SUPPORTING MATREALS. Methods and Materials

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1 SUPPORTING MATREALS Methods and Materials Cell Culture MC3T3-E1 (subclone 4) cells were maintained in -MEM with 10% FBS, 1% Pen/Strep at 37ºC in a humidified incubator with 5% CO2. MC3T3 cell differentiation was induced by replacing with an osteogenic differentiation medium containing 10 mm β-glycerophosphate, 50 μg/ml of ascorbic acid and 15% FBS. RAW264.7 cells were maintained in -MEM with 10% FBS, 1% Pen/Strep containing M-CSF (30 ng/ml) and RANKL (60 ng/ml). A parallel set of MC3T3-E1 or RAW264.7 cells were seeded and cultured in 12 well-tissue culture plate that were used to extract total RNA or proteins, respectively. Lentiviral packaging and transduction TN cells were transfected with Lenti-miR218-2 (PMIRH218-2PA-1) or scramble control plasmid (mir-c, PMIRH000PA-1) plus the ppackh1 Packaging Plasmid mix (System Biosciences) using the Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific). Supernatant was collected 48 hours post transfection and centrifuged at 3,000 rpm for 5 minutes to remove the cell debris. Clarified supernatant was concentrated 10-fold using the Lenti-X concentrator (Clontech), and virus titration was performed using the Global UltraRapid Lentiviral Titer Kit (System Biosciences). For viral transduction, Ocy454, IDG--SW3 or MC3T3 cells were seeded at a density of cells per well in a 24-wells plate and were grown for 12, 24 and 7 days prior to co-culture and/ or treatment experiments. Cells were then transduced with concentrated mir-218 or scramble lentiviral supernatant (M.O.I=4) in the presence of 5 μg/ml polybrene. Biological assays were performed 48 hours post transduction. Electron microscopy Negative staining of exosomes with uranyl acetate. Exosomes were isolated from Ocy454 cells and the pellet was suspended in PBS. Two-µl aliquots of exosomes suspended in PBS were mixed with 15 µl of 2.5% glutaraldehyde for 30min at RT. A 5-µl aliquot of fixed exosomes was deposited onto the formvarand carbon-coated 200 mesh copper grid (Electron Microscopy Sciences, Cat. No. FCF-200-Cu) and allowed to dry completely under a hood. Negative staining with 2% uranyl acetate was then performed as described previously (Mathias et al., 2009). Samples on grids were viewed using Hitachi H7000 (Tokyo, Japan) electron microscope at 75 kv. The electron microscope was equipped with an AMT Advantage HS digital camera (Danvers, MA, USA) and micrographs were recorded digitally. Immunogold labeling of exosomes. A 20-µl aliquot of exosomes was mixed with 20µl 2% paraformaldehyde for 1h at RT. A 5-µl aliquot of fixed exosomes was deposited onto the formvar- and carbon-coated 200 mesh copper grid and allowed to dry completely under a hood. Immunogold labeling was then performed as described previously (Mathias et al., 2009) with some modifications. The modifications included using PBS containing 0.1% gelatin and 0.1% bovine serum albumin (PBS+) with 5% horse serum for blocking and 1% horse serum in PBS+ for antibody dilutions. The marker for tetraspanin in exosome membrane was CD63 antibody raised in rabbits (SBI, Cat. No. EXOAB-CD63A- 1) diluted 1:100. Secondary anti-rabbit antibody was conjugated to 10-nm gold (Electron Microscopy

2 Sciences, Cat. No ) and diluted 1:25. Samples on grids were negatively stained with 2% uranyl acetate. Visualization of Exosome Uptake The Staining of Exosomes: Osteocytic exosomes were isolated as described above, and further purified by being dissolved in PBS and ultracentrifuged at 120, 000 g for 70 min. The exosomes were labeled with PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Sigma-Aldrich) according to the manufacturer s protocol, with minor modifications in the washing process 67. Briefly, the exosomes were re-suspended in PBS and then mixed 1 ml of Diluent C was added. As a control, 1 ml of Diluent C mixed with the same volume of PBS was used. Before being added to the exosomes and the control, 1 ml of Diluent C was mixed with 4 μl of PKH67 dye. The samples were mixed gently for 4 min before 2 ml of 1% BSA was added to bind the excess dye. The samples were then transferred to 300 kda Vivaspin filters (Sartorius Stedim Biotech GmbH, Goettingen, Germany) and centrifuged at 4,000 g. The sample were washed 3 times with 5 ml of PBS before being transferred to new 300 kda Vivaspin filters and washed twice with 5 ml PBS. Uptake of Osteocytic Exosomes: MC3T3 cells or RAW264.7 cells were pre-incubated with 25 g/ml of Texas red transferrin (Life technologies) for 30 minutes at 37 C in medium without FBS to label early endosomes ,000 MC3T3 cells or RAW264.7 cells were then mixed with 10 μg of the PKH67 labeled exosomes or the same volume of the PKH67-PBS control and incubated for 5 or 30 minutes at 37ºC. The uptake of exosomes by these cells was stopped by washing in cold 0.1% sodium azide PBS, followed by fixation in 4% formaldehyde for 15 min and washed twice with PBS before being mounted with Vectashield (Vector Laboratories Inc., Burlingame, USA). The binding of the exosomes to the MC3T3 cells or RAW264.7 cells was visualized with fluorescence microscope (Zeiss Axioplan 2, Carl Zeiss, Jena, Germany). In a parallel study, instead of using Texas red transferrin to label early endosomes, the MC3T3 cells or RAW264.7 cells were directly incubated with PKH67 labeled exosomes or the same volume of the PKH67-PBS control, and then the binding of the exosomes to these cells was visualized with fluorescence microscope with 3% 7-ADD (BD Biosciences) to label nuclei. Western Blot Analysis Proteins from cultured cells and purified exosomes were separated on a denatured SDS polyacrylamide gel before transfer to a polyvinylidene difluoride membrane (Bio-Rad). The blotting membrane was blocked with bovine serum albumin and incubated with various primary antibodies, followed by incubation with horseradish peroxidase-coupled anti-rabbit IgG (1: 5,000; Abcam, ab6721), and then visualized by enhanced chemiluminescence (GE Healthcare). The primary antibodies were goat polyclonal anti-cd63 antibody (1:1,000; System Bioscience, EXOAB-CD63A-1), rabbit polyclonal anti-sost antibody (1:200; Santa Cruz, sc ), mouse monoclonal anti-runx2 antibody (1:500; Santa Cruz, sc ), rabbit polyclonal anti-active -catenin antibody (1 g/ml; Millipore, ), mouse monoclonal anti-p-gsk3 Tyr 279/216 (1:1,000; Upstate, ), and rabbit polyclonal anti- β-tubulin antibody (1:5,000; Abcam, ab6046) as loading controls. Phosphorylation of GSK-3β at Tyr-216 in the Ocy454 cells is more ready to be detected than that of GSK-3 at Tyr For this reason, levels of phosphorylation of GSK-3β at Tyr- 216 were analyzed to reflect GSK enzyme activity in this study. For each protein, images from groups of different treatments were from the same blot to enable the results to be appropriately compared.

3 RNA Isolation and Analysis of Gene Expression by qpcr Total RNA was isolated using the mirvana mirna isolation kit (Invitrogen) according to the manufacturer s instructions. Briefly, the cultured cells were homogenized in 10 volumes of lysis, the homogenate was extracted with acid-phenol:chloroform and the aqueous phase was directly loaded onto a glass fiber filter cartridge column to further purify the RNA. Total RNA will then be eluted with 0.1 mm EDTA preheated at 95 o C. The quality of the RNA from exosomes and cells was assessed with an Agilent Bioanalyzer (Agilent Biotechnologies) using the pico and nano kits 52, 58, 59, 70, respectively. Sizing, quantification and quality control of RNA are performed on chips, using only 1µl of each RNA sample and a few reagents. The gel-dye mix used on the chip is prepared by first filtering the Agilent RNA 6000 gel matrix through a spin filter in a microcentrifuge for 10 minutes at 1500g. 1µl of the RNA 6000 dye concentrate is then added to the filtered gel matrix. The mixture is vortexed and subsequently spun for 10 minutes at room temperature at 13,000g. The gel-dye mix is loaded onto the wells of the chip (9µl in each well), followed by the RNA 6000 Nano Marker (5µl), the RNA ladder (1µl) and finally the RNA sample (1µl). For determination of mrna levels,1 µg of total RNA was used to generate a cdna library with a High Capacity cdna Reverse Transcription Kit (Applied Biosystems, Forster City, CA). For determination of mirna levels, cdna was synthesized from 1µg total RNA using the specific primers provided with the TaqMan mirna assays (Life Technology) according to the protocol provided by the manufacturer (Life Technology protocol # ). The RT primers for the mirna targets to be quantitated plus the ones for U6 snrna (normalizer) were pooled and diluted (1:100 final dilution). After 1:12.5 dilution of cdna libraries into water, mrna or mirna levels for specific mrnas or mirnas were determined by Taqman Assay On Demand probesets obtained from Applied Biosystems or SYBR quantitative PCR (StepOnePlus, Life Technologies) using an ABI 7500 real time PCR machine. Primer sequences are available upon request. Changes in gene expression were calculated using the 2- Ct method using 18S RNA or U6 snrna for normalization, and the control group as reference 69.

4 Fold change Supplemental Figures: 1.2 Exosomal mir * ** ** hours Supplemental Figure 1. Myostatin inhibited levels of osteocyted-derived exosomal mir-218 in a time-dependent manner. Ocy454 cells were differentiated for 12 days and then treated with 100ng/ml myostatin for 24, 48 and 72 hours, followed by extraction of total RNA from exosomes isolated form Cyc454 cells. Levels of mir-218 in exosomes produced by Ocy454 cells were determined by real-time PCR. Data shown are mean values ± SE for 3 separate determinations. * P<0.05 and ** P < Supplemental Figure 2. Repaid uptake of osteocytic exosomes by MC3T3 cells.10 μg of the PKH67-labelled osteocytic exosome were added into per 75, 000 MC3T3 cells and incubated at 37ºC for 5 minutes. The uptake of the fluorescently labeled exosomes by MC3T3 cells was detected with confocal fluorescence microscopy. PKH67 (green) was used to label the exosomes. Texas red transferrin (red) was used to detect the early endosomes of the MC3T3 cells. PKH67 exosomes (green) were rapidly internalized into early endosomes labeled with Texas red transferring (yellow indicates co-localization of green and red). Arrow indicates co-localization; Asterisk indicates no co-localization.

5 Supplemental Figure 3. Repaid uptake of osteocytic exosomes by RAW264.7 cells.10 μg of the PKH67-labelled osteocytic exosome were added into per 75, 000 RAW264.7 cells and incubated at 37ºC for 5 minutes. The uptake of the fluorescently labeled exosomes by RAW264.7 cells was detected with confocal fluorescence microscopy. PKH67 (green) was used to label the exosomes. Texas red transferrin (red) was used to detect the early endosomes of the RAW cells. PKH67 exosomes (green) were rapidly internalized into early endosomes labeled with Texas red transferring (yellow indicates co-localization of green and red). Arrow indicates co-localization; Asterisk indicates no co-localization Supplemental Figure 4. No change of RANKL mrna expression in the osteocyte-derived exosomes after myostatin treatment. As shown in Supplemental Figure 1, Ocy454 cells were differentiated for 12 days and then treated with 100ng/ml myostatin or vehicle for 48 hours, followed by extraction of total RNA from Ocy454 parent cells or the isolation of exosomes released form Cyc454 cells. Levels of RANKL in exosomes produced by Ocy454 cells were determined by real-time PCR. Data shown are mean values ± SE for 3 separate determinations. NS, statistically no significance.