Fig. 1: zVAD-fmk induces necroptosis in BV2 cells at 24 h of incubation.

a BV2 cells were pre-exposed to 100 ng/mL LPS for 24 h and then incubated with the pan-caspase inhibitor zVAD-fmk (25 µM) for additional 24 h. Nec-1 (30 µM) was added 1 h before zVAD-fmk. Cell metabolic activity was determined by the MTS metabolism assay and cell membrane integrity by the LDH activity assay. Results are presented as the mean value ± SEM of three independent experiments performed in duplicates and normalized to control cells. *p < 0.001 vs. control; ^§p < 0.001 vs. LPS; ^Ɨp < 0.001 vs. zVAD-fmk; ^ǂp < 0.001 vs. LPS/zVAD-fmk. b BV2 cells were co-incubated with zVAD-fmk (25 µM) plus Nec-1 (30 µM) for 24 h. Detergent soluble and insoluble fractions were prepared for Western blot analysis of RIP1 and RIP3. β-actin was used as loading control. Representative immunoblots are presented