Effect of baicalein on phosphorylation of IkB-α, expression of NF-kB target genes, and mitochondrial apoptotic pathway. (A) Lysates from U266 cells treated with 50 or 100 μM baicalein for 1 hour were assessed for phosphorylation of IkB-α by Western blotting with anti-phospho-IkB-α antibody. (B) Expression of IL-6 and XIAP genes, targets of NF-kB, was assessed by semiquantitative RT-PCR in U266 cells treated with 50 or 100 μM baicalein for 3 hours. (C) The mitochondrial membrane potential of U266 cells was assessed after treatment with 0, 25, 50, 100, or 200 ng/mL of baicalein (•), baicalin (▴), or wogonin (♦). Values shown in this figure are the mean ± 1 SD of 3 independent experiments on flow cytometric detection of DiOC₆ uptake at indicated time points. ^**P < .01. (D) Cells treated with 12.5, 25, or 50 μM baicalein for 12 hours were subcellularly fractionated. Cytochrome c (Cyto C) in cytosol was assessed by Western blotting with a specific anti-cytochrome c antibody. (E) Lysates from U266 cells treated with 25 or 50 μM baicalein for 12 hours were assessed for cleaved caspase cleavage by Western blotting with antibodies to caspase-9 or -3. (F) Flow cytometric assessment of nucleic translocation of NF-kB (p65). U266 cells were treated with 50 or 100 μM of baicalein for 1 hour.