Figure 3
Khz-cp triggers cytoplasmic and mitochondrial ROS generation. (A) SNU-1 cells were loaded with H2DCF-DA and treated with Khz-cp (diluted 1:100), and the cytoplasmic ROS levels were assessed by flow cytometry. (B) Intracellular ROS levels in SNU-1 cells treated with Khz-cp (diluted 1:100) were analyzed using an Amplex Red hydrogen peroxide assay. (C) The cells were pretreated with NAC (5 mM), DPI (10 μM), or apocynin (Apo; 300 μM) for 1 h. Intracellular ROS levels in SNU-1 cells were analyzed 30 min after Khz-cp treatment by using the Amplex Red hydrogen peroxide assay. (D) SNU-1 cells were transfected with siRNA targeting Nox2 and Nox4. After 48 h, the cells were treated with Khz-cp (1:100, 0.5 h), and ROS generation was measured after 60 min by using the Amplex Red hydrogen peroxide assay. (E) Silencing of Nox2 and Nox4 by siRNA transfection was assessed by RT-PCR. (F) SNU-1 cells were treated with Khz-cp, and the membrane and cytosol fractions were separated using the Compartmental Protein Extraction kit. The expression of the p47phox and p67phox proteins in the membrane and cytosol fractions was analyzed by immunoblotting. (G) SNU-1 cells were loaded with MitoSOX Red for 30 min and treated with Khz-cp. Mitochondrial ROS generation was then assessed by fluorescent microscopy at the indicated time points. (H) SNU-1 cells were pretreated with DPI or apocynin for 1 h, and mitochondrial ROS generation was analyzed 60 min after Khz-cp treatment as in (G). (I) SNU-1 cells were pretreated with 0.5 μM MitoQ or TPP for 30 min. Right panel: Cytoplasmic ROS generation was measured 30 min after Khz-cp treatment by DCF staining and flow cytometry. Left panel: Mitochondrial ROS generation was assessed 60 min after Khz-cp treatment by MitoSOX Red staining and flow cytometry.