inhibitors, additional assays were employed to ensure the functionality and specificity of the inhibitors.

Protein dephosphorylation by Calf Intestinal Alkaline Phosphatase (CIP) and 2-D analysis

Cell extracts (150 µg/ 100 µl) were incubated with 10 units of Calf Intestinal Alkaline

Phosphatase or phosphatase inhibitors at 37 °C for 30 min, and acetone precipitated.

Pellets were solubilized in 2-D rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS,

1% DTT) by sonication, adjusted to 0.5% Biolytes 3-10 (Bio-Rad) and subjected to isoelectric focusing on a 7-cm IPG strip of pH 3-10 NL (Amersham Biosciences,

Piscataway, NJ). Focused IPG strips were incubated in equilibration buffer (50 mM Tris-

HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% SDS) containing 1% DTT followed by incubation in equilibration buffer containing 4% iodoacetamide. Proteins were then resolved by 8% SDS-PAGE and transferred to PVDF.