Vegfr2

The cells were fixed in 4% paraformaldehyde for 15 min. Cells were incubated with primary anti-VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MHY-11, P2X7, P2Y1, P2Y2, and P2Y11 (Santa Cruz Biotechnology, CA, USA) diluted in a ratio of 1 : 100 in antibody dilution buffer containing 1% BSA and 0.2% Triton-X-100 in PBS at 4°C overnight. After rinsing with PBS 3 times, cells were stained with FITC-labeled anti-goat or rabbit antibody, respectively (1 : 100) (Southern Biotech, AL, USA), at RT for 60 min. Cell nuclei were stained with DAPI (Sigma, MO, USA), and the cell cytoskeleton was labeled using rhodamine (1 : 2,000) (Life Technologies, CA, USA). After washing with PBS, fluorescent signals were analyzed with an Axio Observer D1 fluorescence microscope (Carl Zeiss, Germany) or a FW300 confocal fluorescent microscope (Olympus, Japan), respectively.

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).

Western blotting was performed with a SDS-PAGE electrophoresis system. Briefly, protein samples were re-suspended in sample buffer (containing 10% β-mercaptoethanol) and electrophoresed on a 10% SDS-PAGE. Protein were transferred electrophoretically onto a polyvinylidene fluoride (PVDF) membrane and nonspecific binding sites blocked by immersion of membrane into 5% - milk. The membrane was incubated with primary antibodies against Axl, Gas6, ICAM-1, VCAM-1 and VEGFR2 (Santa Cruz, CA, USA), p-Akt, Akt, p-44/42 MAPK, 44/42 MAPK (both from Cell Signaling Technology, Danvers, MA) and GAPDH antibody (Novus Biologicals, Littleton, CO) was used as loading control. A horseradish peroxidase-conjugated mouse anti-goat, goat anti-mouse, goat anti-rabbit antibody (purchased from Jackson, USA) was then added as secondary antibody. Protein was detected by Luminata Classico Western HRP Substrate (Millipore, Bedford, MA). Semi-quantification of relative protein expression was analyzed with Image J.

Cells were lysed in 0.5% Nonidet P-40 lysis buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, Immobilon-P (Millipore Co., Milford, MA, USA). Membranes were blocked with Tris-buffered saline-Blotto/Blotto B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour and subsequently incubated overnight with antibodies directed against α-CaMKII, p-α-CaMKII, p-CREB, CREB, p-c-Jun, c- Fos, Lamin B1, HIF-1α, VEGFR-1, VEGFR-2 or β-actin (Santa Cruz Biotechnology and Cell Signaling Technology, Beverly, MA, USA). Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Pittsburgh, PA, USA) [14 (link)]. Band density was measured using ImageJ software and normalized to β-actin.

Standard western blotting of whole cell lysates was performed with antibodies for Stat3, Stat3 phosphorylated at Tyr705 (Cell Signaling Technologies), VEGF, VEGFR2, and phosphorylated VEGFR2 (Santa Cruz Biotechnology). β-actin (Sigma) was used as a control for loading.

Acrylamide, bovine serum albumin (BSA), bromophenol blue, 7,12-dimethylbenz[a]anthracene (DMBA), hydroxyurea, 2-mercaptoethanol, sodium dodecyl sulphate (SDS) N,N,N′,N′ - tetramethylene diamine (TEMED) and Trizol were purchased from Sigma Chemical Company, St. Louis, MO, USA. Astaxanthin was procured from Bio-Real, Sweden. DMEM-F12 medium, antibiotic solution consisting of penicillin and streptomycin and Alamar blue were from HiMedia Labs, Mumbai, India. Fetal bovine serum of South American origin was from GIBCO, Invitrogen, NY, USA. Power SYBR Green PCR master mix was obtained from Applied Biosystems, California, USA. Antibodies for IL-6, GAPDH, Cyclin D1, PCNA, p21, MMP-2, MMP-9, TIMP-2, RECK, VEGF, VEGFR2, HIF1α, were purchased from Santa Cruz Biotechnology, USA. pJAK-2^tyr1007/1008, JAK-2, pSTAT-3^tyr705, STAT-3 and histone (H2B) antibodies and BrdU, STAT-3^tyr705, total Cyclin D1 and pVEGFR2^tyr1175 ELISA kits were from Cell Signaling Technology, USA. CD-34 antibody was purchased from Novocastra, Germany. Matrigel was from BD Biosciences, USA. All other reagents used were of analytical grade.

Tissues were fixed in 4% PFA, embedded in paraffin, and sectioned at 4–7 µm thickness. Immunostainings were performed using the following antibodies: α-SMA (1:500, Sigma-Aldrich, Cat. no A5228), CD31 (1:50, Dako, Cat. no M0823), VEGFR-1 (1:250, Santa Cruz Biotechnology, Cat no sc-31173), VEGFR-2 (1:250, Santa Cruz Biotechnology, Cat. no sc-6251) and VEGF-A (1:500, Santa Cruz Biotechnology, Cat no sc-7269). Photographs were taken with Olympus AX70 microscope (Olympus Optical, Tokyo, Japan). For whole-mount tissue imaging, 1 mm thick longitudinal sections were cut and stained with CD31 (1:100, Dako, Cat. no M0823) and α-SMA conjugated with Cy3 (1:500, Sigma-Aldrich, Cat. no C6198). For CD31, goat anti-mouse Alexa 488 (1:500; Invitrogen, Cat. no A-11001) was used. Imaging was performed by Nikon A1R multiphoton microscope (MPLSM) and LSM700 Zeiss confocal microscope (LSM).