Figure 3.

Time-dependent expression of NAPDH oxidase, ROS accumulation and signaling pathways in HaCaT cells after treatment with DK223. (A) Time- dependent ROS generation was detected in treated HaCaT cells using the DCF-DA assay. Cells were treated with 2 μM DK223 at different times and then stained with DCF-DA to detect ROS generation, as described previously. H₂O₂ was used as a positive control; (B,C) The expression of NAPDH oxidase 4 was examined by Western blot and RT-PCR, as described previously; (D–F) Signaling pathways were examined by Western blot with the specific antibodies indicated; (G) Cells were pretreated with 10 μM DPI or 20 μM NAC for 2 h before 24 h DK223 incubation. Signaling pathways were examined by Western blot with the specific antibodies indicated; (H) Cells were seeded at adensity of 3 × 10⁴/mL 24 h before the scratch test. Scratch widths were measured 48 h after DK223 treatment only or co-treatment with 10 μM DPI or 20 μM NAC. A histogram shows the results of ImageJ data analysis. Values represent means ± SD of three independent experiments, * p < 0.05 in all under experiments.