Imagelock plate

Cells were seeded into a 96-well ImageLock^TM plate (Essen Bioscience, Ann Arbor, Michigan) then appropriate wells induced by the addition of 50 ng/ml doxycycline to culture media after 24 h. After a further 48 h the Essen Bioscience WoundMaker^TM was used to wound the confluent monolayer of cells, and media replaced after washing. Cellular density in the wound was measured with two hourly images collected with the IncuCyte Zoom (Essen Bioscience). To measure invasion cells were grown to confluence in a 96-well plate coated with 1 mg/ml Basement Membrane Matrix (BMM; Corning Inc, Corning, NY). Cells were induced with doxycycline as above and a wound created as above. After wounding, 50 µl of 3 mg/ml BMM in culture media was plated over the cells. The BMM was gelled at 37 °C for 1 h then covered with culture media +/− doxycycline. Images were collected every 2 h for 96 h to measure wound cell density.

Twenty-four hours prior to transfection, cells were plated in a 96-well ImageLock^TM plate (Essen BioScience Ltd.). The next day the cells were transfected with selected miRNA mimics/inhibitors or appropriate controls. At 24 h post-transfection, all 96 wells were scratched simultaneously in the central axis of the individual wells using the WoundMaker^TM (Essen Bioscience Ltd.). A live-cell imaging system, IncuCyte (Essen BioScience Ltd.) was used to automatically monitor the kinetics of cell migration every 2 hours for a total duration of 26 h during which cells migrate from the scratch edges into the wound area.

Wells of a 96-well imageLock plate (4379, Sartorius) were first coated with 100 µg/ml of Matrigel (Sigma-Aldrich, E609-10 ml). After 1 hr, MDA-MB-231 cells (5.5 × 10⁴) were seeded 24 hr prior to the assay. A wound was then performed in the cell monolayer with the WoundMaker and a new layer of Matrigel (800 µg/ml, 2.55 mm thickness) was deposited onto cells for 1 hr at 37°C to allow polymerization. The top of the cells was covered with complete medium and the invasion potential of cancer cells was evaluated and quantified using IncuCyte ZOOM-based time-lapse microscopy.

10³ breast cancer cells were seeded onto a 96-well ImageLock plate (Sartorius, 4379) and imaged for 24 hr using the IncuCyte ZOOM-based time-lapse microscopy. The acquired time-lapse images were treated with a manual tracking plugin using the ImageJ software. About 100 cells/condition were followed for 30 min in order to determine the accumulated distance and their velocity.

Transfected cells were seeded in 96-well ImageLock plate (4379, Sartorius, Gottingen, Germany) at a density of 3 × 10⁴ cells/well. Wounds were created by IncuCyte WoundMaker Tool (4563, Sartorius, Germany) and cell debris were removed by washing with 1 × PBS. Cells were monitored and analyzed by IncuCyte Live-Cell analysis System (Sartorius, Germany) for up to 3 days.

For the determination of cell proliferation, 1 × 10³ Ctrl or HDLBP KO A549 cells were seeded in 96-well plates. Cell confluency was monitored for 5 days using an IncuCyte S3 system (Sartorius) with 4× magnification for a whole well scan. Confluence masks were generated using the IncuCyte analysis software. Cell viability and DNA content were determined using CellTiter Glo (Promega) supplemented with 1/2000 SYBR Green I (Thermo Fisher) according to the manufacturer’s protocols. Luminescence and fluorescence intensities were measured in a GloMax microplate reader (Promega). For the scratch wound migration analysis, 2.5 × 10⁴ Ctrl, HDLBP KO A549 cells and stable A549 cells expressing HDLBP (±doxycline) were seeded in a 96-well ImageLock plate (Sartorius) for 24 h. The wound areas were created on confluent cell monolayers using a 96-well WoundMaker (Sartorius). Wound healing was monitored using an IncuCyte S3 system at 10x magnification and 1 h intervals. Confluence and wound masks were generated and quantified using the IncuCyte analysis software.

A549 lung cancer cells (4 × 10⁴) were plated in a 96‐well ImageLock plate (Sartorius) and cultured in a 20% O₂, 5% CO₂, and 37°C incubator for 24 h. Then, the 96‐pin IncuCyte WoundMaker was used to create precise and reproducible cell‐free zone in each well. Cells were washed with PBS and then incubated with serum‐free conditioned media collected from drug‐induced senescent fibroblasts for 24 h in the IncuCyte imaging machine. Images were taken every 2 h and analyzed by the IncuCyte software.