Fig. 7: CR8 attenuates etoposide-induced mitochondrial dysfunction and mitochondrial permeabilization in primary neurons. | Cell Death & Disease

Fig. 7: CR8 attenuates etoposide-induced mitochondrial dysfunction and mitochondrial permeabilization in primary neurons.

From: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury

Fig. 7

Neurons were treated with 50 μM of etoposide ± 1 μM CR8. Fractions and whole lysates were collected 24 h later and proteins were separated on SDS-polyacrylamide gels, then immunodetected using antibodies against AIF-1, cytochrome C, cleaved caspase-9, Apaf-1, and Mcl-1. Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared with control untreated levels. In parallel, 6 h after treatment cellular respiration measurements were made with a Seahorse XF24 Extracellular Flux Analyzer before and after sequential additions of oligomycin, FCCP, pyruvate, and antimycin A at the indicated times (representative experiment (a)). CR8 rescues maximal respiration (b) and spare respiratory capacity (c) compared to etoposide treatment alone. n = 4 averages from separate days of experiments; on each day, n = 4 technical replicates for control, n = 5 technical replicates for all other groups. CR8 attenuates the increase in AIF-1 and cytochrome C in the cytosolic fraction and cleavage of caspase-9 in total lysate of rat primary cortical neurons following etoposide treatment (d). Western images (e) quantified in d. No change in Apaf-1 relative to controls was observed until 24 h. Both etoposide and etoposide + CR8 decreased Mcl-1 at 6 h, at 24 h etoposide alone increased relative to etoposide + CR8 (d). n = 3/group for all groups for westerns. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, *p < 0.05 vs. control, &p < 0.05 vs. etoposide + CR8 at the same time point

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