Figure 5.

Loss of NCLX increases _mCa²⁺ overload, aggregate formation, and cell death

(A) Schematic for generation of NCLX knockout cell line (NCLX^−/−) using CRISPR/SpCas9.

(B) NCLX mRNA expression in NCLX^−/− and controls (WT) N2a cells, corrected to the housekeeping gene, Rps13; expressed as fold change versus control, n = 3 for both groups. All data presented as mean ± SEM; ∗∗∗∗p<0.001; two-tailed, unpaired t-test.

(C) Representative traces for basal _mCa²⁺ content.

(D and E) (D) Quantification of _mCa²⁺ content, n = 3 for both groups. All data presented as mean ± SEM; ∗∗p<0.001; two-tailed, unpaired t-test(E). Representative recordings of _mCa²⁺ retention capacity.

(F and G) (F)Percent change in _mCa²⁺ retention capacity versus N2a control cells, n = 3 for both groups. All data presented as mean ± SEM; ∗p<0.05; two-tailed, unpaired t-test(G). Representative images of intracellular protein aggregates in NCLX^−/− and WT cells stained with Proteostat aggresome detection reagent (red) and Hoechst 33,342 nuclear stain (blue), scale bars = 20-μm.

(H) Total aggregates per cell, n = 96 for NCLX^−/− and n = 58 for WT N2a control. All data presented as mean ± SEM; ∗∗∗∗p<0.001; two-tailed, unpaired t-test.

(I and J) NCLX^−/− and WT cells were assessed for plasma membrane rupture, Sytox Green, after treatment with (I). Ionomycin (10–40 μM), (J). thapsigargin (10–50 μM), n = 10 for both groups. All data presented as mean ± SEM; ∗∗∗p<0.001, ∗∗p<0.01, ∗p<0.05; two-way ANOVA with Sidak’s multiple comparisons test.