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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Nephrology (Carlton). 2019 Apr 29;24(6):661–669. doi: 10.1111/nep.13487

Figure 4. Absence of RIPK3 and caspase-8 augments caspase 3 and 9 activities and sensitizes TEC to intrinsic apoptotic death.

Figure 4.

Wild type, RIPK3−/−, and DKO TECs were cultured as described in the method and treated with combination of 4 ng/mL of IL-1β and 120 ng/mL of IFN-γ for 24 hours. TEC were also treated with 1 hour pre-incubation of 50 μM BIP. A. Cell viability was measured and quantified using MTT assay analyses. B. Relative cell death restoration after BIP treatment. Cell viability in cytokines-treated TEC without BIP was used as reference. Each figure represents mean of quadruplicates. Four independent experiments showed a similar response (n=4, *: p < 0.05, **: p<0.01, ****: p<0.0001, Two-Way ANOVA). C. TECs were grown to confluent monolayers in triplicates and treated with recombinant murine IL-1β and IFN- γ with or without BIP for 24 hrs. 70 μl of Caspase-Glo-9 (Promega) was added directly to the TEC cultures. Luminescence emission was detected after 1 hour using a VictorX Light (PerkinElmer). Three independent experiments showed a similar result. D. Cleaved caspase-3 activity was measured using CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent (Essen Bioscience). Fluorescent intensity (caspase 3 activity) was detected using Incucyte ZOOM (Essen Bioscience) live cell imaging at 24 hours (n=3. *: p < 0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, Two-Way ANOVA). Three independent experiments showed a similar result.