Fig. 4: Effects of FOXM1 silencing on CCA cells in response to 5-FU treatment.

FOXM1 expression in HuCCA and KKU-D131 cells was silenced using siRNA (siFOXM1) and non-silencing siRNA (NS-siRNA) was used as control. After 24 h, CCA cells were reseeded and grown overnight. a After treatment with 100 μM 5-FU for 24 h, cells were harvested and the expression levels of FOXM1, E2F1 and TYMS were determined by western blot and RT-qPCR analysis. Western blot was performed using β-tubulin as loading control (upper panel). Expression of FOXM1, E2F1 and TYMS mRNA was also determined by RT-qPCR analysis using L19 as an internal control (lower panel). Data are presented as mean ± SEM (n > 3). RT-qPCR data were analysed by unpaired t tests. Double and triple asterisks (** and ***) indicate significant difference at p < 0.01 and p < 0.001, respectively, from the non-targeting siRNA-treated control cells; ‘ns’ indicates no significant difference. b Effect of FOXM1 silencing on 5-FU sensitivity was also investigated using clonogenic assays. Representative clonogenic images show the effects of FOXM1 silencing on the outcome of 5-FU treatment. Data are presented as mean ± SEM (n = 3) and were analysed by two-way ANOVA. The asterisks (****) indicate significant difference at p < 0.0001 from the non-targeting siRNA-treated control cells; ‘ns’ indicates no significant difference