Calreticulin

The vesicle pellet obtained by use of the Total exosome isolation (TEI) reagent was lysed in 50 μl of RIPA buffer supplemented with complete protease-inhibitors (Roche Diagnostics, Mannheim, Germany) and PhosSTOP phosphatase-inhibitors (Roche Diagnostics). Lysates were sonicated twice (15 s) in a 4°C water bath. Protein concentrations were quantified using the Micro BCA^TM Protein assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Twenty five microgram of vesicles extracted from plasma or CM were for western blotting. Protein expression was assessed using antibodies against CD63, TSG101 (both 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), β-actin, vimentin (1:5000 and 1:500 respectively, Sigma–Aldrich, St.Louis, MO, USA), collagen type I (1:1000, Abcam, Cambridge, UK), and PDGFRβ (1:1000, GeneTex, Irvine, CA, USA). Calreticulin (1:1000, Cell Signaling Technology, Danvers, MA, USA) was used to verify absence of cell contamination. As positive control, a protein cell lysate of the HepG2 cell line was used.

Immunofluorescence staining was performed as previously described^{64 (link)}. Primary antibodies against anti-phospho-histone H2AX (Ser139/Tyr142) (1:100, Cell Signaling Technology, Cat 5438), calreticulin (1:400, Cell Signaling Technology, Cat 12238), and HMGB1 (1:400, Abcam, Cat ab18256) were used. Fluorescence-conjugated secondary antibodies (1:500, anti-rabbit, Dylight 488/594, Abbkine, Cat 23220/23430) and anti-fade fluorescence mounting medium with DAPI (ZSGB-BIO) were then used. The slides were examined with an FV1000 confocal microscope (Olympus) using FV10-ASW software v. 4.0 (Olympus). The fluorescence signals were analyzed using Image-Pro Plus software v. 6.0 (Media Cybernetics).

Antibodies against reticulon 4B (AB-163; Kinasource Ltd, Dundee, UK), reticulon 4A (ab62024; Abcam, Cambridge, UK), HA (MMS-101R-50; Covance, Princeton, NJ), FLAG (F7425; Sigma-Aldrich), calreticulin (2679S; Cell Signalling Technologies, MA) and β-actin (ab8227-50; Abcam) were used as primary antibodies. When indicated, rabbit anti-sheep bridging antibody (313-001-003; Jackson ImmunoResearch Labs Inc., West Grove, PA) was used. Secondary antibodies were Rhodamine Red-X (016-290-084; Jackson ImmunoResearch), Alexa 647 (A31571; Life technologies), Alexa 488 (A-11008; Life Technologies) and 1.4 nm nanogold-conjugated anti-rabbit antibody (Nanoprobes, Stony Brook, NY).

VEGF-A165 was obtained from Sigma-Aldrich (St. Louis, MO; V5765). SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas). Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).