Fig. 2

Fn induces chemoresistance of CRC cells to 5-Fu via upregulation of BIRC3 in vitro. a Cells were incubated with or without Fn and then exposed to serial dilutions of 5-Fu for 48 h. Cell viability was determined by CCK8 assay. Data are presented as the percentage of viable cells (mean ± s.d.). The IC₅₀ of 5-Fu in HCT116 and HT29 cells was approximately 30 μM and 20 μM respectively, as demonstrated by the dotted line. b Cells were incubated with or without Fn and then exposed to IC₅₀ 5-Fu. Cell viability was assessed by CCK8 assay at 0 h, 24 h, 48 h, and 72 h. Data are presented as the percentage of viable cells (mean ± s.d.). c-d Cells were incubated with or without Fn in the presence or absence of IC₅₀ 5-Fu for 48 h. Cleaved of caspase-3 (C-caspase-3), cleaved of PARP (C-PARP) and BIRC3 were detected by Western blot (c). Apoptosis was detected by flow cytometry. Data are presented as the ratio of apoptotic cells (mean ± s.d.). Representative quadrantal diagrams (left) and the corresponding histogram (right) from three independent experiments are shown (d). e-f Cells were transfected with specific siRNA for 24 h and then incubated with or without Fn in culture medium with IC₅₀ 5-Fu for an additional 48 h. Protein levels of BIRC3, C-caspase-3, and C-PARP were measured by Western blot (e). Apoptosis ration was detected with flow cytometry. Data are presented as the ratio of apoptotic cells (mean ± s.d.). Representative quadrantal diagrams (left) and the corresponding histogram (right) from three independent experiments are shown (f). g Cells were incubated with or without Fn and then exposed to gradient concentrations of a SMAC mimetic in the culture medium with IC₅₀ 5-Fu for 48 h. Cell viability was determined by CCK8 assay and the data are plotted. Data are presented as the percentage of viable cells (means±s.d.) from three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001 by unpaired Student t test)