E plate 16

Endothelial cell proliferation was measured by using an xCELLigence RTCA instrument (Roche Diagnostics) and E-plate 16 (a modified 16-well plate, Roche Diagnostics). The E-plate 16 was coated with 0.1% gelatin, loaded with 100 μL cell-free medium, and left in a tissue culture hood for 30 min to reach equilibrium. The E-plate 16 was placed into the RTCA instrument to measure the background impedance. Thereafter, 100 μL cell suspensions with fewer than 3500 cells were added into each well of the E-plate 16, which was then placed in a tissue culture incubator for 30 min to allow cells to settle down before being measured by the RTCA device. The impedance value of E-plate 16 was automatically monitored every 15 min. FGF2 was added at 100 ng/mL.

An xCELLigence Real-Time Cell Analyzer (Acea Biosciences/Roche Applied Science) was used to assess barrier function of HUVEC monolayers. HUVEC 48 h post-transfection were seeded at a density of 60,000 cells/well of the E-plate (E-plate 16, Roche Applied Science), then electrical impedance readings acquired every 2 min for 24 h. Cells attached within the first 7-10 h and were fully confluent by 24 h. Results are reported at 24 h as the percent change in cell index calculated using the following formula: (Cell Index_siRNA−Cell Index_NT)/ABS(Cell Index_NT).
For drug treatments, HUVEC 48 h post-transfection were seeded to E-plates as above and grown to confluence for 24 h, then media was replaced with drugs and readings taken every 30 s. Drug concentrations were as follows: for contractility assays, 0.5 U/ml thrombin (Sigma-Aldrich, T7201-500UN) at 37°C for 15 min. For PI3K activation assays, 20 µM 740Y-P (MedChemExpress, HY-P0175) at 37°C for 1 h.

Possible cytotoxicity of the selected compounds on cell lines used in the invasion assays were evaluated using the xCELLigence System (Roche, Basel, Switzerland). The system monitors cellular events in real time by measuring the electrical impedance (expressed as cell index) generated by cells attached to the bottom of wells with integrated electrodes. 150 μl of MCF-10A neoT, U-87 MG, MMTV-PyMT (5×10⁴ cells/ml) and LPB (3.3×10⁴ cells/ml) cell suspension were seeded in the wells of an E-plate 16 (Roche) according to the manufacturer's instructions. After seeding, the CI was monitored every 15 min. After ∼10 h (MCF-10A neoT and MMTV-PyMT), 14 h (U-87 MG) or 24 h (LPB), when the cells were in their log phase of growth, 50 μl of the compound or 0.1% DMSO was added, and the experiment allowed to run for 72 h. Once every 24 h the medium was replaced with fresh medium containing the inhibitor or suitable control to prevent cell death due to medium depletion. Compounds and their concentrations were: nitroxoline (5 μM) and CA-074 (5 μM) for all cell lines other than MCF-10A neoT cell line, where nitroxoline was used at 2.5 μM. All measurements were performed in quadruplicate.

Barrier function was assessed using the xCELLigence Real-Time Cell Analyzer (RTCA, Acea Biosciences/Roche Applied Science) to measure electrical impedance across HUVEC monolayers seeded onto microelectrodes. HUVEC were seeded to confluency on the microelectrodes of the E-plate (E-plate 16, Roche Applied Science). Electrical impedance readings were taken every 2 min for 5 hr. The percent change in cell index was obtained at the 5 hr timepoint using the following formula: (Cell Index_SUN1-Cell Index_NT)/ABS(Cell Index_NT).

Lipofectamine 2000 transfection reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA), while TRIzol reagent, Takara reverse transcription kit, Takara real-time PCR kit, Dulbecco’s modified Eagle’s medium (DMEM; high glucose), fetal bovine serum (FBS), trypsin-EDTA, miR-449a reverse transcription and PCR primer, U6snRNA reverse transcription and PCR primer, has-miR-449a mini and has-miR-449a inhibitor were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, Guangdong, China). Mouse monoclonal anti-rabbit CDK6 and rabbit monoclonal anti-human β-actin rhesus antibodies were purchased from Abcam Co. (Cambridge, UK), and E-Plate 16 was obtained from Roche (Basel, Switzerland). The MGC-803 cell line was supplied by the Department of Medical Experiments, Guangzhou General Hospital of Guangzhou Military Command.