Plumbagin suppresses CXCR4 through modulation of its mRNA level. A and B, Plumbagin suppresses CXCR4, through lysosomal but not proteosomal degradation. Cells were treated with indicated concentrations of lactacystin or chloroquine for 1 h at 37°C, followed by treatment of 5 μM plumbagin for 6 h. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against CXCR4. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. Representative results of three independent experiments are shown. C, Plumbagin suppresses expression of CXCR4 mRNA in MDA-MB-231 cells. Cells were treated with 5 μM plumbagin for indicated times. Total RNA was isolated and analyzed by RT-PCR assay as described in Materials and Methods. 18S was shown to equal loading of total RNA. Representative results of three independent experiments are shown. D, Plumbagin inhibits NF-κB activation in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were incubated with indicated concentrations of plumbagin for 2 h. The nuclear extracts were assayed for NF-κB activation by TransAM p65 transcription factor assay kit. E, MDA-MB-231 cells were transiently transfected with an NF-κB-luciferase plasmid and then treated with the indicated concentrations of plumbagin for 2 h. Cell supernatants were thereafter collected and assayed for luciferase activity as described in Materials and Methods. Representative results of three independent experiments are shown. Results are expressed as fold activity over the activity of the vector control. Bars indicate standard deviation. * indicates p value < 0.05). F, Plumbagin inhibits binding of NF-κB to the CXCR4 promoter. MDA-MB-231 cells were treated with 5 μM plumbagin for indicated time intervals and the proteins were cross-linked with DNA by formaldehyde and then subjected to ChIP assay using an anti-p65 antibody with the CXCR4 primer. Reaction products were resolved by electrophoresis.