Figure 3.
Figure 3. Combined deletion of A20 and ABIN-1 sensitizes enteroids to TNF-induced cell death. (A) Immunoblot analyses of A20 and ABIN-1 expression in enteroids isolated from villin-ER/Cre+ mice of the indicated genotypes and treated with 4-OHT for 24 h followed by stimulation with 2.5 ng/ml TNF for the indicated time points. (B) Representative confocal microscopy images of PI-stained enteroids from indicated genotypes of mice treated with 4-OHT for 24 h followed by 24 h of 2.5 ng/ml TNF versus mock treatment as indicated. Bars, 75 or 100 µm, as indicated. (C) Quantitation of cell viability of enteroid cultures described in B (mean ± SD). (D) qPCR analysis of TNF mRNA from enteroid cultures with the indicated genotypes after 24 h of 4-OHT treatment (mean ± SD). (E) Dose–response curve of TNF-induced cytotoxicity (mean ± SD). Cell viability of enteroids with the indicated genotypes treated for 24 h with 4-OHT followed by 24 h of TNF at the indicated concentrations versus mock treatment. Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing the effect of each concentration to villin-ER/Cre− or villin-ER/Cre+ enteroids as indicated. (F) Cell viability of organoids of the indicated genotypes treated with 4-OHT for 24 h followed by stimulation with TWEAK, FasL, or TRAIL for 24 h (mean ± SD). For C, E, and F, viability was measured by using the Cell-Titer Glo 3D assay, and data were normalized to mock-treated cells (%Mock). (C–E) Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing the effect to villin-ER/Cre− or villin-ER/Cre+ enteroids as indicated. (F) Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing each stimulation to 4-OHT alone. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are representative of at least two independent experiments.

Combined deletion of A20 and ABIN-1 sensitizes enteroids to TNF-induced cell death. (A) Immunoblot analyses of A20 and ABIN-1 expression in enteroids isolated from villin-ER/Cre+ mice of the indicated genotypes and treated with 4-OHT for 24 h followed by stimulation with 2.5 ng/ml TNF for the indicated time points. (B) Representative confocal microscopy images of PI-stained enteroids from indicated genotypes of mice treated with 4-OHT for 24 h followed by 24 h of 2.5 ng/ml TNF versus mock treatment as indicated. Bars, 75 or 100 µm, as indicated. (C) Quantitation of cell viability of enteroid cultures described in B (mean ± SD). (D) qPCR analysis of TNF mRNA from enteroid cultures with the indicated genotypes after 24 h of 4-OHT treatment (mean ± SD). (E) Dose–response curve of TNF-induced cytotoxicity (mean ± SD). Cell viability of enteroids with the indicated genotypes treated for 24 h with 4-OHT followed by 24 h of TNF at the indicated concentrations versus mock treatment. Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing the effect of each concentration to villin-ER/Cre− or villin-ER/Cre+ enteroids as indicated. (F) Cell viability of organoids of the indicated genotypes treated with 4-OHT for 24 h followed by stimulation with TWEAK, FasL, or TRAIL for 24 h (mean ± SD). For C, E, and F, viability was measured by using the Cell-Titer Glo 3D assay, and data were normalized to mock-treated cells (%Mock). (C–E) Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing the effect to villin-ER/Cre− or villin-ER/Cre+ enteroids as indicated. (F) Statistical significance was assessed by two-way ANOVA with Dunnett’s multiple comparison test comparing each stimulation to 4-OHT alone. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are representative of at least two independent experiments.

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