Then, sections were incubated with the primary antibodies (mouse-anti-PAR clone 10H, 1:300 in blocking solution, 600 μl/slide, LSBio, rabbit-anti-H2A.X, 1:400 in blocking buffer (600 μl/slide), New England Biolabs) at RT for 1h. After rinsing 2x, then washing 3-4x for 5 minutes each in 0.2% Tween 20/TBS, sections were incubated with the secondary antibodies for 45 min at RT (goat-anti-mouse IgG-Alexa488, 1:300 in blocking solution, 500 μl/slide, Molecular Probes; goat-anti—rabbitIgG (H+L)-Alexa568, 1:300 in blocking buffer, Invitrogen). After washing the slides 3x for 5 min each in 0.2% Tween 20/TBS and twice in PBS, cell nuclei were stained using Hoechst dye (10 min at RT, 5 μg/ml Hoechst in PBS). After rinsing twice in PBS, slides were covered with coverslips using ProLong Antifade (Molecular Probes). Sections were viewed in a Leica SP2 confocal laser fluorescence microscope using the 405 nm, 488 nm and 561 nm laser lines to excite Hoechst, Alexa488 (PAR) and Alexa568 (γH2A.X) dyes, respectively. Mineral was visible in phase-contrast mode as dark deposits/structures.

Frozen sections of fetal sheep growth plate were thawed for 10 min, soaked for 5 min in PBS to remove OCT and fixed in 100% cold methanol (−20°C) for 10 min. Then, the sections were permeabilized at RT for 15 min with 0.2% TX100/PBS, then blocked for 30 min with 3% BSA/0.2% TX100/PBS.

In the calcein experiment, lesion mineral deposition was visualized in both transmission and fluorescence mode. Calcein and Hoechst were excited using the 488 nm and 405 nm laser lines respectively and images were acquired using a 20x/NA 0.5 water-immersion objective. In the double immunostaining experiment, images were acquired using a 63x/NA 1.2 water-immersion objective. The 405 nm, 488 nm and 561 nm laser lines were used to excite Hoechst, Alexa488 (PAR) and Alexa568 (H2A.X) dyes, respectively.

For the double immunostaining of PAR and the DNA damage marker γH2A.X, sections were fixed with cold methanol (as above) and subsequently permeabilized with 0.2% Triton X-100/ PBS for 15 min. After blocking non-specific binding sites with blocking buffer (3% BSA/ 0.2% TX100/PBS) for 30 min, sections were incubated with primary antibodies (mouse-anti-PAR antibody, clone 10H, Abcam, 1:300 in blocking buffer and rabbit-anti-H2A.X antibody, 20E3, New England Biolabs, 1:400 in blocking buffer) for 1h. After washing sections 4x for 5 min each in washing buffer (0.2% TX100/PBS), sections were incubated with secondary antibodies (goat-anti-mouseIgG-Alexa488 and goat-anti-rabbitIgG-Alexa568, Invitrogen, both at 1:300 in blocking buffer) for 45 min. Unbound antibodies were removed by washing 3x for 5 min each in washing buffer and 2x in PBS, then nuclei were counter-stained with 10 μg/ml Hoechst for 10 min. Sections were rinsed 2x with PBS and then mounted as above. Samples were viewed using a Leica SP2 confocal fluorescence microscope.

High resolution imaging. Frozen sections of human carotid and coronary artery lesions were thawed, rehydrated for 5 min in 0.9% saline and then fixed for 10 min in 100% methanol cooled to −20°C. Residual methanol was removed by rinsing 2x in PBS. All following staining steps were performed at RT. In the calcein experiment, lesion mineral deposits were stained with the Ca 2+ -binding dye calcein (4 μM in PBS, Sigma C-0875) for 1h; Hoechst 33258 (Sigma B-2883, at 10 μg/ml) was included in the staining solution to counter-stain nuclei. Unbound dyes were removed by washing 3x for 5 min each in PBS and sections were then mounted in ProLong Antifade Gold (Invitrogen).

Low resolution imaging. Human aortic and carotid samples were dissected and fixed into 10% neutral buffered formalin. These samples were then embedded in paraffin wax blocks and cut in 7 μm thick sections. Immunohistochemistry staining for PAR expression was performed using human aortic (n = 13) and carotid (n = 10) samples from normal and diseased patients. Parallel sections of the same samples were processed for von Kossa staining to visualize calcification and counterstained with 0.1% of nuclear fast red solution. The % of calcified area was measured by the threshold method using ImageJ software. For the co-localization of PAR, mineral and elastin in medial calcification by fluorescence microscopy the same histological sections were used. Elastin was visualized by its autofluorescence (405 nm laser line), mineral was imaged in reflectance mode (488 nm laser line) and PAR was visualized in transmitted mode. Samples were viewed in a Leica SP2 laser confocal microscope using using a 63x/NA 0.9 water-immersion objective. In all experiments, images were acquired in sequential mode.

A Bruker 400MHz Avance spectroscopy II spectrometer was used for solid-state 13 C (REDOR) and 31 P NMR measurements, at frequencies of 100.6MHz and 162.1MHz respectively, with standard Bruker double (for 31 P) and triple (for 13 { 31 P} REDOR) resonance, magic-angle spinning (MAS) probes. Samples were packed into disposable high-resolution (HR)-MAS inserts, and loaded into 4 mm zirconia rotors. The rotors were rotated at magic angle at a rate of 10 kHz. Samples were characterized using direct-polarization (DP) 31 P NMR and 13 C{ 31 P} rotational-echo double resonance (REDOR) techniques ( 1 H 90° pulse length 2.5 μs, 31 P 90° pulse length 2.57 μs, 1 H- 31 P CP contact time10ms). Recycle times of 600 s were used for 31 P DP experiments, and 2 s for REDOR experiments. REDOR experiments used REDOR dephasing times of 10 ms. Broadband TPPM decoupling during signal acquisition for all experiments.) 13 C spectra were referenced to the glycine C α signal at 43.1 ppm relative to TMS at 0 ppm. 31 P spectra were referenced to the hydroxyapatite 31 P signal at 2.8 ppm relative to 85 wt% H 3 PO 4 at 0 ppm.

The mineralized cultures were washed with PBS and then Calcein AM (1 μM) in PBS was added to the cultures and incubated for 25 minutes in humidified incubator. PBS was added to negative controls instead of calcein. After incubation the cultures were washed three time with PBS. For positive controls the cultures were treated with 2mM H 2 O 2 in PBS for 5 min. The cultures were washed in PBS and fixed with −20°C methanol for 10 minutes. Then cells were washed three times with PBS and permeabilized with 0.4% Tween 20 in PBS for 15 min at room temperature. The cells were washed twice in PBS and blocked with BlockAid blocking solution (Thermo) for 30 minutes at room temperature. After blocking, cells were incubated with primary antibody, mouse monoclonal anti-PAR (clone 10H) antibody (Abcam) / 300 μl/well) diluted (1:300) for overnight at 4°C. Then, cells were washed three times with 0.2% Tween 20 in PBS and fluorescent dye-labeled secondary antibody (goat-anti mouse IgG (H+L)-Alexa594 antibody (Abcam), (1:500 diluted)) in BlockAid was applied for 1 hour at room temperature. The cells were washed three times in 0.2% Tween20 in PBS then cells were rinsed twice in PBS. Afterward, PBS was removed glass coverslips were carefully mounted in Fluoroshield mounting media with DAPI (4’,6-diamidino-2-phenylindole, Abcam).

MC3T3-E1 cells were seeded on sterile 8-well chamber slide (LabTech) at a cell density of 1x10 4 cells per well in α-MEM (Life Technologies) growth medium. After three days, growth medium was replaced by mineralization medium and was changed every three days till day thirty when the mineralized nodules are wide spread in culture. bVSMC culture mineralized at day twenty.

The fraction of PAR staining which was non-nuclear, was assessed by thresholding the images of PAR and DAPI staining in ImageJ and calculating the fraction of PAR-positive pixels that did not overlap with DAPI-positive pixels.

Confocal Imaging was performed on Leica TCS SP8 confocal microscope (Leica Microsystems). To avoid in-between channel crosstalk all images were taken in frame sequential mode. Schedule for the dyes was as follows: DAPI excitation 405/ emission 410-540 nm, calcein ex496/em500-520 nm and Alexa-594 ex594/ em600-700 nm. Multiphoton imaging was performed on LaVision BioTec TriM Scope II (LaVision BioTec GmbH, Germany) equipped with the Inside Deepsee laser light source (Spectra Physics, CA, USA). Excitation of 1140 nm allowed tri-photon excitation of DAPI (emission collected below 495 nm), generation of second harmonic (SHG) of collagen (collected 495-560 nm) and two-photon excitation of Alexa-594 (collected above 560 nm).

All in vitro experiments using cultured VSMCs or MC3T3 –E1 cells were performed with 3-5 experimental replicates. At least 3 independent experiments were performed for each procedure.

Extracellular matrix synthesis for PAR binding assessment ( Figures 3 and S3

Extracellular matrix (ECM) was synthesized by growing hVSMCs to confluency on gelatin coated plates and coverslips. The cells were then extracted and the ECM used for western blotting or immunofluorescent staining.

6-well plates and coverslips were prepared by incubating with 0.2% gelatin solution in PBS at 37°C for 1 hour. The solution was removed and the gelatin was crosslinked by adding 1% glutaraldehyde in PBS and incubating at room temperature for 30 minutes. The wells and coverslips were washed with PBS and the non-crosslinked glutaraldehyde was quenched by incubating with 1 mol/l ethanolamine for 30 minutes. Cells were seeded onto the plates (50000 cells for a 6-well plate and 20000 cells for a coverslip) and after 24 hours the media was changed to complete media supplemented with 50 μg/ml l-ascorbic acid. The media was changed every 48 hours and after 7 or 18 days the cells were extracted from the ECM.

Extraction buffer (0.1% Triton X-100, 20 mmol/l NH4OH in PBS) was added to the cells and incubated for 5 minutes at 37°C. The lysed cells were removed and PBS was added to the ECM overnight at 4°C to remove remaining cell debris. The ECM was rinsed with PBS and either fixed with 3.7% PFA for immunofluorescent staining or scraped in lysis buffer (0.1% Triton X-100, 150mM NaCl in 0.1M Tris-HCl) for IP and slot blot analysis.

Immunofluorescent staining Immunofluorescent staining of the ECM was performed to visualize the localization of PAR. Localization was studied by counterstaining with fibronectin (FN), a late endosomal marker (CD63) and calcification (Osteoimage). Following fixing with 3.7% PFA, blocking was done for 1 hour at RT with 3% BSA (in PBS). The primary antibodies were diluted in blocking solution and incubated with the coverslips for 1 hour at RT. After washing, the secondary antibody, diluted in blocking solution, was added and incubated for 1 hour at RT in the dark. 4’,6-diamidino-2-phenylindole (DAPI) was diluted in PBS and incubated with the coverslips for 5 minutes, to stain nuclei. Following washing, coverslips were mounted with Mowiol mounting medium and left to dry in the dark at RT overnight. Slides were then stored at 4°C. Images were taken using a Leica TCS SP5 confocal microscope. Quantification was performed on n > 3 images per condition. PAR area/integrated density was analyzed using ImageJ. The images were converted to RGB stacks and measured using the auto threshold method. The default threshold method was selected, and percentage area and integrated density were measured. The percentage area is calculated by dividing the number of pixels that have been highlighted by the threshold by the total number of pixels. The integrated density is the product of the area and mean gray value (the sum of the gray values of the pixels highlighted by the threshold, divided by the total number of pixels).

Slot-blot analysis of PAR and PARylated proteins Cell, ECM, apoptotic body or vesicle lysates were harvested and diluted in TBS. Samples were applied to a Bio-Dot SF Microfiltration Apparatus (Bio-Rad) and blotted onto PVDF membrane that had been pre-soaked in TBS using a Welch Vacuum system (Model 2515) at 5 inHg pressure. Membranes were washed in TBS and then stained with Ponceau S solution and imaged prior to blocking with 5% milk in TBST for and immunodetection of PAR using either 10H anti-PAR antibody or Anti-pan-ADP-ribose binding reagent.

Boronate-IP of PARylated proteins Lysates were mixed with m-aminophenylboronic acid agarose for 1 hour at room temperature. Following this incubation, beads were washed twice in SDS wash buffer (1% SDS, 100mM HEPES (pH 8.5), 150mM NaCl) and twice in non-SDS wash buffer (100mM HEPES (pH 8.5), 150mM NaCl). Proteins were eluted from beads by boiling in 1 x sample buffer for 10 minutes and analyzed by western blot.

13C, 15N-labeling ( In vitro VSMC extracellular matrix withC,N-labeling ( Figure S3 A) Bovine VSMCs were cultured in a T-175 flask containing 25 mL complete Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) with addition of 10% fetal calf serum (First Link), 30 μg/ml L-ascorbic acid 2-phosphate (Sigma) and 10 ml/l L-glutamine-penicillin-streptomycin (200 mM L-glutamine, 10,000 units/ml penicillin, and 10 mg/ml streptomycin in 0.9% sodium chloride; Sigma). All supplements were filter sterilized (0.22 μm filter, Appleton Woods) before addition. After cells were confluent, labeled (U-13C, 15N) glycine (Cambridge Isotope Laboratories), (U-13C, 15N) lysine (Cambridge Isotope Laboratories) and (U-13C) glucose (Cambridge Isotope Laboratories) were added after filter sterilization (0.22 μm) to a final concentration of 60 mg/L, 292 mg/L and 4.5 g/L respectively in the complete medium. The culture was incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The culture medium with isotope-labeled supplements was renewed every 2 days. When cells produced a dense matrix which started to peel off the surface of the tissue culture flask, the medium was removed and the cells were washed twice with 10 mL phosphate buffered saline (1x PBS, Invitrogen).

Recovery of extracellular matrix Flasks containing matrix plus cells were placed in a freezer at −80°C for 24 hours and the cells were lysed by thawing the flasks at room temperature for 30 minutes. The debris produced by cell lysis was removed by repeated washes with PBS. The decellularized ECM was dislodged by gently swirling the flask in the presence of 20 mL PBS. The matrix collected in PBS was transferred to a fresh 50 mL centrifuge tube. The ECM was lyophilized overnight. Any adventitious DNA, RNA and PAR was removed by daily incubation with 40 mM aqueous MgCl 2 followed by thorough washing, for two days. The samples were stored at −20°C until NMR analysis.

13C, 15N-labeling ( In vitro fetal sheep osteoblast extracellular matrix withC,N-labeling ( Figure S3 B) Osteoblasts were cultured to confluence in T-175 flasks containing 25 mL BME complete medium. Labeled (U-13C5, 15N) proline (Cambridge Isotope Laboratories) and (U-13C2, 15N) glycine (Cambridge Isotope Laboratories) were added after filter sterilization (0.22 μm filter) to a final concentration of 46 mg/L and 30 mg/L respectively and 1g/L of U-13C-glucose (Cambridge Isotope Laboratories). The cultures were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The culture medium with isotope labeled supplements was renewed every 2 days until the cells and matrix began to detach from the culture flask, by which time enough ECM had formed for SSNMR. Samples from more than 20 batches using the final optimized protocol were prepared using isotope-enriched amino acids and similarly for samples incorporating U-13C-glucose and all characterized by SSNMR to ensure reproducibility of results.

Recovery of ECM The matrix was harvested after 9 days of culture, when the cells produce a dense matrix which started to peel off the surface of the tissue culture flask. The medium was removed and the cells were washed with 20 mL 1 x phosphate buffered saline (1X PBS). The flask was placed in a freezer at −80°C for 24 hours and the cells were lysed by thawing the flasks at room temperature for 30 minutes. The debris produced by cell lysis was removed by repeated washes with PBS. The decellularized ECM was dislodged by gently swirling the flask in the presence of 20 mL PBS. The matrix collected in PBS was transferred to a fresh 50 mL tube and centrifuged at 1200 rpm for 5 min at room temperature. The supernatant was poured off and the ECM dehydrated in an oven at 37°C overnight. The samples were stored at −20°C until NMR analysis. ECM of mouse tissue was used directly in SSNMR experiments without extraction, purification, or excessive processing where possible. Tissues used in each experiment are described below in the SSNMR section.