We have evaluated the cleavage and the consequent activation of both caspase 9 and 3 with western blotting using specific antibodies that recognize only the intact forms of the two enzymes. We have found that 5-FU increased the cleavage of caspase 3 in H9c2 cells and the latter was potentiated in presence of LF. These effects were paralleled by a decrease of pro-caspase 9 expression (activation index). On the other hand, DOXO increased the cleavage of caspase 3 and 9 after 24 h from the beginning of treatment but the latter returned to basal level after 48 h (Figure 4). Moreover, the Lazertinib mw different treatments caused no significant changes

of the levels of pro-caspase 3 and 9 in HT29 cell line (Figure 5). Figure 4 Effects of the different treatments on caspase activation

in H9c2 cells. H9c2 cells were treated with 5-FU alone or combined with LF or DOXO alone for 48 h at the concentrations inhibiting the 50% of the proliferation of the cardiocytes as previously indicated in Table 1. Thereafter, the expression of caspase 3, 7 and 9 were evaluated after blotting with specific antibodies that recognise both the full and the cleaved forms of the proteins, as described in “”Materials and Methods”". Expression of the house-keeping protein α-tubulin, used as loading control, was also evaluated. Amine dehydrogenase The experiments were performed at least three different times and the results were always similar. CTR, untreated cells; 5-FU, cells treated Selinexor concentration with 5-FU alone; 5-FU + LF,

cells treated with 5-FU in combination with LF; DOXO, cells treated with DOXO alone. Figure 5 Effects of the different treatments on caspase activation in HT29 cells. HT-29 cells were treated with 5-FU alone or combined with LF or DOXO alone for 48 h at the concentrations inhibiting the 50% of the proliferation of the colon cancer cells as previously indicated in Table 1. Thereafter, the expression of caspase 3 and 7 were evaluated after blotting with specific antibodies that recognise the full form of the proteins, as described in “”Materials and Methods”". Expression of the house-keeping protein α-tubulin, used as loading control, was also evaluated. The experiments were performed at least three different times and the results were always similar. CTR, untreated cells; 5-FU, cells treated with 5-FU alone; 5-FU + LF, cells treated with 5-FU in combination with LF; DOXO, cells treated with DOXO alone. These results were consistent with the data derived from FACS analysis; in fact, the treatment with 5-FU and LF induced a stronger apoptotic effect on cardiocytes cell line if compared with that one recorded in colon adenocarcinoma cell line.