Incucyte analysis software

For cell growth assay, SW1222 cells were reverse transfected with CTL-siRNA or FORCP-siRNA (20 nM) for 48 hr, then reseeded onto 48-well plates at 8000 cells per well and loaded into Incucyte live image system (ESSEN Bioscience). Images were taken every 4 hr for 6 days and cell growth was determined by Incucyte Analysis Software (ESSEN Bioscience). For cell viability assay, HCT116 cells stably expressing WT FORCP or Mutant FORCP were generated using Lentivirus package and transducing system. Cells were seeded onto 96-well plates at 1000 cells per well. Cell viability was determined using CCK-8 assay (Dojindo Laboratories). For colony formation on plastic, or soft agarose assays, cells were reseeded in a six-well plate at a density of 1000 cells per well. After 2 to 3 weeks, colonies were fixed with ice-cold 100% methanol for 5 min, stained with crystal violet and colonies were counted and analyzed using ImageJ. For SW1222 and LS180 cells, after transfection with CTL and FORCP siRNAs for 48 hr, cells were treated with or without ER stress agents, DTT (2 nM) or TM (2 µg/ml) for 2 hr, medium containing drug was removed and replaced with fresh medium. Cells were then seeded onto 96-well plates for cell viability assay or six-well plates for colony formation assays as described above.

MDCK wild-type cells were seeded in a 48-well plate (Thermo Fisher Scientific, Waltham, MA, USA) and allowed to reach 70% confluency. Subsequently, the cells were transfected with p60AmotL2-GFP or GFP-ctrl plasmid using the GenJet™ In Vitro DNA transfection reagent, as previously mentioned. Following transfection, the cells were transferred to the Incucyte^® Live-Cell Analysis System (Essen Bioscience, Ann Arbor, MI, USA). Live images were captured at 2-h intervals over a 48-h period using bright field and GFP imaging with 20× objective lenses. The imaging process was performed inside an incubator. To analyze the extruding cell numbers, Incucyte^® analysis software (Version 2021B, Essen Bioscience, Ann Arbor, MI, USA) was used.