Figure 5. Phox-I1 and Phox-I2 show undetectable toxicity and site effects.

(A) Apoptosis analysis by FACS of HL-60 cells treated with compound or vehicle control for 2 hours prior to 7-AAD and Annexin V staining. (B) HL-60 cells from A were harvested and lysates were immunoblotted for levels of pPAK, and actin was used as a control for loading. (C) F-actin reorganization in freshly isolated fMLP-stimulated primary murine neutrophils was analyzed. Representative images (left panel) and quantification (right panel) are displayed. Treatment with Analog 13 is included as a “dead analog” that possesses no intrinsic ROS inhibitory activity, and nocodazole is included as a positive control for actin disruption. Cells were exposed to a 10μM dose of Phox-I1, Phox-I2, and Analog 13, and 200nM nocodazole. (D) The effect of Phox-I on NOX4 mediated ROS production was tested in primary murine neutrophils transfected with a NOX4 expressing plasmid. 10 uM Phox-I1 was applied to the cells for 30 min prior to ROS assay by luminol chemiluminescence in the presence of HRP. The fMLP-stimulated ROS activity in the presence or absence of 10 uM Phox-I1 was measured in parallel. (E) The antioxidant abilities of these lead compounds were tested by prestimulating dHL-60 cells with fMLP for 30 minutes prior to treatment with Phox-I1 or Phox-I2. Levels of superoxide were analyzed by DCFDA assay and FACS. NAC, apocyanin, DPI, and NSC23766 treated cells served as controls. (F) For affinity assay, DMSO-differentiated HL-60 cells were treated with standard effective dose of indicated compound for 2 hours, washed, and allowed to recover in normal media for 4 hours or 2 hours prior to fMLP stimulation and DCFDA ROS production assay by FACS analysis. (G) For stability assay, DMSO-differentiated HL-60 cells were treated with 20μM dose of compound for the indicated time period prior to fMLP stimulation and DCFDA ROS production assay by FACS analysis. 30 minute and 18 hour time periods are not displayed because they revealed no ROS inhibition.