Figure 4
Figure 4. Resveratrol inhibits constitutively active NF-κB in MM cells. Resveratrol suppressed NF-κB in a time-dependent and a dose-dependent manner (A) in U266, (B) in MM1.S, and (C) RPMI 8826 cells. (A, left) U266 cells (2 × 106/mL) were treated with 50 μM resveratrol for 0, 3, 6, 12, and 24 hours and then tested for NF-κB by EMSA. (A, right) U266 cells (2 × 106/mL) were treated with the indicated concentrations of resveratrol for 24 hours and then tested for NF-κB by EMSA. (B, left) MM1.S cells (2 × 106/mL) were treated with 50 μM resveratrol for 0, 3, 6, 12, and 24 hours and then tested for NF-κB by EMSA. (B, right) MM1.S cells (2 × 106/mL) were treated with the indicated concentrations of resveratrol for 24 hours and then tested for NF-κB by EMSA. (C, left) RPMI 8826 cells (2 × 106/mL) were treated with 50 μM resveratrol for 0, 3, 6, 12, and 24 hours and then tested for NF-κB by EMSA. (C, right), RPMI 8826 cells (2 × 106/mL) were treated with the indicated concentrations of resveratrol for 24 hours and then tested for NF-κB by EMSA. (D) NF-κB DNA binding was specific, and the activated complex consisted of p50 and p65 subunits. The results shown are representative of 3 independent experiments. (E) To measure IKK activity, U266 cells (4 × 106/mL) were incubated with 50 μM resveratrol for the indicated times, after which whole-cell lysates were prepared and immunoprecipitated with an antibody against IKK-α and analyzed with an immunocomplex kinase assay. (Bottom) IKK protein levels were assessed by fractionating whole-cell extracts on SDS-PAGE and examining them with Western blot using anti-IKKα and anti-IKKβ antibodies. (F-G) To assess phosphorylation of IκBα (F) and p65 (G), U266 cells (2 × 106/mL) were treated with 50 μM resveratrol for indicated times and subjected to cytoplasmic fractionation. Then, 30-μg extracts were resolved on 7.5% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes. The cytoplasmic fraction was probed for phospho-IκBα (F), and the nuclear fraction was probed for phospho-p65 (G). The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (H) Resveratrol induced translocation of activated nuclear p65 from the nucleus to the cytoplasm. U266 cells (2 × 106/mL) were incubated with medium (left) or with 50 μM resveratrol (right) for 24 hours and then analyzed for the intracellular distribution of p65 by immunocytochemistry (original magnification ×200). The results shown are representative of 3 independent experiments.

Resveratrol inhibits constitutively active NF-κB in MM cells. Resveratrol suppressed NF-κB in a time-dependent and a dose-dependent manner (A) in U266, (B) in MM1.S, and (C) RPMI 8826 cells. (A, left) U266 cells (2 × 106/mL) were treated with 50 μM resveratrol for 0, 3, 6, 12, and 24 hours and then tested for NF-κB by EMSA. (A, right) U266 cells (2 × 106/mL) were treated with the indicated concentrations of resveratrol for 24 hours and then tested for NF-κB by EMSA. (B, left) MM1.S cells (2 × 106/mL) were treated with 50 μM resveratrol for 0, 3, 6, 12, and 24 hours and then tested for NF-κB by EMSA. (B, right) MM1.S cells (2 × 106/mL) were treated with the indicated concentrations of resveratrol for 24 hours and then tested for NF-κB by EMSA. (C, left) RPMI 8826 cells (2 × 106/mL) were treated with 50 μM resveratrol for 0, 3, 6, 12, and 24 hours and then tested for NF-κB by EMSA. (C, right), RPMI 8826 cells (2 × 106/mL) were treated with the indicated concentrations of resveratrol for 24 hours and then tested for NF-κB by EMSA. (D) NF-κB DNA binding was specific, and the activated complex consisted of p50 and p65 subunits. The results shown are representative of 3 independent experiments. (E) To measure IKK activity, U266 cells (4 × 106/mL) were incubated with 50 μM resveratrol for the indicated times, after which whole-cell lysates were prepared and immunoprecipitated with an antibody against IKK-α and analyzed with an immunocomplex kinase assay. (Bottom) IKK protein levels were assessed by fractionating whole-cell extracts on SDS-PAGE and examining them with Western blot using anti-IKKα and anti-IKKβ antibodies. (F-G) To assess phosphorylation of IκBα (F) and p65 (G), U266 cells (2 × 106/mL) were treated with 50 μM resveratrol for indicated times and subjected to cytoplasmic fractionation. Then, 30-μg extracts were resolved on 7.5% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes. The cytoplasmic fraction was probed for phospho-IκBα (F), and the nuclear fraction was probed for phospho-p65 (G). The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (H) Resveratrol induced translocation of activated nuclear p65 from the nucleus to the cytoplasm. U266 cells (2 × 106/mL) were incubated with medium (left) or with 50 μM resveratrol (right) for 24 hours and then analyzed for the intracellular distribution of p65 by immunocytochemistry (original magnification ×200). The results shown are representative of 3 independent experiments.

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