Figure 5

CD40 ligation by mCD40L down-regulates Trx-1 protein levels in carcinoma but not normal epithelial cells. (a) Trx-1 protein expression in EJ carcinoma cells was monitored by immunoblotting at the indicated time points following CD40 ligation. Although Trx-1 expression increased in cultures following resumption of growth after sub-culture (as seen in the controls, ‘–’), CD40 ligation by mCD40L fully blocked Trx-1 expression (as seen in ‘+’ samples). CK18 detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) to ensure equal loading. Results are representative of three independent experiments. (b) Immunoblotting experiments show that CD40 ligation triggers the phosphorylation of ASK-1 at 3 h which coincides with a reduction in Trx-1 expression (the results represent co-cultures of EJ cells with mCD40L-expressing 3T3CD40L and control 3T3Neo fibroblasts). 3T3CD40L- (3T3CD40L) and 3T3Neo-only (3T3Neo) cultures showed no ASK-1 phosphorylation and EJ cells cultured alone (EJ alone) showed little (basal) ASK-1 phosphorylation. β-actin detection was used to ensure equal loading. Results are representative of two independent experiments. (c) Trx-1 protein expression was determined in normal (NHU) and malignant epithelial cells (EJ and HCT116) following treatment with mCD40L (24 h) by immunoblotting. In contrast to malignant cells where CD40 ligation induced Trx-1 reduction, NHU cells expressed low levels of Trx-1 and CD40 appeared to have no effect on Trx-1 expression. CK18 (EJ) and CK8 (HCT116) detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) to demonstrate equal loading. Of note, as NHU cells naturally express higher levels of CK18 in comparison to malignant (EJ) cells, and to ensure that any differences in basal Trx-1 protein expression were not artefactual, correct loading was confirmed for urothelial cells by detection of β-actin, the levels of which were similar in both cell types. Results are representative of two independent experiments. (d) Using immunoblotting, the levels of Trx-1 protein were determined in EJ-TRAF3-KD and their isogenic controls (EJ-Con) following treatment with mCD40L (6 h). In contrast to control cells where CD40 ligation induced a reduction in Trx-1 expression, EJ-TRAF3-KD cells maintained normal Trx-1 expression. CK18 detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) to ensure equal loading. Results are representative of two independent experiments.