Functional Assessments of lincRNA-p21

HN6 and Cal27 cells were transfected with siRNA or plasmids of lincRNA-p21 for 24 h, and then seeded in the plates. As described in our previous study [25 (link)], cell proliferation experiments were performed using the Cell Counting Kit (CCK8; Dojindo, Kumamoto, Japan) assays. The colony-forming assay was performed to monitor the cloning capability of HN6 and Cal27 cells [22 (link)]. Cell migration and invasion assays were performed using a Transwell technique with uncoated polycarbonate inserts (Millipore, Darmstadt, Germany) or BioCoat™ inserts (BD Biosciences, Franklin Lake, NJ, USA). Medium without FBS (2 × 10⁴ cells/200 μl) were added into the upper portion of a migration (uncoated insert) or invasion (matrigel-coated insert) chamber, with 500 μl DMEM containing 10% FBS added into the lower chamber. After crystal violet staining, the crossed cells were dissolved in 33% of acetic acid. OD 570 nm absorbance was measured.