Article Figures & Data
Figures
- FIGURE 1.
TUNEL assay of cell death in chB6-transfected FL5.12 cell lines. Apoptotic cell death was measured by TdT-mediated incorporation of dUTP into fragmented DNA (TUNEL). FL5 cells stably transfected with chB6.1 or chB6.2 cDNA were cultured in the presence or the absence of IL-3 for 16 h. Abs to chB6.1 or chB6.2 were added during the last hour of culture. Cells (105) were spun onto glass slides and stained via the TUNEL method. Counts were performed in triplicate, with data presented as the mean ± SD, and the values shown are representative of nine independent experiments for chB6.1 transfectants (A) and six independent experiments for chB6.2 transfectants (B). Control TUNEL reactions in the absence of TdT enzyme yielded no staining in any culture condition.
- FIGURE 2.
Influence of Bcl-xL on chB6-induced cell death in FL5.12 cell lines. A, Representative Western blot confirming overexpression of 32-kDa Bcl-xL in murine FL5.12 cell lines. All lanes were loaded with 25 μg protein. Lane 1, Lysate from FL5.12 cells transfected with chB6.1. Lane 2, Lysate from FL5.12 cell line transfected with chB6.1 and a vector containing Bcl-xL. Lane 3, Lysate from FL5.12 cell line transfected with vector containing Bcl-xL (provided by Dr. L. Boise, University of Miami). Native Bcl-xL is a 32-kDa protein seen as the lower band in each cell lysate, but clearly in higher concentration in cells transfected with Bcl-xL. Parallel experiments were performed with lysates from cell lines expressing chB6.2 with similar results. B and C, FL5 transfectants expressing only chB6 or chB6 and Bcl-xL were cultured in the presence or the absence of IL-3. Allele-specific Ab to chB6 was administered at 15 h of incubation. At the indicated intervals samples were withdrawn, and cell viability was determined by trypan blue exclusion. Counts were performed in triplicate, with data presented as the mean ± SD. Data are representative of nine independent experiments for chB6.1 and six independent experiments for chB6.2.
- FIGURE 3.
TUNEL assay of cell death in FL5.12 cell lines overexpressing Bcl-xL. Cells were cultured for 16 h in the presence or the absence of IL-3, and Abs to chB6 were added during the last hour of culture. At 16 h of culture 105 cells were spun onto glass slides, and apoptotic cells were detected with TUNEL assay. For each experiment three fields containing at least 200 cells each were counted for each treatment. Data from three independent experiments were pooled and are presented as the mean ± SD.
- FIGURE 4.
Caspase 8 cleavage in cell lines treated with anti-chB6 Ab. A, Representative Western blot for determination of caspase 8 cleavage. Ratio of cleaved (18 kDa) to uncleaved (55 kDa) caspase 8, as detected by densitometry, in cell lines expressing chB6.1 with or without supertransfected Bcl-xL (B) or chB6.2 with or without supertransfected Bcl-xL (C). Cells were cultured with or without IL-3 for 16 h, with allele-specific Ab added during the last hour of incubation. Caspase 8 cleavage was determined by Western blot, and the ratio of cleaved (18 kDa) to uncleaved (55 kDa) caspase 8 was determined by densitometric analysis. The data in B are presented as the mean of two experiments. In C, the data are presented as the mean ± SD of three independent experiments. ∗, p < 0.05; ∗∗, p < 0.1 (by independent t test, compared with cultures containing IL-3 without anti-chB6 Ab).
- FIGURE 5.
Caspase 3 cleavage in cell lines treated with anti-chB6 Ab. A, Representative Western blot for determination of caspase 3 cleavage. Ratio of cleaved (20 kDa) to uncleaved (32 kDa) caspase 3, as detected by densitometry, in cell lines expressing chB6.1 with or without supertransfected Bcl-xL (B) or chB6.2 with or without supertransfected Bcl-xL (C). Cells were cultured with or without IL-3 for 16 h, with allele-specific Ab added to samples during the last hour of culture. Caspase 3 cleavage was determined by Western blot, and ratio of cleaved to uncleaved caspase 3 was determined by densitometric analysis. Data are presented as the mean ± SD of three independent experiments.
- FIGURE 6.
PARP inactivation in cells treated with anti-chB6 Ab. A, Representative Western blot for determination of PARP cleavage. The ratio of cleaved (89 kDa) to uncleaved (112 kDa) PARP, as detected by densitometry, was determined in cell lines expressing chB6.1 with or without supertransfected Bcl-xL (B) or chB6.2 with or without supertransfected Bcl-xL (C). Cells were cultured for 16 h with or without IL-3. Abs were added to the indicated cultures at 15 h. PARP cleavage was determined by Western blot analysis, and the ratio of cleaved to uncleaved PARP was determined by densitometric analysis. Data are presented as the mean ± SD of three independent experiments.
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