Nis element ar 3

All Western blot and Northern blot images were processed with Adobe Photoshop CS4 for all linear adjustments and cropping. All images captured on Nikon Eclipse Ti microscope were analyzed and processed with Nikon NIS ELEMENT AR 3.1 software. Image cropping was done using Adobe Photoshop CS4.

Single-cell migration was evaluated to determine the migration rate of keratinocytes under subconfluent conditions. As described in the previous section, time-lapse images were captured from five randomly selected positions per well. Cell migration was analysed using NIS Element AR 3.1 software (Nikon, Minato City, Tokyo, Japan). At least 150 cells from each condition were chosen randomly and tracked every 20 min for 1 h (36 h after seeding). The migration rate of the cells was evaluated every 20 min using Equation (3).
$$\mathrm{Migration} \mathrm{rate}=\frac{{\sqrt{{({\mathrm{x}}_2{-\mathrm{x}}_1)}^2+{({\mathrm{y}}_2-{\mathrm{y}}_1)}^2}}^{}}{{\mathrm{t}}_2-{\mathrm{t}}_1}$$
where (x₁, y₁) and (x₂, y₂) indicate the centre of the cell evaluated by the software at time t₁ and t₂, respectively. The overall migration rate for a cell was evaluated by averaging migration rate for three 20 min segments.

The cells in each group were washed three times with PBS, and the intracellular lysosome was extracted by lysosome extraction kit (Sigma-Aldrich, St. Louis, MO, USA). Then, 3 µmol/L Fluo-3AM solution was immediately added, and incubated in a incubator at 37°C for 30 min. After aspirating the working fluid, lysosome was rinsed with PBS for three times, and PBS was added to balance for 10 min, and finally for observation. By using Ca²⁺ analyzer, the image and time was obtained with real-time monitoring via NIS-Element AR3.0 (Nikon) software system, with a period of 6 min. Lysosomal Ca²⁺ intensity was expressed as R340/380 (Wavelengths at 340 and 380 nm, respectively). Each experiment was detected at least 10 cells, baseline was also detected (baseline fluctuates within a relatively small range), with each experiment repeating at least three times.