American Society of Hematology

Figure 5

$Figure 5. Resveratrol inhibits constitutively active and IL-6–inducible STAT3 activation in MM cells. Resveratrol suppressed phospho-STAT3 levels in a time-dependent (A) and dose-dependent (B) manner. (A) U266 cells (2 × 106/mL) were treated with 50 μM resveratrol for the indicated times; after which whole-cell extracts were prepared; and 30-μg portions of those extracts were resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (B) U266 cells (2 × 106/mL) were treated with the indicated concentration of resveratrol for 3 hours, after which Western blotting was performed as described for panel A. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (C) Resveratrol had no effect on STAT5 or phospho-STAT5 protein levels. U266 cells (2 × 106/mL) were treated with 50 μM resveratrol for the indicated times. Whole-cell extracts were prepared, fractionated on SDS-PAGE, and examined by Western blot using antibodies against phospho-STAT5 and STAT5. (D) Resveratrol causes inhibition of translocation of STAT3 to the nucleus. U266 cells (1 × 105/mL) were incubated with or without 50 μM resveratrol for 3 hours and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry (original magnification ×200). (E-F) Resveratrol down-regulates IL-6–induced phospho-STAT3. (E) RPMI 8226 cells (2 × 106/mL) were treated with IL-6 (10 ng/mL) for the indicated times, whole-cell extracts were prepared, and phospho-STAT3 was detected by Western blot as described in “Materials and methods.” The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. Arrowheads indicate pSTAT3 and STAT3, respectively. (F) RPMI 8226 cells (2 × 106/mL) were treated with 50 μM resveratrol for the indicated times and then stimulated with IL-6 (10 ng/mL) for 15 minutes. Whole-cell extracts were then prepared and analyzed for phospho-STAT3 by Western blotting. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. The results shown are representative of 3 independent experiments.$

Resveratrol inhibits constitutively active and IL-6–inducible STAT3 activation in MM cells. Resveratrol suppressed phospho-STAT3 levels in a time-dependent (A) and dose-dependent (B) manner. (A) U266 cells (2 × 10⁶/mL) were treated with 50 μM resveratrol for the indicated times; after which whole-cell extracts were prepared; and 30-μg portions of those extracts were resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (B) U266 cells (2 × 10⁶/mL) were treated with the indicated concentration of resveratrol for 3 hours, after which Western blotting was performed as described for panel A. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (C) Resveratrol had no effect on STAT5 or phospho-STAT5 protein levels. U266 cells (2 × 10⁶/mL) were treated with 50 μM resveratrol for the indicated times. Whole-cell extracts were prepared, fractionated on SDS-PAGE, and examined by Western blot using antibodies against phospho-STAT5 and STAT5. (D) Resveratrol causes inhibition of translocation of STAT3 to the nucleus. U266 cells (1 × 10⁵/mL) were incubated with or without 50 μM resveratrol for 3 hours and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry (original magnification ×200). (E-F) Resveratrol down-regulates IL-6–induced phospho-STAT3. (E) RPMI 8226 cells (2 × 10⁶/mL) were treated with IL-6 (10 ng/mL) for the indicated times, whole-cell extracts were prepared, and phospho-STAT3 was detected by Western blot as described in “Materials and methods.” The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. Arrowheads indicate pSTAT3 and STAT3, respectively. (F) RPMI 8226 cells (2 × 10⁶/mL) were treated with 50 μM resveratrol for the indicated times and then stimulated with IL-6 (10 ng/mL) for 15 minutes. Whole-cell extracts were then prepared and analyzed for phospho-STAT3 by Western blotting. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. The results shown are representative of 3 independent experiments.

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