Figure 6

CHOP, but not Noxa, is critically involved in DMC-induced ER dilation and subsequent cell death. (a) MDA-MB 435S cells transfected with CHOP or Noxa siRNA were further treated with 20 μ M DMC for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. (b) MDA-MB 435 cells were transfected with CHOP or Noxa siRNA and their knockdown was confirmed by western blotting of CHOP or Noxa. Effect of CHOP or Noxa knockdown on ubiquitinated proteins was examined by western blotting using anti-ubiquitin antibody. β-Actin expression was analyzed to confirm equal loading of the protein samples. (c) The sublines expressing the fluorescence selectively in ER (YFP-ER cells/435S) or mitochondria (YFP-Mito cells/435S) were transfected with CHOP siRNA and further treated with 20 μ M DMC for 16 h. Cells were observed under a fluorescence microscope. Bars, 20 μm. (d) The changes in the widths of mitochondria-derived vacuoles and the ER-derived vacuoles by CHOP knockdown were quantitatively measured in YFP-Mito cells and YFP-ER cells treated with 20 μ M DMC for 16 h using AxioVision Rel. 4.8 software. CHOP knockdown significantly reduced the DMC-induced increase in the width of the ER. Results were repeated in three other experiments. In each experiment, 50 cells were scored as described in Materials and Methods section. (e) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) and then treated with 20 μ M DMC for 16 h. Treated cells were processed for immunocytochemistry of CHOP, PDI and COX IV. Bars, 20 μm. (f) Tumors in nude mice treated with DMC (50 mg/kg) or curcumin (50 mg/kg) were collected after 25 days of treatments and tissue extracts were prepared for western blotting using anti-ubiquitin and anti-CHOP antibody. Western blotting of α-tubulin was examined to verify equal loading