Supplementary Material: Suppression of angiogenesis by atmospheric pressure plasma in human aortic endothelial cells

Bomi Gweon1+, Hyeonyu Kim1++, Kijung Kim2, Mina Kim1, Eunyoung Shim1, Sunja Kim3, Wonho Choe2,a), Jennifer H. Shin1,4,a)

1Department of Mechanical Engineering,Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea

2Department of Physics, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea

3Department of Physics, Dong-A University, 37 Nakdong-Daero 550beon-gil,Saha-gu, Busan 604-714, Republic of Korea

4Graduate school of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea

+ Present address: School of Public Health, Harvard University, Boston, Massachusetts 02115, USA

++ Present address: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge 02139, Massachusetts, USA

a) Authors to whom correspondence should be addressed. Electronic mail: ,

1. Cell culture and APP treatment

2.1 Cell culture

Human Aortic Endothelial cells (HAECs, Lonza #CC-2535, passage 7 - 9) were cultures in Endothelial cell growth medium-2 (EGM-2, Lonza #CC-3162) and maintained in humidified 37°C incubator (5% CO2 and 95% of air). HAECs were routinely sub-cultured using trypsin-EDTA (Lonze #CC-5012) and trypsin neutralization solution (TNS, Lonza #CC-5002). Cells were seeded on a glass bottom petri-dish (ϕ = 35 mm, SPL #100351) and the glass surface (22 x 22 mm) was coated with 0.5ug/ml fibronectin (Gibco, 1 h) before cell seeding. HAECs were seeded by 1800 cells/cm2in density and incubated for 1 day before atmospheric pressure plasma (APP) treatment. The petri-dish with cells had its medium removed and refilled (2.5 ml)right before the plasma treatment to keep the medium height (≈ 3 mm) in consistent level and to have fresh medium for the following 24 h and 72 h incubation.

2.2 Plasma treatment

Fig. S1 Schematic of the plasma applicator [Petri dish image from SPL life science]

Schematic of the atmospheric pressure plasma (APP) applicator is shown in Fig. S1. 970 V and 50 kHz were chosen for the discharge condition and 2 slpm (standard liter per minutes) of helium gas was supplied. APP was treated for 20 min in room temperature and then incubated for 24 h or 72 h in the environment controlled incubator (37°C, 5% CO2).

2. Assays and analysis

2.1 Proliferation assay

To measure the proliferation rate, both in APP treated and control sample, cells were allowed to grow in incubator up to 72 h. Not like the normal culture condition, where we normally change medium every 2 days, culture medium was not change for 72 h. Cellular population change was observed by the phase contrast images of the control and the plasma treated samples. Six to seven images were taken right after plasma treatment, after 24 h and after 72 h of the plasma treatment and the cell number were measured manually with ImageJ tool (National Institute of Heath, Bethesda, MD, USA).

2.2 Flow cytometry

To analyze the cell cycle, HAECs were trypsinized from glass surfaceat each time point (0 h, 24 h, 72 h) and suspended in hypotonic solution (0.2% Triton X-100, 0.1 mM EDTA,1 mM Tris–HCl(pH 8.0), 3.4 mM sodium citrate) to be stained with propidium iodide (5 lg/ml). DNA contents were analyzed using flow cytometry(FACS Canto II, Becton Dickinson, San Jose, CA, USA). The proportions of cells in G0/G1, S, and G2/Mphase weredetermined usingFlowJo software (Tree Star, Ashland, OR,USA).

2.3 Real time - PCR

Total RNA wasobtained from control and APP treated HAECs withNucleoSpin RNAII kit (MN, Düren, Germany) following the protocol provided by manufacturer. Using iScriptTM cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), RNA about 1μg of its amount was reversely transcribed to cDNA. Polymerase chain reaction (PCR) was performed using iQ SYBR Green supermix (Bio-Rad Laboratories) in a real-time PCR detection system (Bio-Rad CFX96,Bio-Rad Laboratories). The reaction protocol were set first on 95°C (3 min) and then40 cycles of 95°C (15 sec) and 60°C (20 sec). We used p21 and p53primersfor our real-time PCR analysis:p21 reverse 5’-GGCGTTTGGAGTGGTAGAAA-3’, p21 forward 5’-GGACAGCAGAGGAAGAC-3’, p53 reverse 5-GAATGTCAGTCTGAGTCAGGC-3, p53 forward 5’-CCAGCCAAAGAAGAAACCAC-3’, Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reverse 3’-GAAGATGGTGATGGGATTT-5’, GAPDH forward 5’-GAAGGTGAAGGTCGGAGTC-3’.

2.4 Immunofluorescence imaging

Cells were washed with PBS and fixed by 3.7% formaldehyde (Sigma, St Louis, MO, 20 min). After fixing, cells were washed with PBS (Phosphate buffer saline, Gibco) three times and then cells were permeabilizedby 0.2% Triton X-100 (Sigma,15 min).After another three times washing, cells were blocked by the blocking solution 3% bovine serum albumin (BSA/PBS, Gibco, 60 min).Primary antibody of phosphorylated form of histone 2AX (H2AX) at serine 139 (γ-H2AX, 1:400; Cell signaling, Danvers, MA, USA) were loaded and kept in 4°C overnight. Again the cells were washed with PBS three times, and cells were incubated for 1 h with rabbit Alexa Fluor 488-conjugated secondary antibodies (1:200; Molecular Probes, Eugene, OR, USA) in blocking buffer. Also, to see the cell shape change and the cytoskeletal re-arrangement, actin stabilization agent Alexa 568-phalloidin (1:50; Molecular Probes) was used. For the nuclei staining 4', 6-diamidino-2-phenylindole (1:50000; Molecular Probes, 3 min) was used. Vectashield® (Vector Laboratories, Burlingame, CA, USA) mounting solution was used to minimize photo-bleaching.The fluorescence image was taken with confocal microscope (LSM710Carl Zeiss, Germany) and inverted fluorescence microscope (Zeiss LSM 510).

2.5 Migration assay

HAECs were treated by plasma for 20 min in room temperature and were incubated in 37o for 24 h. We took the cell images for 2 h in 5 min interval to track down the cell path. The time sequential image stack was analyzed by ImageJ tool (National Institute of Heath, Bethesda, MD, USA) to measure the motility of HAECs. To compare the cellular motility between plasma treated cells and control one, total path lengthwas obtained for both cases.

2.6 Tube assay

HAECs were treated by plasma for 20 min in room temperature and were incubated in 37oC for 24 h. After 24 h, cells were trypsinized and these collected HAECs were transferred to Matrigel(BD Matrigel™ Basement Membrane Matrix, BD Biosciences #356234) coated 48 well to test the tube formation ability. Per well 1x105 cells were seeded. For the control cells same protocol was adopted. For this analysis it was inevitable to detach the cells from the original glass substrate to a new gel. To keep the plasma induced effects persistent, we usedplasma treated media after re-plating on Matrigel.