Figure 5.

Effects of NBDHEX on the interaction between GSTP1-1 and TRAF2, JNK phospho-activation and cell cycle progression/cell death. (a) U-2OS cells were treated with 5 μM NBDHEX 48 h after plating and stained with specific antibodies at 1, 3 or 6 h of treatment, to detect the TRAF2–GSTP1-1 complex (PLA: red dots) and the phospho-activated form of JNK (diffuse red fluorescence, see arrow). The intrinsic fluorescence (green) of NBDHEX was localized in the cell cytoplasm (see arrow). (b) Analysis of the amount of TRAF2–GSTP1-1 complex and (c) of phospho-JNK. NBDHEX induces a rapid dissociation of the TRAF2–GSTP1-1 complex (a and b) paralleled by the phosphorylation of JNK (a and c). (d) NBDHEX causes a sustained cell cycle arrest in the G2/M phase followed by apoptosis. (e) U-2OS cells were treated with NBDHEX (5 μM), the autophagy inhibitor CQ (2.5–10.0 μM) or NBDHEX-CQ combinations. Cell survival was assessed after 48 h by the SRB assay; the effect of NBDHEX on cell survival was not modified by simultaneous treatment with autophagy-inhibiting, non-cytotoxic doses of CQ. (f) Cell lysates from U-2OS cultures, untreated or treated with CQ (2.5–40.0 μM dose range) for 24 h, were subjected to immunoblot analysis with an anti-LC3 antibody recognizing both the cytosolic LC3-I and the autophagosome-associated LC3-II. β-actin was used to ensure equal loading and transfer of samples