Figure 4
mCD40L-induced ROS release is essential for apoptosis and is driven by TRAF3- and Nox-dependent signalling. (a) The levels of ROS in EJ and HCT116 carcinoma cells were measured by H2DCFDA labelling, which was detected spectrophotometrically and expressed as fold change relative to H2DCFDA-labelled untreated cells. Unlike cells treated with soluble agonist (10μg/ml cross-linked G28-5), mCD40L-treated cells showed rapid induction of ROS (3 h) which was blocked by the ROS scavenger/antioxidant NAC (30 mM). Bars represent mean fold increase in ROS ±s.d. (n=6). In addition to spectrophotometric measurements, ROS induction by CD40 ligation was also detected by H2DCFDA labelling and flow cytometry, and representative results for HCT116 cells from at least two experiments are shown on the overlay histogram on the right to compare controls (Con) versus mCD40L-treated cells (mCD40L). (b) mCD40L-mediated apoptosis in EJ and HCT116 carcinoma cells is blocked by both the antioxidant NAC (30 mM) and the pharmacological Nox inhibitor DPI (0.25 or 0.125 μM in EJ and HCT116, respectively). Bars represent mean fold change ±s.d. (n=6). (c) Immunoblotting experiments demonstrate that ligation of CD40 in EJ (as shown in EJ-Con cells) and HCT116 cells triggers phosphorylation of the Nox subunit p40phox. The experiments also show that CD40-mediated phosphorylation of p40phox is attenuated in TRAF3 shRNA-expressing cells (EJ-TRAF3-KD) in comparison to isogenic controls (EJ-Con). Cytokeratin detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) in EJ derivatives (CK18) and HCT116 (CK8) cells, respectively, to ensure equal loading. Results are representative of at least two independent experiments. (d) Blockade of Nox by the pharmacological inhibitor DPI (0.125 μM) completely inhibited mCD40L-mediated induction of Bak (24 h) in HCT116 cells. CK8 detection was used to assess equal loading. Results are representative of two independent experiments. (e) Western blotting shows that pharmacological inhibition of Nox by DPI (0.25 μM) blocks activation of ASK-1 (3 h) and subsequently Bax induction (24 h) by CD40 in EJ cells. CK18 detection confirmed equal loading. Results are representative of three experiments. (f) Nox blockade during CD40 ligation in HCT116 and EJ cells by treatment with DPI (as described in panels D and E, respectively), attenuated the induction of TRAF3 by mCD40L. Cytokeratin detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) in EJ (CK18) and HCT116 cells (CK8), respectively, to ensure equal loading. Results are representative of two independent experiments.
