Fx 5000 tension system

Human umbilical endothelial cells (HUVECs) were purchased from the American Tissue and Cell Culture (ATCC, MD, USA) and cultured in an F-12K cell culture medium containing 10% fetal bovine serum (FBS), 1% penicillin, and 0.03 mg/mL endothelial cell growth supplement. After 5 h stabilization, cells underwent 12 or 24 h mechanical stretch, and then they were collected for subsequent experiments. Control cells were cultured under the same condition in collagen I-coated Bio flex 6-well plates without mechanical stretch.
For a mechanical stretch, HUVECs were switched to serum-free DMEM for 5 h, followed by a cyclic mechanical stretch for 24 h with the following parameters: 10% stretching, 1 cycle/s using the FX-5000 Tension System (Flexcell, NC, USA). In addition, cells were incubated with 50 µM latrunculin A (actin cytoskeleton inhibitor) or/and nocodazole (microtubule inhibitor) that were dissolved in dimethyl sulfoxide (DMSO). DMSO administration was used as the control and then subjected to mechanical stretch treatment.

The protocol of astrocyte stretch injury model was similar to those described previously [22 (link)]. Equiaxial stretch (20% strain, 1.0 Hz frequency) was applied to cultured astrocytes for 1 h, by a Flexcell^® FX-5000™ Tension System (Flexcell, USA) with specific designed 6-well plates (BioFLEX^®). The culture media of astrocytes in stretch injury group and control group were collected at this time and will be used as conditioned medium. Again, to clarify the effects and signaling relationship of S100B, RAGE, ADAM17 on stretch injury, ONO-2506 (100 µM) [18 (link)], FPS-ZM1 (100 nM) [23 (link)] or TAPI-1 (50 µM) [24 (link)] were, respectively, applied for 6 h after stretch injury. To further verify the effect of S100B on astrocyte injury, exogenous S100B (1 µM) [8 (link)] was directly administrated to normal astrocytes for 6 h. The cell viability and relevant parameters were detected as indicated.

HUVECs were plated on collagen I - 0.2% gelatine-coated Bioflex plates (BF-3001C, Flexcell International Corporation). Gene knockdown was preformed as previously described. Cells were incubated in transfection media for 24 hr, and allowed to recover in fresh complete media for 4 hr. Afterwards cells were incubated for 24 hr in serum starvation media (0,1%BSA in EBM2 pure media) to form a confluent, quiescent monolayer. Cyclic stretch (0.25 Hz, 15% elongation) was then applied for 24 hr using a Flexcell FX-5000 Tension System. Control cells were placed in the same incubator but not on the Flexcell device (static conditions). EdU pulsing was performed after 20 hr of the 24 hr stretch period. At the end of the experiment cells were fixed in 4% PFA and EdU staining was performed according to the manufacturer’s protocol (Click-It EdU C10340 Life Technologies). Nuclei were labelled with DAPI. Three regions of interested were acquired per sample in a Carl Zeiss LSM700 scanning confocal microscopes (Zeiss, Germany). Quantification of proliferation was done using a CellProfiler pipeline. Percentage of S phase cells was determined as percentage of EdU positive nuclei over the total number of nuclei.

CI-muMECs (murine microvascular endothelial cells; #INS-CI-1004, InSCREENeX) were cultured in gelatin-coated culture flasks (Thermofisher Scientific) in high-glucose DMEM containing 20% FBS, 0.5% endothelial cell growth supplement from bovine neural tissue, 1% sodium pyruvate, 1% non-essential amino acids and antibiotics (all Sigma-Aldrich). For experiments, cells were plated in collagen type I BioFlex plates (Flexcell^® International) and transfected with control or zyxin siRNA (Qiagen) using MATra siRNA reagent (IBA) according to the manufacturer’s instructions. Following 72 h of culture, transfected cells were either exposed to cyclic stretch (15% elongation, 0.5 Hz) using a Flexcell^® FX-5000™ Tension System or incubated under static conditions for 6 h. Cells were then harvested and processed for protein analysis or RNA isolation.

The rate of release of exosomes is influenced by biomechanical forces acting on the tumor cells, and to quantify this, an experimental setup depicted in Figure 1 was used. The murine TNBC cell line 4T1.2 (an aggressive clone derived from 4T1) was obtained from Dr. Robin L. Anderson's laboratory (Peter McCallum Cancer Center, Australia). 4T1.2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 10 mM HEPES (MP Biomedicals, Santa Ana, CA). Prior to the exposure to tensile strain, the 4T1.2 cells were stained with the lypophilic dye PKH26 Red Fluorescent Cell Linker (Sigma, St. Louis, MO), per the manufacturer's instructions. 2.5 × 10⁵ 4T1.2 were seeded on collagen coated 6 well UniFlex culture plates (Flexcell International Corporation, Burlington, NC) and cultured to confluence. Once confluent, the media was changed to exosome depleted growth media and the plates were subjected to 10% uniaxial oscillatory strain at 0.3 Hz for 48 h, 10% constant strain for 48 h, or no strain for 48 h using a FlexCell FX-6000 or FX-5000 Tension System.