Epiphot

Cell motility was assessed using a scratch assay modified from Lee et al. [34 (link)]. Prior to differentiation, cells were allowed to proliferate to achieve a confluence greater than usual (>70%). After serum starvation, culture media was removed and cells were subjected to two washes. A sterile 10 μl pipette tip was used to scratch the cell monolayer in 3 straight lines. Cells were washed twice in media to remove debris and 200× baseline images were taken with inverted microscopy (Nikon Epiphot). 24 h later, vehicle or PA (100, 500, 750 μM) treatment was applied. After that, cells were fixed in 10% formalin and stained with Crystal Violet dye. Images were taken by inverted microscopy. The number of cells that migrated into the scratch was counted in experimental vs. control conditions by Image J (v1.46; National Institutes of Health, USA). Viability of cells was quantified by a Crystal Violet method [35 (link)] in SPECTRA-Fluor Plus (TECAN, Austria). The results were expressed in arbitrary units normalized to the Crystal Violet viability data (N = 2 experiments, 8 fields/treatment).

LOM images were taken using microscopes Neophot32 (Karl Zeiss, Jena, Germany), Epiphot (Nikon, Tokyo, Japan) and DSX1000 (Olympus Corp., Tokyo, Japan). SEM was performed using the Jeol JSM-7600F scanning electron microscope (JEOL, Tokyo, Japan) equipped with detectors for EDS (Oxford X-Max 50 mm², Oxford Instruments, Abington, UK) and EBSD (HKL Nordlys, Oxford Instruments, Abington, UK). Quantitative EDS point analysis on the Beraha etched samples was performed with a Tescan Lyra3 scanning electron microscope (Tescan, Brno, Czech Republic). The results of the EBSD analysis in the IPF maps and the grain size map were processed using the ATEX software (Version 3.x, Metz, France) [14 ]. The Bruker D8 Discover powder X-ray diffractometer equipped with Co anode and Lynx Eye detector was used for phase identification and quantification. (Bruker, Billerica, MA, USA)