Figure 3.
Figure 3. An inducible form of Bcl10 conditionally enhanced the survival of WEHI-231 cells. (A) Cells stably expressing a Bcl10-ER fusion protein were treated with ethanol (solvent) or varying concentrations of the inducer, estradiol (E2), and stimulated with 10 μg/mL anti-IgM for 48 hours. The numbers of viable cells (PI-) were then determined in triplicate by flow cytometry. Examination of WEHI-231 cells expressing ER (ligand binding domain only) via short-term retroviral infection revealed no effect of E2 on BCR-induced apoptosis (data not shown). Shown in insert is an anti-HA immunoblot of the HA-tagged Bcl10-ER in the stable expressor clone of WEHI-231 and in a “nonexpressor,” that is, a clone derived from drug selection of cells infected with the Bcl10-ER-expressing virus showing an undetectable level of Bcl10-ER expression. (B) Bcl10 activation prevented BCR-induced cell-cycle arrest. Bcl10-ER-expressing cells (4 × 105 cells/mL) were stimulated with anti-IgM (10 μg/mL) and/or treated with E2 (1 μM) to activate Bcl10 for 16 hours, a time prior to onset of apoptosis. The cell-cycle status of the cultures was analyzed by BrdU incorporation (1 hour), anti-BrdU/PI staining, and 2-parameter flow cytometry. The percentage of S-phase cells (ie, BrdU-incorporating cells, see insert) in a culture was used to assess the proliferation of the culture. Shown is a representative of 3 different experiments. (C) Bcl10 CARD induced growth arrest. Cells stably expressing Bcl10 CARD-ER (N4) (1 × 105 cells/mL) were treated with E2 (1 μM) for 4 or 16 hours and then subjected to cell-cycle analysis as in panel B. One of 3 representative experiments is shown. No effects of E2 treatment for 4 hours were detected (data not shown). (D) Prolonged activation of the Bcl10 CARD-ER protein led to apoptosis. Cells expressing Bcl10 CARD-ER, Bcl10-ER, and nonexpressing cells (1 × 105 cells/mL) were treated with E2 (1 μM) or ethanol for 16, 24, 48 hours and then stained with fluorescein-labeled annexin V to detect cells with externalized phosphatidylserine. PI- cells were gated upon to reveal dying but not dead cells. Data shown are triplicate determinations from the 48-hour time point; no effects of E2 treatment on annexin-V staining were detected at the 16- or 24-hour time points. Error bars indicate SEM.

An inducible form of Bcl10 conditionally enhanced the survival of WEHI-231 cells. (A) Cells stably expressing a Bcl10-ER fusion protein were treated with ethanol (solvent) or varying concentrations of the inducer, estradiol (E2), and stimulated with 10 μg/mL anti-IgM for 48 hours. The numbers of viable cells (PI-) were then determined in triplicate by flow cytometry. Examination of WEHI-231 cells expressing ER (ligand binding domain only) via short-term retroviral infection revealed no effect of E2 on BCR-induced apoptosis (data not shown). Shown in insert is an anti-HA immunoblot of the HA-tagged Bcl10-ER in the stable expressor clone of WEHI-231 and in a “nonexpressor,” that is, a clone derived from drug selection of cells infected with the Bcl10-ER-expressing virus showing an undetectable level of Bcl10-ER expression. (B) Bcl10 activation prevented BCR-induced cell-cycle arrest. Bcl10-ER-expressing cells (4 × 105 cells/mL) were stimulated with anti-IgM (10 μg/mL) and/or treated with E2 (1 μM) to activate Bcl10 for 16 hours, a time prior to onset of apoptosis. The cell-cycle status of the cultures was analyzed by BrdU incorporation (1 hour), anti-BrdU/PI staining, and 2-parameter flow cytometry. The percentage of S-phase cells (ie, BrdU-incorporating cells, see insert) in a culture was used to assess the proliferation of the culture. Shown is a representative of 3 different experiments. (C) Bcl10 CARD induced growth arrest. Cells stably expressing Bcl10 CARD-ER (N4) (1 × 105 cells/mL) were treated with E2 (1 μM) for 4 or 16 hours and then subjected to cell-cycle analysis as in panel B. One of 3 representative experiments is shown. No effects of E2 treatment for 4 hours were detected (data not shown). (D) Prolonged activation of the Bcl10 CARD-ER protein led to apoptosis. Cells expressing Bcl10 CARD-ER, Bcl10-ER, and nonexpressing cells (1 × 105 cells/mL) were treated with E2 (1 μM) or ethanol for 16, 24, 48 hours and then stained with fluorescein-labeled annexin V to detect cells with externalized phosphatidylserine. PI- cells were gated upon to reveal dying but not dead cells. Data shown are triplicate determinations from the 48-hour time point; no effects of E2 treatment on annexin-V staining were detected at the 16- or 24-hour time points. Error bars indicate SEM.

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