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First published online 28 June 2005
doi: 10.1242/jcs.02440

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Cell-cycle-dependent resistance to Bacillus thuringiensis Cry1C toxin in Sf9 cells

Department of Plant Sciences, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

View larger version (173K): [in a new window]   Fig. 1. Sf9 cells gain temporary Cry1C-tolerance during mitosis and early G1 phase. Log-phase Sf9 cells were incubated with 500 ng/ml Cry1C and photographed every 3 minutes. (A,B) Two microscopic fields with dividing cells (indicated by arrows) that were Cry1C-insensitive and progressed in their divisions. Inserts show the magnified regions (x2.3) to their right. After division and in the presence of Cry1C, daughter cells remained alive at least 300 minutes (A) or up to 22 hours (B). The other non-dividing cells gradually died within 180 minutes. A mean value of dividing cells in percent (1.58±0.95) was estimated by counting 10 different cell cultures.

View larger version (29K): [in a new window]   Fig. 2. G2-M-arrested Sf9 cells survived treatment with Cry1C. Normal Sf9 cells and G2-M-arrested Sf9 cells were exposed to increasing concentrations of Cry1C for 4 hours. Dead and viable cells were counted (three repeated experiments). Dose-dependent mortality curves (A) were used to estimate LC50 (B). The control curve, data from untreated cells exposed to a mixture of Cry1C (increasing concentrations) and nocodazole (10 µg/ml), indicates that nocodazole has no effect on Cry1C-activity.

View larger version (122K): [in a new window]   Fig. 3. Sf9 cells gradually regain Cry1C-sensitivity while progressing through the cell-cycle and recovering from G2-M arrest. (A) Normal (unarrested) Sf9 cells (left) and Sf9 cells released from the G2/M arrest after nocodazole had been washed off (right) and (B) G2-M-arrested cells. All cells were exposed to 300 ng/ml Cry1C for 18 hours. Dying cells with a disrupted plasma membrane were detected by staining nucleic acids with ethidium homodimer-1 (red fluorescence). (C) Time course of Cry1C-induced cell death in the three treatments shown in A and B. Most of the untreated Sf9 cells (90%) died within 6 hours of Cry1C-exposure (). Together with cell-cycle progression in cells released from G2-M arrest, a gradual sensitivity to Cry1C was regained, detected by dead red-stained cells that started to appear after 8-9 hours of Cry1C exposure (). Very low cell mortality was observed in G2/M-arrested cells following application of Cry1C ().

View larger version (57K): [in a new window]   Fig. 4. Comparison of lipid rafts of untreated and G2-M-arrested Sf9 cells. Lipid rafts were isolated from unarrested and G2-M arrested Sf9 cells (lanes 2-4 and 5-7, respectively). The lipid raft fractions of G2-M cells were concentrated 30-fold after isolation. (A) Immonoblot analysis of proteins from lipid-raft- and soluble-membrane fractions probed with anti-human Cav-1 Ab. Lane 1 shows lipid raft proteins isolated from the human HT-29-MDR cell line as a control of caveolin-1 presence (Lavie et al., 1998). Lanes 2-7 represent fraction 4, 5 and 6 of the sucrose gradient that contained lipid rafts. Lanes 8 and 9 correspond to the soluble membrane fraction (fraction 11). Caveolin-1 oligomers are indicated by arrowheads. (B) Silver staining of an identical gel. Lane 1 shows protein-size marker. (C) Comparison of caveolin-immunodetected patterns of Sf9 cells (lanes 1 and 3) and human HT-29-MDR cells (lanes 2 and 4). Western blot analysis of total protein extracts incubated with anti-human Cav-1 Ab. Lanes 1 and 2 show caveolin monomers and oligomers of Sf9 cells and HT-29-MDR cells, respectively. Lanes 3 and 4 show that the caveolin bands disappear from the same blot upon probing with the same amount of anti-human Cav-1 Ab that was incubated earlier with an equivalent amount of HT-29-MDR total proteins under the same conditions to pre-absorb the anti Cav-1 Ab.

View larger version (55K): [in a new window]   Fig. 5. Reduced Cry1C binding to G2-M-arrested Sf9 cells. (A,C) Cry1C (1 µg/ml in A and 2.5 µg/ml in C) was incubated with normal and G2-M-arrested cells for 5 or 90 minutes. After washing off the non-bound toxin, total proteins (9 µg/lane) were separated by SDS-PAGE and Cry1C binding was detected by western analysis using anti-Cry1C Ab. (Lane 1) Cry1C marker, 100 ng. (Lane 2) Sf9 cells with no Cry1C in the binding reaction. (Lane 3) Sf9 cells after 5 minutes incubation with Cry1C). (Lane 4) G2-M cells after 5 minutes incubation with Cry1C. (Lane 5) Sf9 cells after 90 minutes incubation with Cry1C. (Lane 6) G2-M cells after 90 minutes incubation with Cry1C. Quantification of band-intensity (indicated below the corresponding lanes in arbitrary units) showed that G2-M cells bound 10 times less Cry1C than the untreated cells. (B) Blot shown in (A) probed with anti-actin Ab as a loading control.

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