Figure 2
JNK/AP-1-induced Bak and Bax proteins are essential in CD40-mediated apoptosis. (a) mCD40L-mediated apoptosis in EJ carcinoma cells is dependent on the activation of the JNK kinase and the AP-1 transcription factor as indicated by the blockade and attenuation of cell death by the JNK inhibitor SP600125 (25 μM) and the AP-1 inhibitor NDGA (25 μM), respectively, in comparison to vehicle control (CON). Bars represent mean fold change ±s.d. (n=5). (b) Immunoblotting analysis demonstrates that the induction of Bak and Bax protein expression following CD40 ligation (24 h) is dependent on JNK and AP-1, as the use of both JNK inhibitor SP600125 (25 μM) and AP-1 inhibitor NDGA (25 μM) abrogates the induction of both Bak and Bax, in comparison to the untreated controls (CON). CK18 detection served as a loading control. Results are representative of three independent experiments. (c) Retrovirus transduction was used to prepare EJ cell derivatives EJ-Bak-KD and EJ-Bax-KD that stably express anti-Bak and anti-Bax shRNAs, respectively, as well as their isogenic controls (EJ-Con). Successful knockdown was confirmed by Western blotting for Bak and Bax in the appropriate cell lines following CD40 ligation (24 h), as Bak and Bax up-regulation is prevented in the EJ-Bak-KD and EJ-Bax-KD lines, respectively, in comparison to the control shRNA-expressing EJ-Con cells. CK18 detection served as a loading control. Results are representative of two independent experiments. (d) CD40-mediated apoptosis is abrogated by Bak and Bax knockdown as indicated by the blockade of caspase-3/7 activation in the EJ-Bak-KD and EJ-Bax-KD lines, in comparison to EJ-Con. Bars represent mean fold caspase activity ±s.d. (n=5).
