Figure 6.

MAPK and Akt are upstream and NF-κB is downstream of TS. (a) Inhibition of NF-κB using the peptide inhibitor SN-50. Phospho-p65 status was used to check the inhibition of NF-κB using SN-50. Western blot analysis was carried out using whole cell lysate from MDA-MB-231 cells pre-treated with SN-50 and subsequently to 5-FU. (b) 5-FU induced NF-κB activation in MDA-MB-231-Neo cells, while it failed to induce the same in MDA-MB-231-IκB-α DM cells. Nuclear extracts prepared after exposing Neo and IκB-α DM cells to 10 μM 5-FU were subjected to electrophoretic mobility shift assay (EMSA) to check activation of NF-κB. (c) 5-FU failed to induce IκBα degradation and p65 phosphorylation in NF-κB-inhibited MDA-MB-231 cells. Western blot analysis was carried out after treating Neo and IκB-α DM cells to 10 μM 5-FU and expression of phospho-p65 and IκB-α was checked. (d and e) Effect of NF-κB inactivation on 5-FU-induced TS activation. NF-κB expression was inhibited in MDA-MB-231 cells either by SN-50 or by transient transfection using IκB-α DM plasmid. The cells were then treated with 5-FU for 1 h and subjected to western blotting using TS antibody. (f and g) Effect of inhibition of Akt and MAPKs on 5-FU-induced TS expression. MDA-MB-231 cells were pre-treated with LY294002 (10 μM), U0126 (10 μM), SP600125 (25 μM) and SB203580 (20 μM) for 30 min, and subsequently exposed to 5-FU for 48 h and western blotted against TS antibody. (h and i) Inhibition of 5-FU-induced NF-κB activation and nuclear translocation in TS-silenced cells. MDA-MB-231 cells were transiently transfected with control and TS siRNA and then treated with 10 μM 5-FU for 30 min and checked for phosphorylation of p65 by western blot and NF-κB DNA-binding activity by EMSA. (j) Effect of 5-FU-induced phosphorylation status of Akt and MAPKs upon silencing of TS. MDA-MB-231 cells were transiently transfected with control and TS siRNA and then treated with 10 μM 5-FU for 30 min and checked for phosphorylation of Akt and MAPKs by western blotting. (k) Effect of 5-FU induced NF-κB DNA-binding activity upon silencing of Akt. MDA-MB-231 cells pre-treated with LY294002 (5 μM) were treated with 5-FU for 1 h, nuclear extracts were prepared and EMSA was performed. (l) Effect of 5-FU-induced NF-κB DNA-binding activity upon silencing of MAPKs. Nuclear extracts were prepared from MDA-MB-231 cells pre-treated with U0126 (10 μM), SP600125 (50 μM) or SB203580 (40 μM), followed by 5-FU treatment for 1 h and EMSA was performed. The experiments were repeated at least three times to confirm reproducibility