Figure 4. CuES induced astrocyte toxicity is not mediated by cuproptosis.

All experiments utilized 2 μM copper. Experiments conducted in astrocyte cultures utilized 200 nM ES while those conducted in cultures containing neurons and astrocytes utilized 300 nM ES. Inhibition of mitochondrial respiration with (A) 300 nM antimycin A (AA), a complex III inhibitor, is insufficient to rescue CuES toxicity in primary astrocytes (F(1.052, 2.105)=103.1, repeated measures one-way ANOVA, p=0.0080, Sidak’s post hoc, control vs CuES, p=0.0054; CuES vs CuES + AA, p=08162, n=3 biological replicates) as is (B) inhibition with 300 μM of the complex IV inhibitor sodium azide (NaAz) (F(1.418, 2.83)=75.11, repeated measures one-way ANOVA, p=0.0037, Sidak post-hoc, control vs CuES, p=0.0128; CuES vs CuES + NaAz, p=0.5748, n=3 biological replicates). (C) Treatment with FDX1 siRNA does not rescue cultures from CuES toxicity as evidenced by significant LDH release (F(1.244, 3.733)= 20.55, repeated measures one-way ANOVA, p=0.0114, Sidak post-hoc, control vs CuES, p=0.0041; CuES vs CuES + NTC siRNA, p=0.9713; CuES vs CuES + CuES + FDX1 siRNA, p=0.9914, n=4 biological replicates), which (D) cannot be explained by a lack of knockdown in siRNA treated cultures (t(3)=6.143, ratio paired two-tailed t-test, p=0.0087, n=4 biological replicates).