Other pro-apoptotic genes such as programmed cell death 6 (Pdcd6), SIVA1 apoptosis-inducing factor (Siva1), BH3 interacting domain death agonist (Bid), cell death-inducing DFFA-like effector a (Cidea) and apoptosis antagonizing transcription factor (Aatf) increased their expression from 5 to 14 days.
The filters were incubated with antibodies that recognise the pro and cleaved forms of the apoptotic proteins PARP (Pharmingen, San Diego, CA, USA), caspase 3, caspase 8, caspase 9 (Upstate Biotechnology Inc., Lake Placid, NY, USA), Bid (Cell Signaling, Beverly, MA, USA), and actin (Santa Cruz, Santa Cruz, CA, USA).
Primary antibodies used were; anti-VDAC1 (Abcam ab14734), anti-PARP (E-Bioscience 14-6666-92), anti-Caspase-8 (Alexis 804-242) anti-BAX (Cell Signaling #2774), anti-COX4 (Cell Signaling #4850), anti-BID (Cell Signaling #2002), anti α-tubulin (Abcam ab15246).
Treatment of HeLa Bid kd cells and control HeLa cells with Tunicamycin resulted in a similar induction in protein levels of the ER resident molecular chaperones Grp78 and Grp94, which are known ER stress response target genes (Fig. 5 A), but also resulted in similar levels of caspase activation (Fig. 5 B, E), apoptosis and cell death (Fig. 5 F).
In contrast to death receptor-induced apoptosis, HeLa Bid kd cells were not protected from caspase activation and cell death after exposure to the protein kinase inhibitor Staurosporine (STS), demonstrating that these cells did not acquire a general resistance against apoptosis activation (Fig. 4 A, B).
In HeLa Bid kd cells, procaspase-3 processing, cleaved PARP levels, and the accumulation of Annexin V-positive cells were significantly reduced.
In our study, puma was potently activated by Oxaliplatin in HeLa control and Bid kd cells, and transient RNA interference against puma was able to significantly reduce apoptosis in both cell lines.
We could not observe significant differences in puma mRNA upregulation between HeLa control and HeLa Bid kd cells (Fig. 11 A), indicating that the stress signaling pathways mediating puma expression were similarly activated in control and Bid kd cells, and that the induction of puma occurred independently of Bid.
The knockdown of puma led to a further decrease of apoptosis in the HeLa Bid kd cells, indicating that both BH3-only proteins cooperate in mediating apoptosis triggered by Oxaliplatin.
Transient RNA interference against puma was able to reduce puma mRNA expression by 47.58+/−8.7% and 64.61+/−3.2% after 24 h and by 59.71+/−1.1% and 69.67+/−0.1% after 48 h in the HeLa control and HeLa Bid kd cells, respectively (n = 3 experiments; normalized to β-actin mRNA expression and compared to cultures transfected with a scrambled siRNA sequence).
