A, BxPC-3 cells were treated with 0-20 μM BITC for 24h or with 10μM BITC for different time intervals. Cells were lysed and 40 μg protein (whole cell or nuclear) was resolved by SDS-PAGE. Each blot was stripped and reprobed with β-actin for cytosolic and lamin B for nuclear proteins to ensure equal protein loading. Each experiment was repeated three times with similar results. B, The NF-kB luciferase assay was determined in control and BITC treated BxPC-3 cells using luminometer with the dual luciferase substrate system and luciferase activity was normalized with Renilla luciferase as internal control. C, EMSA was performed using nuclear protein obtained from BITC treated BxPC-3. In addition, NF-κB/p65 DNA binding by ELISA method was determined using 5 μg of nuclear protein from control and BITC treated cells and assayed for the presence of activated p65 using antibody specific for p65 following binding to NF-κB consensus sequence using TransAM NF-κB/p65 ELISA kit. D, BxPC-3 cells were treated with 0-20 μM BITC for 24h or treated with 10 μM BITC for varying time intervals and the whole cell lysates were subjected to SDS-PAGE. The membranes were probed with cyclin D1 antibody. Each blot was stripped and reprobed with β-actin to ensure equal protein loading. Each experiment was repeated three times with similar results. BxPC-3 cells were also transiently transfected with cyclin D1 promoter luciferase construct and Renilla luciferase plasmid as a control, followed by treatment with 10μM BITC or DMSO for 24 h. Cell lysates were subjected to luciferase activity and normalized with control Renilla luciferase. Data represents means ± SD of three independent experiments each performed in triplicates. *Statistically different when compared with control, P < 0.05.

A, BxPC-3 cells were transfected with Flag, Flag-HDAC1 or Flag-HDAC3 plasmid with/without NF-κB luciferase plasmid and Renilla luciferase as control for 24h followed by treatment with 10μM BITC for 24h. Whole cell lysate from transfected and/or treated cells were subjected to SDS-PAGE and the immunoblots were probed with HDAC1 and HDAC3 antibody. Equal loading was determined with β-actin antibody. The relative NF-κB luciferase activity was performed in BxPC-3 cells as described in Figure 1 and normalized with Renilla luciferase as a control. Data represents means ± SD of three independent experiments. Nuclear lysates from transfected and BITC treated BxPC-3 cells were used to determine relative DNA binding of p65 by ELISA kit as described above. Data represents means ± SD of three independent experiments. *Statistically different when compared with control, P < 0.05. B, BxPC-3 cells were transiently transfected with Flag, Flag-HDAC1 and Flag-HDAC3 plasmid DNA with or without cyclin D1 luciferase reporter plasmid and Renilla luciferase as internal control followed by treatment with 10μM BITC for 24 hours. Cell lysates were resolved using SDS-PAGE and the membrane was probed with cyclin D1 antibody. For equal loading the membrane was blotted with β-actin antibody. Relative cyclin D1 reporter luciferase activities was performed in transfected cells as described above and normalized against Renilla luciferase as a control. Cyclin D1 was further immunoprecipitated from control and BITC treated BxPC-3 cells and the lysate was immunoblotted for HDAC1 and HDAC3. Data represents means ± SD of three independent experiments. C, Cells were transiently transfected with Flag, Flag-HDAC1 and Flag-HDAC3 plasmids as described above and treated with10μM BITC or DMSO for 24h and analyzed for full length caspase-3 and cleaved PARP. Each experiment was repeated three times with similar results. D, HDAC overexpressing cells after BITC treatment were subjected to Sulforhodamine B cell survival assay. Values are means ± SD of three independent experiments. *Statistically different when compared with control, P < 0.05.

A, Capan-2 cells were treated with 0-20 μM BITC for 24h and whole cell lysate was subjected to SDS-PAGE and the membranes were probed with p300/CBP, p21^WAF1 and β-actin antibody. B, NF-κB luciferase activity, DNA binding and cyclin D1 luciferase activity was performed in control and BITC treated Capan-2 cells transfected with Flag, Flag-HDAC1 or Flag-HDAC3 plasmid DNA with/without NF-κB luciferase plasmid and Renilla luciferase as described in Figure 3. Data represents means ± SD of three independent experiments. *Statistically different when compared with control, P < 0.05. C, HDAC overexpressing cells treated with BITC were subjected to Sulforhodamine B cell survival assay. Values are means ± SD of three independent experiments. *Statistically different when compared with control, P < 0.05. D, HPDE-6 cells were treated with different concentrations of BITC for 24h and whole cell lysates were subjected to SDS-PAGE. Representative immunoblots show the effect of BITC treatment on the expression of phospho-NF-κB/p65 Ser536, NF-κB/p65, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7 and HDAC8. Each blot was stripped and reprobed with anti-β-actin to ensure equal protein loading. Each experiment was repeated three times with similar results.

A, BxPC-3 cells were treated with different concentrations of BITC for 24h or treated with 10μM BITC for varying time intervals and the whole cell lysates were subjected to SDS-PAGE. Representative immunoblots show the effect of BITC treatment on the expression of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7 and HDAC8. Each blot was stripped and reprobed with anti-β-actin to ensure equal protein loading. Each experiment was repeated three times with similar results. B, HDAC1 and HDAC3 activity was determined in the nuclear lysates of DMSO and BITC treated BxPC-3 cells using HDAC Fluorescent Activity Assay kit. *Statistically different when compared with control, P < 0.05. C, Effect of SAHA was determined in BxPC-3 cells and compared with the effects of BITC. Cells were exposed to 0-100μM SAHA or BITC for 24h and evaluated for NF-kB, cyclin D1 and HDAC1/3 expression by western blotting and cell survival by SRB assay. D, BxPC-3 cells were treated with 0-20 μM BITC for 24h. Whole cell lysates were subjected to SDS-PAGE and the membranes were probed with p300/CBP, p21^WAF1 and β-actin antibody. Each experiment was repeated three times with similar results.

A, Capan-2 cells were treated with 0-20 μM BITC for 24h or with 10μM BITC for different time intervals. Proteins were resolved by SDS-PAGE and immunobloted with p-NF-κB/p65 (Ser536) and NF-κB/p65 antibodies. Each blot was stripped and reprobed with β-actin antibody to ensure equal protein loading. Each experiment was repeated three times with similar results. B, Control and BITC treated cells were transfected with NF-κB luciferase reporter plasmid as described in Fig 1. The luciferase assay was performed with the dual luciferase substrate system and luciferase activity was normalized with Renilla luciferase as internal control. In addition, NF-κB/p65 DNA binding was determined using TransAM NF-κB/p65 ELISA kit. Data represents means ± SD of three independent experiments each performed in triplicates. *Statistically different when compared with control, P < 0.05. C, Capan-2 cells were treated with 0-20 μM BITC for 24h or treated with 10 μM BITC for varying time intervals and the whole cell lysates were subjected to SDS-PAGE. The membranes were probed with cyclin D1antibodies. Control and BITC treated cells were also transiently transfected with cyclin D1 promoter luciferase construct and subjected to luciferase activity as described in Fig 1. Data represents means ± SD of three independent experiments each performed in triplicates. *Statistically different when compared with control, P < 0.05. D, Capan-2 cells were treated with different concentrations of BITC for 24h or treated with 10μM BITC for varying time intervals, and whole cell lysates were subjected to SDS-PAGE. Representative immunoblots show the effect of BITC on the expression of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7 and HDAC8. Each blot was stripped and reprobed with anti-β-actin to ensure equal protein loading. Each experiment was repeated three times with similar results.

Tumor sections from control and 12μmol BITC treated mice were analyzed for the expression of NF-kB, cyclin D1, HDAC1 and HDAC3 by immunohistochemistry. Each section was analyzed under the microscope at 200 x magnification.