After permeabilizing with 0.3% TX-100, samples were blocked with 4% BSA and incubated with primary antibodies: PDI, CHOP (Cell Signaling), C/EBP β and ATF4 (Santa Cruz Biotechnology).
Cardiomyocytes were stained with monoclonal antibodies to ATF6 (Abcam), cytochrome c, CHOP (Cell Signaling), polyclonal antibodies to XBP1 (Santa Cruz) and sarcomeric α-actinin (Abcam), followed by incubation with labeled secondary antibodies (Alexa fluor 488, Alexa fluor 594, Alexa fluor 633; Molecular Probes, Eugene, OR, USA), as described earlier.
Membranes were incubated with anti- β-actin (Sigma, Madrid, Spain), anti-Bim (Cell Signalling, IZASA), anti-CHOP (Cell Signalling) or anti-phospho (Thr180/Tyr182 -p38MAPK (Cell Signalling) primary anThr180/Tyr182 -p38MAPKdilutions indiCelld by the manufacturer.
Membranes were blocked with 10% dry milk in TBS solution for 2 h at room temperature (RT) and then probed overnight at 4 °C or for 2 h at RT with primary antibodies: anti-PARP, anti-caspase-3, anti-LC3, anti-BiP or anti-Akt (Cell Signaling, Danvers, MA, USA) or anti-p62, anti- β-actin (Sigma-Aldrich) in 5% dry milk or 5% BSA for anti-CHOP (Cell Signaling) in TBS.
As shown in Additional file 1: Figure S2, we found that WZ35 also induced ROS accumulation, CHOP expression, cell apoptosis and G2/M arrest in DU145 cells.
Next, we investigated the role of CHOP in cell death mediated by PDT+EGF-SubA in DU-145 cells.
To ascertain the role of CHOP in cell death induction, we transiently transfected empty vector (ctr) or dominant-negative form of CHOP (DN-CHOP) in U2OS cells.
As expected, the knock-down of CHOP increased cell viability, especially in OASF.
Given the impact of CHOP on cell death, this effect likely explains the protective role of PON2, at least in part and in response to ER stress.
It has been shown that CHOP-induced cell death is associated with the translocation of Bax from the cytosol to the mitochondria and that CHOP-induced cell death can be prevented by the overexpression of Bcl-2 or the knockdown of Bax [ 52].
Additional parallel proapoptotic signaling pathways must also exist given the continued sensitivity of Perk and Chop null cells to ER protein misfolding [9], [13].
