Ckx53 inverted light microscope

The migration ability of all four mesothelioma cells was analyzed using a wound scratch assay. Serum starved mesothelioma cell lines grown to 80% confluence were incubated overnight with 5 pmol PVT1/NC siRNA in collagen-coated 24-well plates at 37°C in a humidified incubator supplied with 5% CO₂. Wounds were created by scratching the cells with 1-ml micropipette tips. The wells were washed twice to remove floating cells. Images of the gap area (wound) were captured every 24 h (for ACC-MESO-1 every 12 h) using a CKX53 inverted light microscope equipped with a DP21 digital camera (Olympus Corporation), and the gap area was further analyzed using T Scratch software version 1.0 downloaded from https://github.com/cselab/TScratch (27 (link)).

PVT1 small interfering (si)RNA (Lincode Human PVT1 siRNA - SMARTpool; cat. no. R-029357-00-0005) and its negative control (NC) siRNA (Lincode Non-targeting Pool; cat. no. D-001320-10-05) were purchased from GE Healthcare Dharmacon, Inc. Forkhead box M1 (FOXM1) siRNA (FOXM1 Silencer Select Pre-designed siRNA; cat. no. 4427037 ID# s5248) and its NC siRNA (Silencer Select Negative Control No. siRNA; cat. no. 4390843) were purchased from Thermo Fisher Scientific, Inc. Cells cultured until attaining 70–80% confluency, were transfected with 50 nM of PVT1/NC siRNA, 25 nM of FOXM1/NC siRNA, or both, using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc.) in Opti-Mem Reduced Serum Medium (Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator supplied with 5% CO₂ according to the manufacturer's recommended protocols. The images of morphological change of the transfected mesothelioma cells were captured at 0 and 72 h using a CKX53 inverted light microscope with a DP21 digital camera (Olympus Corporation).