Ix70 phase contrast microscopy

A172 cells were pretreated with 0, 0.25, 0.5, 1, 2 and 4 mM of SB for 48 h. The cell motility assay was performed for 16 h using 24-well Bio-Coat cell migration chambers (BD Biosciences) using 0.5% FBS as a chemoattractant, following a previously described protocol (13 (link)). The cell invasion assay was performed using Matrigel-coated polyethylene terephthalate membranes for 16 h. Cells at a density of 2×10⁵ cells/well were inserted into the upper chamber. The migrating cells on the lower side of the filter were fixed with 70% ethanol at room temperature for 1 h, prior to being stained with Giemsa solution (Wako Pure Chemical Industries, Ltd.) at room temperature for 10 min and counted using Olympus IX70 phase contrast microscopy with a ×10 objective lens. For cell area analysis, cell images were captured using Olympus IX70 phase contrast microscopy with a ×10 objective lens. Cell periphery was circled then the inner area was calculated with ImageJ 1.38e software. A minimum of 100 cells/each experiment were measured.

The β-gal staining assay was performed using a Senescence β-Galactosidase Staining kit (cat. no. 9860; Cell Signaling Technology, Inc., Danvers, MA, USA). Briefly, A172 cells were cultured with 0, 0.25, 0.5, 1, 2 or 4 mM SB, or 25, 50 or 100 nM TSA at 37°C for 4 days in 35 mm plastic culture plates, and fixed with 10% formaldehyde at room temperature for 1 h. Plates were rinsed twice with PBS, subsequently the β-gal staining solution containing X-gal as a substrate was added and plates were incubated at 37°C overnight in a dry incubator. β-gal-positive cells were then counted using Olympus IX70 phase contrast microscopy with ×10 objective lens and the percentage of positive cells was calculated.