Figure 1. Inhibitory effects of GAC 17:1 on constitutively activated STAT3 in U266 cells. (A) The chemical structure of GAC 17:1 and GAC 15:1; (B,C) U266 cells and PBMCs (1 × 104 cells/well) were treated with various concentrations of GAC 17:1 for 24 h and cell viability was determined by MTT assay, ** p < 0.01 and *** p < 0.001 indicates significant differences from the control group; (D) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) or GAC 15:1 (50 μM) for 3 h. Whole-cell extracts were prepared then equal amount of lysates were subjected to western blot analysis for p-STAT3 (Tyr705), STAT3, and β-actin; (E) U266 cells (5 × 105 cells/well) were incubated with GAC 17:1 (30 or 50 μM) for 3 h. Thereafter, equal amounts of lysates were analyzed by western blot analysis using antibodies against for p-STAT3 (Tyr705), STAT3, and β-actin; (F,G) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) for the indicated time. Lysates from the cells were analyzed using western blot analysis for p-STAT3 (Tyr705), STAT3, p-JAK1 (Tyr1022/1023), JAK1, p-Src (Tyr416), and Src; (H) After 3 h of GAC 17:1 (50 μM) treatment, the cells were fixed and permeabilized. STAT3 (green) and p-STAT3 (red) were immunostained with rabbit anti-Stat3 and goat anti-p-STAT3 respectively followed by FITC-conjugated secondary antibodies, then nucleus (blue) was stained with DAPI. The fourth panels show the merged images of the first, second and third panels; (I) U266 cells (5 × 105 cells/well) were incubated with the GAC 17:1 (50 μM) for 1.5 or 3 h. Nuclear extracts were prepared for detection of STAT3 binding activities and testing was performed by EMSA.

Figure 1. Inhibitory effects of GAC 17:1 on constitutively activated STAT3 in U266 cells. (A) The chemical structure of GAC 17:1 and GAC 15:1; (B,C) U266 cells and PBMCs (1 × 104 cells/well) were treated with various concentrations of GAC 17:1 for 24 h and cell viability was determined by MTT assay, ** p < 0.01 and *** p < 0.001 indicates significant differences from the control group; (D) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) or GAC 15:1 (50 μM) for 3 h. Whole-cell extracts were prepared then equal amount of lysates were subjected to western blot analysis for p-STAT3 (Tyr705), STAT3, and β-actin; (E) U266 cells (5 × 105 cells/well) were incubated with GAC 17:1 (30 or 50 μM) for 3 h. Thereafter, equal amounts of lysates were analyzed by western blot analysis using antibodies against for p-STAT3 (Tyr705), STAT3, and β-actin; (F,G) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) for the indicated time. Lysates from the cells were analyzed using western blot analysis for p-STAT3 (Tyr705), STAT3, p-JAK1 (Tyr1022/1023), JAK1, p-Src (Tyr416), and Src; (H) After 3 h of GAC 17:1 (50 μM) treatment, the cells were fixed and permeabilized. STAT3 (green) and p-STAT3 (red) were immunostained with rabbit anti-Stat3 and goat anti-p-STAT3 respectively followed by FITC-conjugated secondary antibodies, then nucleus (blue) was stained with DAPI. The fourth panels show the merged images of the first, second and third panels; (I) U266 cells (5 × 105 cells/well) were incubated with the GAC 17:1 (50 μM) for 1.5 or 3 h. Nuclear extracts were prepared for detection of STAT3 binding activities and testing was performed by EMSA.

Figure 2. Inhibition of IL-6 inducible STAT3 activation and the induction of PTEN and SHP-1 proteins by GAC 17:1 in multiple myeloma cells. (A) MM1.S cells (5 × 105 cells/well) were pretreated with GAC 17:1 (50 μM) for 3 h and then stimulated with IL-6 (10 ng/mL) for 15 min to induce activation of STAT3. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705) and STAT3; (B) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) for the indicated time and washed with PBS to remove GAC 17:1 before resuspension in fresh medium. Cells were removed at indicated times and lysed to prepare the whole-cell extract, then analyzed using Western blot analysis for p-STAT3 (Tyr705) and STAT3; (C) U266 cells (5 × 105 cells/well) were treated with 50 μM of GAC 17:1 for 3 h following pretreatment of the indicated concentrations of pervanadate for 30 min. Whole-cell extracts were prepared and immunoblotted with antibodies for p-STAT3 (Tyr705) and STAT3; (D) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) for 1.5 or 3 h. Whole-cell extracts were prepared, equal amounts of lysates were analyzed by western blot analysis using antibodies against PTEN and SHP-1. And total RNA was extracted and examined for expression of PTEN and SHP-1 by RT-PCR; (E) U266 cells were transfected with scrambled or PTEN or SHP-1-specific siRNA (50 nM). After 24 h, cells were treated with GAC 17:1 (50 μM) for 3 h and the whole-cell extracts were subjected to western blot analysis for PTEN, SHP-1, p-STAT3 (Tyr705), and STAT3. β-Actin was used as a loading control.

Figure 2. Inhibition of IL-6 inducible STAT3 activation and the induction of PTEN and SHP-1 proteins by GAC 17:1 in multiple myeloma cells. (A) MM1.S cells (5 × 105 cells/well) were pretreated with GAC 17:1 (50 μM) for 3 h and then stimulated with IL-6 (10 ng/mL) for 15 min to induce activation of STAT3. Whole-cell extracts were prepared and immunoblotted with antibodies for phospho-STAT3 (Tyr705) and STAT3; (B) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) for the indicated time and washed with PBS to remove GAC 17:1 before resuspension in fresh medium. Cells were removed at indicated times and lysed to prepare the whole-cell extract, then analyzed using Western blot analysis for p-STAT3 (Tyr705) and STAT3; (C) U266 cells (5 × 105 cells/well) were treated with 50 μM of GAC 17:1 for 3 h following pretreatment of the indicated concentrations of pervanadate for 30 min. Whole-cell extracts were prepared and immunoblotted with antibodies for p-STAT3 (Tyr705) and STAT3; (D) U266 cells (5 × 105 cells/well) were treated with GAC 17:1 (50 μM) for 1.5 or 3 h. Whole-cell extracts were prepared, equal amounts of lysates were analyzed by western blot analysis using antibodies against PTEN and SHP-1. And total RNA was extracted and examined for expression of PTEN and SHP-1 by RT-PCR; (E) U266 cells were transfected with scrambled or PTEN or SHP-1-specific siRNA (50 nM). After 24 h, cells were treated with GAC 17:1 (50 μM) for 3 h and the whole-cell extracts were subjected to western blot analysis for PTEN, SHP-1, p-STAT3 (Tyr705), and STAT3. β-Actin was used as a loading control.

Figure 3. Apoptotic effects of GAC 17:1 in U266 cells. (A) U266 cells were treated with GAC 17:1 (50 μM) for 24 h. The cells were harvested, washed with a cold PBS buffer and digested with RNase A. Cellular DNA was stained with propidium Iodide and a flow cytometric analysis was done to determine the cell cycle distribution; (B) U266 cells were treated with GAC 17:1 (50 μM) for 24 h and the cells were incubated with Annexin-V/FITC and propidium iodide, then analyzed by a flow cytometer; (C) Apoptosis in U266 cells was detected by TUNEL assay. After treatment, the cells were stained with a TUNEL assay reagent and then analyzed under a flow cytometer; (D) After 24 h of GAC 17:1 (50 μM) treatment, cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a confocal microscope; (E) U266 cells were treated with GAC 17:1 (50 μM) for 24 h. The cells were washed with a PBS and treated with 5 μM of TMRE (tetramethylrhodamine, ethyl ester) and then analyzed by a flow cytometer to detect MMP activity.

Figure 3. Apoptotic effects of GAC 17:1 in U266 cells. (A) U266 cells were treated with GAC 17:1 (50 μM) for 24 h. The cells were harvested, washed with a cold PBS buffer and digested with RNase A. Cellular DNA was stained with propidium Iodide and a flow cytometric analysis was done to determine the cell cycle distribution; (B) U266 cells were treated with GAC 17:1 (50 μM) for 24 h and the cells were incubated with Annexin-V/FITC and propidium iodide, then analyzed by a flow cytometer; (C) Apoptosis in U266 cells was detected by TUNEL assay. After treatment, the cells were stained with a TUNEL assay reagent and then analyzed under a flow cytometer; (D) After 24 h of GAC 17:1 (50 μM) treatment, cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a confocal microscope; (E) U266 cells were treated with GAC 17:1 (50 μM) for 24 h. The cells were washed with a PBS and treated with 5 μM of TMRE (tetramethylrhodamine, ethyl ester) and then analyzed by a flow cytometer to detect MMP activity.

Figure 4. Suppression of STAT3 regulated gene products by GAC 17:1 in multiple myeloma cells. (A) U266 and MM.1S cells (1 or 2 × 104 cells/well) were incubated with the GAC 17:1 (30 or 50 μM) for the indicated time intervals. MTT assay was performed to investigate the effect of GAC 17:1 on cell proliferation; (B–D) U266 cells (3 × 105 cells/well) were incubated with the GAC 17:1 (30 or 50 μM) for 24 h. Lysates from the cells were analyzed using western blotting against Bcl-2, Bcl-xL, Survivin, IAP-1, COX-2, Cyclin D1, VEGF, MMP-9,MMP-2, Caspase-3, and PARP. In addition, Total RNA was isolated, and analyzed by real time PCR for Bcl-2, Bcl-xL, and Cyclin D1. Data represents means ± S.D., *** indicates p < 0.001; (E) MEF cells were transfected pMXs-STAT3C or pMXs-STAT3gw and incubated for 24 h. The cells were treated with GAC 17:1 (100 μM) for 3 h (left panels) or 24 h (right panels). The lysates from the cells were analyzed by Western blotting using antibodies against p-STAT3 (Tyr705) and STAT3 (left panels) or PARP and β-actin (right panels) respectively.