C0775

To evaluate the colony-forming unit (CFU) capacity, cSM-MPCs of normal (n = 8) and OA (n = 12) donors were plated in 58 cm² Petri dishes (664160, CELLSTAR®, Greiner Bio-One) at three cell densities, i.e. ± 0.25-, 0.5-, and 1 × 10³ cells/Petri dish (equivalent to ± 4-, 8-, and 17 cells/cm²). After 10–14 days in humidified, normoxic conditions (5% CO₂/21% O₂) at 37 °C, normal culture conditions, cells were stained with 0.5% crystal violet (C0775, Sigma-Aldrich) in 100% methanol (MC1060092511, Merck Millipore) for 30 min. Colonies containing > 50 cells were counted in the appropriate plating density, i.e. the density wherein the individual colonies were not overlapping, and displayed as the percentage of the total seeded cell number.

Equal numbers of PANC-1 or PANC-1R cells (1000/well) were seeded into 6-well plates and cultured for 2 weeks in the medium. After washing with phosphate-buffered saline (PBS), the cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (C0775, Sigma-Aldrich) for 30 min at room temperature. The number of colonies was counted under a light microscope.

Cell viability after normogravity and microgravity treatments was assessed with Clonogenic Assay and Trypan Blue Exclusion Assay. In the colony assay, at the end of treatments, cells (5 × 10²) were plated in 100 mm dishes in culture medium with 10% FBS to perform the clonogenic assay. At the end of treatments, plates were washed twice with a phosphate buffered saline solution (PBS; 79382; Sigma-Aldrich–MERCK, St. Louis, MO, USA) and fixed with 4% formaldehyde solution in PBS (F8775; Sigma-Aldrich–MERCK, St. Louis, MO, USA) at rt. After 20 min, dishes were washed twice in PBS and stained for 5 min with 0.5% crystal violet (C0775; Sigma-Aldrich–MERCK, St. Louis, MO, USA). Finally, cells were washed with distilled water and air-dried. The colonies were counted the following day. In trypan blue exclusion assay, cells were harvested and stained with 0.4% trypan blue (T8154; Sigma-Aldrich–MERCK, St. Louis, MO, USA). The cell suspension was applied to a haemocytometer and counted with a phase contrast microscopy (NIKON Eclipse TE2000U, Nikon Netherlands, Amsterdam, The Netherlands).

In vitro migration and invasion assay was performed in 24-well plates using 8.0-µm pore polycarbonate trans-well inserts (BD Corning). 5 × 10⁴ cells were suspended in serum-free DMEM and overlaid in the upper chamber of each trans-well. DMEM medium containing 10% FBS was added to the lower chamber as a chemoattractant. The inserts were then incubated at 37 °C in a humidified atmosphere containing 5% CO₂. After 36 and 48 hours of incubation, cells that failed to penetrate the pores of the filter were removed by cotton swabs and the migrated cells were fixed and stained with 0.1% crystal violet (C0775, SIGMA, USA). Images of crystal violet stained cells were acquired with the help of an IX-70 inverted microscope (Olympus, Tokyo, Japan) equipped with a digital camera. Quantification of migrated cells from at least 5 different microscopic fields per sample was done using ImageJ 1.48 v software. The migratory activity was calculated as the average number of migrated cells in each assay from three independent experiments. Further, migratory activity was also assayed by dissolving cells that invaded the filter in methanol and measuring absorbance at 570 nm using a spectrophotometer.

Cell Counting Kit‐8 (CCK‐8: Dojindo Laboratories, Rockville, MD, USA) assay was used to assess cell viability. Cells were seeded in 6‐well plates starting at 2 × 10⁵ cells per well and incubated for 24 h. After siRNA transfection for 48 h, a mixture of CCK‐8 solution and RPMI‐1640 medium supplemented with 10% FBS was added to each well and incubated in 5% CO₂ at 37 °C for 10 min. The absorbance at 450 nm was assessed by a microplate reader. For crystal violet staining, the cells were fixed with cold 100% methanol for 5 min at −20 °C and stained with 0.1% crystal violet solution (C0775, Sigma‐Aldrich, Merck KGaA, Darmstadt, Germany) for 5 min at room temperature after siRNA transfection for 48 h. Then, the cells were observed under a microscope (CELENA^® S Digital Imaging System, Logos Biosystems, Anyang, Korea).