μ slides 4 well

After CPTH6 treatment, cells seeded on coverslips were fixed and permeabilized before immunofluorescence. For analysis of F-actin filament, cells were incubated with phalloidin conjugated-rhodamine (Thermo Fisher Scientific). DNA was counterstained with 0.02 μg.ml^− 1 DAPI (Sigma-Aldrich). Preparations were examined under an Olympus AX70 microscope using a 100X/1.35 NA objective. Images were acquired using a TCH-1.4ICE camera (Tucsen, Fujian, CHINA) controlled by ISCapture and processed by Photoshop CS. NIH ImageJ 1.3 software to quantify fluorescence intensity over the cell area from images acquired under identical exposure settings. For live microscopy experiments, cells were seeded at 80–90% confluency in μ-slides 4-well (Ibidi, Martinsried, DE), exposed to CPTH6 1 h later wound generation by 10 μl tip and time–lapse recording were conducted in a microscope stage incubator (Basic WJ, Okolab, Naples, IT) using an Eclipse Ti inverted microscope (Nikon, Tokyo, JA). Images were acquired over 48 h at 15 min intervals for phase contrast. Videos and still images were processed and analyzed using NIS-Elements AR 4.0. Cell migration ability was determined as previously described [22 ].