Fig. 1. Growth inhibitory effects of IP6 on DU145 cells Fig. 1. Growth inhibitory effects of IP6 on DU145 cells . For the studies assessing the effect of IP6 on exponentially growing DU145 cell growth, 5000 cells/cm ² were plated in 60 mm dishes and next day, cells were fed with fresh medium either with vehicle (distilled water) alone or 0.25–4 mM of IP6 in medium. After 24, 48 and 74 h of these treatments, total cells were counted using hemocytometer. The cell growth data shown were mean ± SE of three independent plates; each sample was counted in duplicate. * P < 0.05, ** P < 0.01, ^$P < 0.001 versus control. From: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes Carcinogenesis. 2003;24(3):555-563. doi:10.1093/carcin/24.3.555 Carcinogenesis | © Oxford University Press

Fig. 2. Effect of IP6 on cell cycle progression in DU145 cells Fig. 2. Effect of IP6 on cell cycle progression in DU145 cells. Cells were cultured in RPMI 1640 supplemented with 10% FBS, and treated with either vehicle or 0.25–4 mM concentrations of IP6. After 24 h of these treatments, cells were collected, washed with PBS, digested with RNase and then cellular DNA stained with propidium iodide as detailed in ‘Materials and methods’. Flow cytometric analysis was then performed for cell cycle distribution. The percentage of cell distribution data for each treatment group shown is the mean of two plates and representative of three independent experiments. From: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes Carcinogenesis. 2003;24(3):555-563. doi:10.1093/carcin/24.3.555 Carcinogenesis | © Oxford University Press

Fig. 3. Effect of IP6 on cell cycle regulators in DU145 cells Fig. 3. Effect of IP6 on cell cycle regulators in DU145 cells. Cells were cultured in RPMI 1640 medium with 10% FBS, and treated with 0.5, 1 and 2 mM doses of IP6 for 24 and 48 h as described in ‘Materials and methods’. At the end of treatments, total cell lysates were prepared and subjected to SDS–PAGE followed by western immunoblotting. Membranes were probed with anti-Cip1/p21, Kip1/p27, CDK2, 4, and 6, cyclin D1, D2, D3, E and A, and β-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system. The experiments were repeated three times with similar results. From: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes Carcinogenesis. 2003;24(3):555-563. doi:10.1093/carcin/24.3.555 Carcinogenesis | © Oxford University Press

Fig. 4. Effect of IP6 on cyclin-CDKI complex, and CDK and cyclin-associated kinase activities. Cells were cultured in RPMI 1640 medium with 10% FBS, and treated with either vehicle or 2 mM doses of IP6 for 24 h as described in ‘Materials and methods’. At the end of treatments, total cell lysates were prepared. ( A ) In binding study, cyclin D1 and E were immunoprecipitated using specific antibody from 200 mg of total protein lysates followed by SDS–PAGE and western immunoblotting. Membranes were probed for Cip1/p21, Kip1/p27, cyclin D1 and E. ( B and C ) Densitometric data of bound levels of Cip1/p21 and Kip1/p27 with cyclin D1 and E, presented as fold change of control after IP correction. ( D ) Effect of IP6 on CDK2, 4 and 6, and cyclin D1 and E-associated kinase activities. Treatments and cell culture conditions were the same as those in binding studies. CDK2 and cyclin E kinase activities were determined by in-bead histone H1 kinase assay using immunoprecipitated CDK2 and cyclin E from total cell lysates (200 μg protein) using specific antibody; and CDK4, 6 and cyclin D1-associated kinase activities were determined by in-bead RB–GST fusion protein kinase assay using immunoprecipitated CDK4, 6 and cyclin D1 from total cell lysates (200 μg protein) using specific antibodies as described in ‘Materials and methods’. After the assay, the labelled substrates were subjected to SDS–PAGE, and the gel was dried and exposed to X-ray film. The bottom panel of each band indicates the fold change in band intensity compared with that of control at each treatment time. The experiments were repeated 2–3 times with similar results. IB, western immunoblotting; IP, immunoprecipitation. From: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes Carcinogenesis. 2003;24(3):555-563. doi:10.1093/carcin/24.3.555 Carcinogenesis | © Oxford University Press

Fig. 5. Effect of IP6 on Rb-related proteins, E2F transcription factor and Rb-related proteins-F2F complex in DU145 cells. ( A ) Cells were cultured in RPMI 1640 medium with 10% FBS, and treated with 0.5, 1 and 2 mM doses of IP6 for 24 h as described in ‘Materials and methods’. At the end of treatments, total cell lysates were prepared in Rb-lysis buffer and subjected to SDS–PAGE (8 or 12% gel) followed by western immunoblotting as detailed in ‘Materials and methods’. Membranes were probed with anti-pRb/p107, pRb2/p130, E2F4 and β-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system. ( B ) In binding studies, only 2 mM dose of IP6 for 24 h was used as described in ‘Materials and methods’. pRb/p107 and pRb2/p130 were immunoprecipitated using specific antibodies from 400 μg of total protein lysates followed by SDS–PAGE and western blotting. In pRb/p107 IP, membrane was probed for E2F4 and pRb/p107 protein levels and in pRb2/p130 IP, membrane was probed for E2F4 and pRb/p130; and ( C ) bound level of E2F4 was extrapolated by densitometric analysis after IP correction for both Rb-related proteins. Experiments were repeated twice with similar results. From: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes Carcinogenesis. 2003;24(3):555-563. doi:10.1093/carcin/24.3.555 Carcinogenesis | © Oxford University Press

Fig. 6. Apoptotic effect of IP6 on DU145 cells Fig. 6. Apoptotic effect of IP6 on DU145 cells. Cells were grown in 10% FBS condition and treated with 1–4 mM IP6 for 48 and 72 h. ( A ) At the end of treatments, total cells were collected and stained with annexin V/PI as mentioned in ‘Materials and methods’ followed by flow cytometric analysis. Data presented are mean ± SE of percent annexin V/PI stained cells of two independent samples in each treatment group. ( B ) In similar treatments as detailed above, total cells were collected and cell lysates were prepared as described in ‘Materials and methods’. SDS–PAGE and western blot analysis were performed for PARP, caspase 3 and β-actin using specific antibodies as described in ‘Materials and methods’. The experiments were repeated with similar results. * P < 0.05. From: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes Carcinogenesis. 2003;24(3):555-563. doi:10.1093/carcin/24.3.555 Carcinogenesis | © Oxford University Press