PMC

(A). T24 cells were treated with the indicated concentration of vitamin K2 and the apoptosis was evaluated with Annexin V-FITC/PI dyes and measured by Flow cytometry. (B). The quantification of apoptotic death in vitamin K2-treated T24 cells. (C). Flow cytometry showed that vitamin K2 induced the apoptotic death in another two human bladder cancer J82 and EJ cells. (D). The effect of vitamin K2 on apoptosis in T24 cells was determined by TUNEL method using a detecting kit. Scale bar: 100μm (E). Western blots indicated that vitamin K2 induced activation of caspase-3 and cleavage of PARP in T24 cells. (F) Vitamin K2 inhibited the caspase-3-dependent viability of T24 cells by MTT assays. 10μM Z-DEVD-FMK, a caspase-3 inhibitor, was pretreated for 1 hours before exposure of 100 μM vitamin K2 to T24 cells for 24 hours. (G). Z-DEVD-FMK, a caspase-3 inhibitor, remarkably attenuated the apoptosis in vitamin K2-treated T24 cells. Cell apoptosis was evaluated with Annexin V-FITC/PI dyes and measured by Flow cytometry. * P<0.05, ** P<0.01 and *** P<0.001.

(A). Vitamin K2 dose-dependently reduced the viability of human bladder cancer T24, J82 and EJ cells. Cells were treated different concentration of vitamin K2 for 24 hours, respectively and cell viability was measured by MTT assays. (B). Vitamin K2 time-dependently decreased the viability of T24, J82 and EJ cells. Cells were treated with 100 μM vitamin K2 for 0, 6, 12, 18 and 24 hours, respectively, and cell viability was evaluated by MTT assays. Data represent the mean ± SEM of three different experiments with triplicate sets in each assay. * P<0.05, ** P<0.01 and *** P<0.001 vs vitamin K2-untreated group.

(A). T24 cells were treated with the indicated concentration of vitamin K2 for 24 hours, then the total proteins were isolated from the cells and the phosphorylation of JNK/p38 was analyzed by western blots. (B). Western blots indicated that vitamin K2 at the concentration of 100 μM induced sustained phosphorylation of JNK/p38 in T24 cells. (C). T24 cells were treated with 40 μM SP600125(SP), a pharmacological inhibitor of JNK activation, for 1 hour before treatment with 100 μM vitamin K2 for 24 hours and cell viability was evaluated by MTT assays. (D). T24 cells were treated 40 μM SP600125(SP) for 1 hour before treatment with 100 μM vitamin K2 for 12 hours and apoptotic death was assessed by flow cytometry. (E). T24 cells were treated with 10 μM SB203580(SB), a pharmacological inhibitor of p38 activation, for 1 hour before treatment with 100 μM vitamin K2 for 24 hours, then cell viability was assessed by MTT assays. (F). T24 cells were pre-treated with 10 μM SB203580(SB) for 1 hour, then treated with 100 μM vitamin K2 for 12 hours and apoptotic death was determined by flow cytometry. (G). T24 cells were treated with 40μM SP600125(SP) for 1 hour prior to treatment with 100 μM vitamin K2 for 24 hours. The total proteins extracted from the cells were assessed by western blots. (H). T24 cells were treated with 10 μM SB203580(SB) for 1 hours before treatment with 100 μM vitamin K2 for 24 hours, the total protein was evaluated by western blots. * P<0.05, ** P<0.01 and *** P<0.001.

(A). T24 cells were treated with the indicated concentration of vitamin K2 for 24 hours and the intracellular ROS generation was evaluated using the DCFH-DA probe by flow cytometry. M reflects the positive DCF fluorescence (B). Quantification of the intracellular ROS generation in vitamin K2-treated T24 cells. (C). J82 and EJ cells were treated with the indicated concentration of vitamin K2 for 24 hours and intracellular ROS generation was assessed by flow cytometry. (D and E). T24 cells were treated with 5mM antioxidant N-acetyl cysteine (NAC) for 1 hour before the treatment with or without 100 μM vitamin K2 for 24 hours and the apoptotic death was determined by flow cytometry. (F). T24 cells were treated with 5mM antioxidant NAC for 1 hour before the treatment with or without 100 μM vitamin K2 for 12 hours and intracellular ROS generation was evaluated using the DCFH-DA probe by flow cytometry. (G). Activation of caspase-3 and cleavage of PARP were analyzed by western blots after T24 cells were treated with 5mM NAC for 1 hour before the treatment with or without 100 μM vitamin K2 for 24 hours. * P<0.05, ** P<0.01 and *** P<0.001.