Woundmaker

Cells were grown to a semi confluent monolayer and were mechanically scratched (wound) using a standard 200 μL pipette tip. Suspension cells were washed away with media. Along the scratch, prefixed points were selected for representative photographs at 0 h and 24 h after initialization of the wound using a phase-contrast microscope.
Scratch assay was also performed using the Incucyte Live Cell SX5 system (Sartorius Canada, ON) as specified in manufacturer’s instructions. Briefly, 786-O cells were transduced with SMOC2 (vSMOC2) or empty vector, cultured and plated at different densities in triplicate, in an ImageLock 96 well plate. Cells were serum starved for 2 h before scratches were made in the monolayer of cells. A wound maker (Sartorius Canada, ON) was used to create identical scratches in the evenly distributed population. Plate was photographed every hour for 43 h. Data was analyzed with the Incucyte software. This system measures scratch closure in real time and automatically calculates wound confluence and density.

2 × 10⁴ cells were seeded into 96‐well plates to form a monolayer after 8 h of incubation. The monolayer was then lined out using Woundmaker (Sartorius, Göttingen, Germany), with an even gap in the middle. The cells were washed in PBS (Shanghai BasalMedia Technologies Co., Ltd., Shanghai, China) before being cultured in RPMI‐1640 with 5% FBS (Gibco) for 48 h at 37°C in an incubator. Images were taken every 2 h in the middle of the well. The IncuCyte S3 platform (Sartorius) was used to measure the wound distance.
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Cells were plated and treated with recombinant WNT5B, LGK‐974 or MTX on Corning 96‐well flat‐bottomed plates. The growth rate was assessed using an IncuCyte (Sartorius) to image cells every 2−4 h until cells reached confluence.
Cell migration was assessed by wound healing. Cells were plated and allowed to grow to confluence and subjected to a single scratch across the surface of the cell growth area using a wound maker (Sartorius). Cells were washed, placed in the IncuCyte, and imaged every 2 h for 24 h. The wound closure rate was analysed as a measure of migration in the adherent population of cells.

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.

HCT116 and LoVo cells were seeded at a density of 5 × 10⁴ cells/well (100 µL/well) in each well of the Image-lock 96-well plate (Sartorius, Göttingen, Germany). The cells were allowed to settle at ambient temperature for 10 min, and then the plates were kept in a 37 °C incubator with 5% CO₂ overnight. The next day the Image-lock plate was carefully removed from the incubator, and a 96-well Wound-maker (Sartorius) was used to create wounds in all wells simultaneously. Immediately after making the wound, cells were washed twice with PBS and replenished with a medium containing DMSO, TNFα, ML-60218 alone, or TNFα and ML-60218. The cells were then kept in the IncuCyte Live cell imaging system and monitored for 72 h at 3 h intervals. Cell migration was analysed using IncuCyte 2019B Rev2 software (Sartorius).

A 96-well ImageLock plate (Sartorius 4806) was coated with gelatin-based coating solution (Cell Biologics 6950) for 30 min at 37 °C. Cells were seeded onto gelatin-coated wells at 1.5e4 cells/well, grown to a confluent monolayer, in DMEM with 10% FBS, 4.5 g/ml glucose supplemented, then scratched using a 96-well Woundmaker (Sartorius). Following the scratch, media was immediately changed to serum-free media containing Incucyte Caspase-3/7 Red Dye for apoptosis (Sartorius 4707) with no supplements and either PBS, IFNα (Biolegend 752802), TNFα (Peprotech315-01A-20ug), or both IFNα and TNFα. Images were taken every 2 h with both phase and red on an Incucyte (Sartorius) to observe both wound closure and apoptosis. For immunofluorescent staining, cells were treated similarly after being grown on gelatin-coated glass coverslips in a 6-well plate. The coverslips were then scratched with a sterile pipette tip before fixation and staining at aforementioned indicated time points.