A, The structure of AKBA. B, Left, AKBA inhibits constitutive STAT3 activation in U266 cells. U266 cells (1×10⁶/ml) were treated with the indicated concentrations of AKBA for 4 h, after which whole-cell extracts were prepared, and 30 µg of protein was resolved on 10% 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. Right, AKBA suppresses phospho-STAT3 levels in a time-dependent manner. U266 cells (1×10⁶/ml) were treated with the 50 µM AKBA for the indicated times, after which Western blotting was performed as described previously. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. C, AKBA suppresses STAT3 nuclear translocation. U266 cells (1×10⁶/ml) were treated with the 50 µM AKBA for 4 h. the samples cytospinned, and immunocytochemistry performed with STAT3 antibody. D, AKBA inhibits constitutively active STAT3 in U266 cells. Left, U266 cells (2×10⁶/ml) were treated with the indicated concentrations of AKBA for 4 h and analyzed for nuclear STAT3 levels by EMSA. Right, U266 cells (2×10⁶/ml) were treated with 50 µM AKBA for the indicated durations and analyzed for nuclear STAT3 levels by EMSA.

A, AKBA suppresses the activation of JAK2 in a dose- and time-dependent manner. U266 cells (4×10⁶/ml) were treated with AKBA at the indicated doses (left) or with 50 µM AKBA for the indicated time intervals (right). Whole-cell extracts were immunoprecipitated with antibody against JAK2 and analyzed by western blot. The same samples were analyzed for JAK2 protein. B, AKBA suppresses the IL-6 induced JAK-2 kinase activity. MM1.S cells (4×10⁶/ml) were pre-incubated with AKBA at the indicated doses for 4h and treated with IL-6 for 10 min. Whole cell lysate were prepared and anlaysed the JAK-2 activity by immunocomplex kinase assay. C, AKBA suppresses phospho-Src levels in a dose- and time-dependent manner. U266 cells (2×10⁶/ml) were treated with the indicated doses of AKBA (left) or with 50 µM AKBA for the indicated times (right), after which whole-cell extracts were prepared and 30µg aliquots of those extracts resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for phospho-Src antibody. The same blots were stripped and reprobed with Src antibody to verify equal protein loading. D, Pervanadate reverses the phospho-STAT3 inhibitory effect of AKBA. U266 cells (2×106/ml) were treated with the indicated concentration of pervanadate and 50 µM AKBA for 4 h, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved on 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3 and STAT3.

A, AKBA suppresses STAT3-regulated proliferative, survival and antiangiogenic gene products. U266 cells (2×10⁶/ml) were treated with 50 µM AKBA for indicated time intervals, after which whole-cell extracts were prepared and 30 µg portions of those extracts were resolved on 10% SDS-PAGE, the membrane sliced according to molecular weight and probed against cyclin D1, Bcl-2, Bcl-xl, Mcl-1 and VEGF antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. B, AKBA suppresses cell proliferation in multiple myeloma. U266 and MM1.S cells were plated in triplicate, treated with 10 and 50 µM AKBA, and then subjected to MTT assay on days 0 to 3 to analyze proliferation of cells. C, AKBA causes significant accumulation of cells in the G1 phase. U266 cells (2×10⁶/mL) were synchronized by incubation overnight in the absence of serum and then treated with 50 µM AKBA for the indicated times, after which the cells were washed, fixed, stained with PI, and analyzed for DNA content by flow cytometry. D (left panel), AKBA induces caspase-3 activation. U266 cells were treated with 50 µM AKBA for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against caspase-3 antibody. The same blot were stripped and reprobed with β-actin antibody to show equal protein loading. C (right panel), AKBA causes PARP cleavage. U266 cells were treated with 50 µM AKBA for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot against PARP antibody. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. The results shown are representative of three independent experiments.

A, AKBA-induced inhibition of STAT3 phosphorylation is reversible. U266 cells (1 × 10 ⁶cells) were treated with 50 µM AKBA for the indicated durations (left panel) or treated for 1 h and washed with PBS two times to remove AKBA before resuspension in fresh medium (right panel). Cells were removed at indicated times and lysed to prepare the whole-cell extract. 30µg of whole-cell extracts were resolved on 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, probed for the phosphorylated STAT3 (pSTAT3), and stripped and reprobed for STAT3 antibodies. B, AKBA downregulates IL-6–induced phospho-STAT3. MM1.S cells (2×10⁶/mL) were treated with IL-6 (10 ng/ml) for indicated times, whole-cell extracts were prepared, and phosphorylated 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.

A, AKBA induces the expression of SHP-1 in U266 cells (left panel). U266 cells (2×10⁶/ml) were treated with AKBA for 4h with different concentrations of AKBA, after which whole-cell extracts were prepared, and 30-µg portions of the extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for SHP-1 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. AKBA induces SHP-1 gene expression (right panel). U266 cells (4 × 10⁶/ml) were treated with different concentrations of AKBA for 4h, and total RNA was extracted and examined for expression of SHP-1 by RT-PCR. GAPDH was used as an internal control to show equal RNA loading. B, AKBA-induced SHP-1 activation is transient. U266 cells (2 ×10 ⁶cells/ml) were treated with 50 µM AKBA for 4h washed with PBS two times to remove AKBA before resuspension in fresh medium. Cells were removed at indicated times and lysed to prepare the whole-cell extract. 30µg of whole-cell extracts were resolved on 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, probed for SHP-1, and stripped and reprobed for β-actin antibody. C, Effect of SHP-1 knockdown on AKBA-induced expression of SHP-1. SCC4 cells (1 × 10⁵/mL) were transfected with either scrambled or SHP-1-specific siRNA (50 nmol/L). After 48 h, cells were treated with 50 µmol/L AKBA for 4 h and whole-cell extracts were subjected to Western blot analysis for SHP-1. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading (left) and transfection with SHP-1 siRNA reverses AKBA-induced suppression of STAT3 activation. The same whole-cell extracts were subjected to phospho-STAT3 and STAT3 (right). D, Effect of SHP-1 knockdown on AKBA-induced inhibition of pSTAT3. SCC4 cells (1 × 10⁵/mL) were transfected with either scrambled or SHP-1-specific siRNA (50 nmol/L). After 48 h, cells were treated with 50 µmol/L AKBA for 4 h and whole-cell extracts were subjected to Western blot analysis for pSTAT3. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading.

A, Knockdown of SHP-1 inhibited the apoptotic effect of AKBA. SCC4 cells (1 × 10⁵/mL) were transfected with either scrambled or SHP-1-specific siRNA (50 nmol/L). After 48h, cells were treated with 50 µmol/L AKBA for 48 h and analaysed the percentage of apoptosis by Live/Dead assay. B, Transfection of constitutive active STAT3 induces pSTAT3. A293 cells (5 × 10⁵/mL) were transfected with constitutive STAT3 plasmid as described in methods. The cells were harvested 24 h after transfection and the trasfection was confirmed by western blot analysis. C, Forced expression of constitutive STAT3 rescues A293 cells from AKBA induced cytotoxicity. A293 cells (5 × 10⁵/mL) were transfected with constitutive STAT3 plasmid. 24 h after transfection the cells were treated with different concentration of AKBA for 48h and then the cytotoxicity was determined by Live/Dead assay.