Cell cultures
All used cell lines were obtained from ATCC, and mycoplasma was regularly tested. The list of cell lines included mice endothelial cells MS1, human breast cancer-derived MDA-MB-231, Cal51, MCF7, and BT549 cell lines, human osteosarcoma-derived U-2-OS cell line, human cervical cancer-derived SiHa and HeLa S3 cell lines, human prostate cancer-derived PC3 cell line, human lung cancer-derived A549 cell line, and human primary foreskin fibroblasts BJ. All cell lines were cultivated in DMEM (Lonza) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich). Fetal serum for MS1 cell line cultivation was thermally inactivated (56°C, 30 min.) before addition to media.
Determination of cellular elongation index
The determination of cell elongation was carried out using image analysis 22. Cells in microphotographs in the transmitted light mode of observation were localized by the "Analyze Particles" function in ImageJ 23. An ellipse was circumscribed to each cell. The elongation index was calculated as the ratio of the major to the minor axis of such an ellipse.
Experimental workflow for the shear stress generator usage
All tested cell lines were seeded on a 150 cm2 cultivation flask for 24 hours before applying continuous fluid shear stress. Seeding numbers were optimized to reach approximately 80% confluency at the beginning of the experiment. The cultivation flask was located on the clip-like platform horizontally, and the device was placed in a standard cell culture CO2 incubator. Cultivation parameters were 37°C, 5% CO2, and 100% humidity. The device controller was set to reach rotor oscillations (i.e., flask movement) of ± 35° within 2.47s, which corresponds to fluid shear stress mean value 1.8 dyne/cm2, median 1.4 dyne/cm2, and range 0 – 10.5 dyne/cm2. Depending on the experiment, the cells were exposed to fluid shear stress conditions for 18, 24, 48, or 72 hours. Adherent cellular fractions for analysis of MS1 cells were harvested by trypsinization.
Quantitative polymerase chain reaction (qPCR)
Total mRNA was extracted using the Qiagen extraction kit according to the manufacturer's instructions, and the extraction yield was quantified by Nanodrop (Implen). Reverse transcription to obtain cDNA was performed according to the protocol previously described 24. qPCR was performed on LightCycler 480 Instrument II (Roche). Master mix for qPCR (total volume 12,5 µl) was prepared according to the recommended protocol for Platinum Taq DNA Polymerase used for PCR reaction (Invitrogen, cat.no 15966005). Eva Green (Biotium) intercalation chemistry was used to detect fluorescence signals. Primers used for qPCR were custom synthesized (Eurofins): VCAM-1 fw: 5´-TCTTACCTGTGCGCTGTGAC-3'; rw: 5´-GACCTCCACCTGGGTTCTCT-3', ICAM-1 fw: 5´-CTTCCAGCTACCATCCCAAA-3', rw: 5´-CTTCCAGGGAGCAAAACAAC-3', ACTB fw: 5´-GCGCAAGTACTCTGTGTGGA-3'; rw: 5´-CCGGACTCATCGTACTCCTG -3'. Each reaction was performed twice using 1 µl of cDNA. qPCR cycling parameters were denaturation at 95°C for 10 min., 45 cycles of amplification at 95°C for 15 s, 60 °C for 20 s, and 72°C for 25 s. Relative quantification of gene expression was calculated using the 2-ΔΔCp method. The graphical processing and statistical significance calculation were performed in GRAPHPAD Prism 8.0.1.
Western blot analysis
Cells exposed to fluid shear stress were lysed directly into 1x Laemmli sample buffer. The proteins in the samples were quantified with Bradford assay (ThermoFisher Scientific) according to the manufacturer's protocol. An equal amount of proteins were separated on commercial gradient 4-15% Mini Protean TGX precast gel (BIO-RAD) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine milk in Tris-buffered saline containing 0.1% Tween 20 for 30 minutes at room temperature. Membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 h at room temperature. Secondary antibodies were visualized by ELC detection reagent (ThermoFisher Scientific). Primary antibodies used in this study: SMC1 (Abcam ab9262; 1:2000), p-Akt(ser403)(Cell Signaling 9271S; 1:1000), Akt (Cell Signalling 9272S; 1:1000), p-JNK (Santa Cruz sc-6254;1:250), JNK (Santa Cruz sc-7345;1:250), p44/42-MAPK (ERK1/2)(thr202/tyr204) (Cell Signaling 4370S; 1:500), p44/42-MAPK (ERK1/2)(Cell Signaling 4696S; 1:1000). Secondary antibodies used in this study: goat-anti mouse IgG-HRP (GE Healthcare; 1:1000) and goat-anti rabbit (GE Healthcare; 1:1000).
Colony formation assay (CFA
Cells released by continuous shear stress were collected and centrifuged. Only live cells were counted based on trypan blue staining (Sigma Aldrich). CFA was performed by seeding 300 cells per well of 6-well plates and cultivated under standard cultivating conditions. The cells were grown for eight days until discreet colonies were visible in a microscope. Fixation of colonies was done with 70 % ice-cold ethanol, and colonies were further stained with crystal violet (Sigma Aldrich). After washed with H2O and air-drying, the colonies were covered with edible white powdered sugar to increase the contrast. Wells were scanned using a table scanner, and colonies were counted with custom-made software analysis. The graphical processing of obtained data and statistical significance calculation were performed in GRAPHPAD Prism 8.0.1.
Flow cytometry analysis
Cells (0,5 .106) were fixed with cold 4% formaldehyde for 15 minutes. After fixation, the cellular suspension was resuspended in PBS+ 5 % FBS +2mM EDTA with 0.5 µg/ml DAPI. Samples were analyzed using a BD FACSverse flow cytometer.