Figure 1.
Scheme showing the three major ER-stress sensors: PERK, ATF6 and IRE1 and the link to the targets assayed in this report.
Activated PERK phosphorylates eIF2α to attenuate protein translation but allowing the expression of ATF4-dependent genes, CHOP and ASNS, involved in redox- and apoptosis-related pathways. Cleaved -active- ATF6 leads to induction of molecular chaperones (GPR78) and of the transcription factor XBP1. IRE1 activation leads to XBP1 splicing, transcriptional activation of chaperones and stimulation of protein degradation through ErdJ4, which is a component of the ER-associated degradation (ERAD) system.
Table 1.
Primer sequences used for quantitative PCR.
Figure 2.
Tunicamycin-induced ER stress in SH-SY5Y cells.
A) SH-SY5Y cells were treated for 48 h with the indicated concentrations of tunicamycin and cell viability was determined using a LDH release assay. Bars represent the percentage of LDH release over that obtained in untreated cells: *p<0.05; ***p<0.0001 vs untreated cells. B–C) The transcriptional activity of different sensors was used to monitor the induction of ER stress by tunicamycin in SHSY-5Y (B) and HEK-293T (C) cells. Bars represent the fold change (mean ± SEM) in gene expression normalized to the control untreated cells: *p<0.05; **p<0.005; vs control untreated cells.
Figure 3.
PBA and PVA protect SH-SY5Y cells against tunicamycin induced ER stress.
SH-SY5Y cells were exposed to tunicamycin (500 nm) for 24 h in the absence (medium) or in the presence of PBA (panel A) or PVA (panel B). Cell viability was determined using a LDH release assay and the results are expressed as the means ± SEM. Bars represent the percentage of LDH release over that obtained in untreated cells. **p<0.005; ***p<0.0001 vs tunicamycin treated cells (Medium). Panel C. Immunoblot of procaspase 3 and cleaved caspase 3. A representative image is shown and the bar diagram represents the ratio of cleaved versus total protein (mean ± SEM) in the different conditions and normalizad to the ratio in absence of tunicamycin. ##p<0.005 vs untreated cells; **p<0.005 vs tunicamycin treated cells.
Figure 4.
PBA and PVA neutralize the ER stress sensors induced by tunicamycin.
SH-SY5Y cells were treated as described in the Materials and Methods and the expression of GPR78 (A), spliced XBP1 (B) and ERdJ4 (C) was determined by RT-PCR. The bars represent the expression (mean ± SEM) normalized to that of the corresponding 36b4 internal control: *p<0.05; **p<0.005 vs tunicamycin treated cells.
Figure 5.
Western blots of SHSY-5Y cells treated with PBA or PVA probed with anti-phospho eIF2α (A) and anti-ATF4 (B).
The bars represent the ratio of eIF2α or anti-ATF4 versus β-actin expression and referred to the ratio in tunicamycin-treated cells (mean ± SEM). *p<0.05, vs tunicamycin treated cells.#p<0.05 vs untreated cells.
Figure 6.
Induction of CHOP and ASNS expression in SHSY-5Y tunicamycin ER stressed cells is reversed by PBA or PVA treatment.
Cells were treated for 24 h with tunicamycin and with PBA or PVA for the times indicated. The expression of CHOP and ASNS was determined by RT-PCR and normalized to that of the corresponding 36b4 internal control. Bars represent the mean ± SEM of the relative change with respect to tunicamycin treated cells: **p<0.005 vs tunicamycin treated cells.