PMC

Effect of ethanol on ER stress. SH-SY5Y cells were cultured in serum-free medium and treated with ethanol (Et; 0 or 400 mg/dl) for specified times. Cell lysates were collected, and the expression of markers for ER stress was examined with immunoblotting. To ensure equal loading, the blots were stripped and reprobed with an antiactin antibody. Tunicamycin was used as a positive control for inducing ER stress. The experiment was replicated three times.

Effect of ethanol on intracellular ROS concentrations. A: SH-SY5Y cells were exposed to ethanol (Et; 0 or 400 mg/dl) for specified times, the intracellular ROS concentrations were detected by flow cytometry as described in Materials and Methods. Each data point (±SEM; bar) is the mean of three experiments. B: SH-SY5Y cells were exposed to ethanol (0 or 400 mg/dl) with/without NAC (10 mM) for 2 hr, then treated with tunicamycin (Tun; 0 or 3 µg/ml) for 6 hr. The intracellular ROS concentrations were detected by flow cytometry. *Significant difference from Ct or Tun-treated groups.

Effect of ethanol and tunicamycin on the survival of CHOP null mouse embryonic fibroblasts (CHOP−/− MEFs). CHOP−/− and CHOP+/+ MEFs were pretreated with ethanol (Et; 0 or 400 mg/dl) with/without NAC (10 mM) for 12 hr, then exposed to tunicamycin (Tun; 0 or 3 µg/ml) for 48 hr. Cell viability was determined by MTT as described in Materials and Methods. The number of viable cells was expressed as a percentage of untreated controls. Each data point (±SEM; bar) is the mean of three experiments. *Significant difference from paired groups of CHOP+/+ MEFs.

Effect of CHOP siRNA on ethanol- and tunicamycin-mediated cell death. A: SH-SY5Y cells were transfected with either CHOP siRNAs (320 and 321) or control siRNA (Ct-siRNA) for 24 hr. After that, cells were exposed to tunicamycin (Tun; 0 or 3 µg/ml) for 6 hr. The expression of CHOP was examined with immunoblotting. B: After transfection with CHOP siRNA or a control siRNA (Ct-siRNA) for 48 hr, cells were pretreated with ethanol (0 or 400 mg/dl) with/without NAC (10 mM) for 12 hr and then exposed to tunicamycin (Tun; 0 or 3 µg/ml) for 48 hr. Cell number was determined by counting under the microscope and expressed as a percentage of the untreated control. Each data point (±SEM; bar) is the mean of three experiments. *Significant difference from paired untreated groups or groups treated with Ct-siRNA.

Effect of ethanol on tunicamycin- and thapsigargin-induced ER stress. A: SH-SY5Y cells were pretreated with ethanol (Et; 0 or 400 mg/dl) with/without antioxidant NAC (10 mM) for 12 hr and then treated with tunicamycin (Tun; 0 or 3 µg/ml) for specified times. Cell lysates were collected, and the expression of markers for ER stress was examined by immunoblotting. The blots were stripped and reprobed with an antiactin antibody. B: The notations are the same as in A except that cells were treated with thapsigargin (Tha; 0 or 1 µM). C: Primary cultures of cerebellar granule neurons were pretreated with ethanol (0 or 400 mg/dl) for 12 hr and then exposed to tunicamycin (Tun; 0 or 3 µg/ml) for specified times. The expression of ER stress markers was examined as described above. D: The notations are the same as in C except that cells were treated with thapsigargin (Tha; 0 or 1 µM). The experiment was replicated three times.

Effect of ethanol on tunicamycin- and thapsigargin-induced cell death. A: SH-SY5Y cells were pretreated with ethanol (Et; 0 or 400 mg/dl) with/without antioxidants (10 mM NAC or 5 mM GSH) for 12 hr and then exposed to tunicamycin (Tun; 0 or 3 µg/ ml) for 48 hr. The number of viable cells was determined by counting under the microscope and expressed as a percentage of the untreated control. Each data point (±SEM; bar) is the mean of three experiments. B: The notations are the same as in A except that cells were treated with thapsigargin (Tha; 0 or 1 µM). C: Primary cultures of cerebellar granule neurons were pretreated with ethanol (0 or 400 mg/dl) and/or NAC for 12 hr, then exposed to tunicamycin (Tun; 0 or 3 µg/ml) for 48 hr. The number of viable cells was examined as described above. D: The notations are the same as in C except that cells were treated with thapsigargin (Tha; 0 or 1 µM). *Significant difference from Tun- or Tha-treated groups.