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First published online May 24, 2004
doi: 10.1242/10.1242/jcs.01126

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Endoplasmic reticulum stress-induced programmed cell death in soybean cells

Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35131 Padova, Italy

View larger version (37K): [in a new window]   Fig. 1. Expression of ER proteins in response to CPA treatment. Protein extracts from untreated and CPA-treated soybean cells were analyzed by western blotting using anti-BiP (A), anti-CRT (B) and anti-caspase 12 (C) antibodies. Quantitative analyses were carried out as described in Materials and Methods and are represented by histograms: white bars, untreated cells (Co); grey bars, CPA-treated cells (CPA). In C the quantitative data refers to the procaspase 12-like (proc 12) protein band. Cells were treated with 50 µM CPA for 24 hours. The values for untreated cells were taken as 100%. Data are mean±s.d. of three independent experiments. Statistically significant at *P<0.05 and **P<0.005.

View larger version (34K): [in a new window]   Fig. 2. Ca2+ responses of soybean cells to 50 µM CPA treatment. (A) Black trace, [Ca2+]cyt elevations induced by CPA in soybean cells grown in normal culture medium; grey trace, [Ca2+]cyt elevations induced by CPA in soybean cells incubated in Ca2+-free medium containing 100 µM EGTA. (B) Black trace, [Ca2+]cyt elevations induced by CPA in soybean cells grown in normal culture medium; grey trace, [Ca2+]cyt elevations induced by CPA in soybean cells pre-treated for 1 hour with 200 nM bafilomycin A1. Traces are representative of three independent experiments, which gave very similar results. Arrow, time of CPA administration.

View larger version (21K): [in a new window]   Fig. 3. Effect of CPA on viability of soybean cells. Exponentially growing cells were treated with CPA (50 µM) for 24 hours (grey bars). Pre-treatment with 10 µM BAPTA-AM was carried out 1 hour before the 24-hour CPA treatment. Cells cultured without CPA were taken as a control (white bars). Data are means±s.d. of three independent experiments. Statistically significant at *P<0.05.

View larger version (13K): [in a new window]   Fig. 4. H2O2 accumulation in the culture medium in response to CPA treatment. Co, untreated cells; CPA, CPA-treated cells (50 µM). H2O2 concentration is normalized per g fresh weight. Traces represent the mean±s.d. of three independent experiments.

View larger version (20K): [in a new window]   Fig. 5. Cytochrome c release. Cytochrome c accumulation in the cytosol was assessed by western blot analysis: control, cytosolic extracts from untreated cells; CPA, cytosolic extracts from cells incubated with 50 µM CPA for 24 hours.

View larger version (18K): [in a new window]   Fig. 6. Caspase 9-like activity in soybean cells treated with CPA. White bar, untreated cells; grey bar, cells treated for 24 hours with 50 µM CPA; black bar, cells treated for 24 hours with 50 µM CPA in the presence of the caspase 9 inhibitor Ac-LEHD-CHO (20 µM). Caspase 9-like activity in untreated cells was taken as 100%. Data are means±s.d. of three independent experiments. Statistically significant at **P<0.005.

View larger version (20K): [in a new window]   Fig. 7. Assay for caspase 3 enzymatic activity in soybean cell-suspension culture. White bar, untreated cells; grey bar, cells treated for 24 hours with 50 µM CPA; black bar, cells treated for 24 hours with 50 µM CPA in the presence of the caspase 3 inhibitor Ac-DEVD-CHO (20 µM). Caspase 3-like activity in untreated cells was taken as 100%. Data are means±s.d. of three independent experiments. Statistically significant at **P<0.005.

View larger version (86K): [in a new window]   Fig. 8. Changes in nuclear morphology in response to CPA treatment. Nuclei of 24-hour CPA-treated cells were stained with Hoechst 33342 and propidium iodide and processed for fluorescent microscopy. (A-C) Untreated cells. (D-I) 50 µM CPA-treated (24 hours) cells. (E,F) Early PCD nucleus. (H,I) Late PCD nuclei. Pictures represent typical examples. cc, chromatin condensation; cw, cell wall; nu, nucleus; pm, plasma membrane. The arrow indicates the detachment of the plasma membrane from the cell wall in CPA-treated cells.

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