Skip to main content
. 2023 Jan 5;110(1):146–160. doi: 10.1016/j.ajhg.2022.12.003

Figure 2.

Figure 2

NAE1 abundance and neddylated to non-neddylated cullin ratio are reduced in individuals with NAE1 variants

(A) Western blots showing NAE1, neddylated, and non-neddylated cullin 1/cullin 3 abundance in dermal fibroblasts of healthy control subjects (2 healthy controls [C1 and C2]; 2 biological replicates per healthy control [C1.1, C1.2, C2.1, C2.2]; 6 technical replicates each), heterozygous carriers (father and mother of individual 1; 5 technical replicates each), and the fibroblasts of individual 1 with bi-allelic NAE1 genetic variants (6 technical replicates). Bar graphs represent the average band intensity of all technical replicates per donor ± SEM. All band intensities were normalized to total protein content. Quantification was performed using ImageJ. One way ANOVA, Post-hoc: Dunnet’s multiple comparisons test (∗∗p < 0.01, ∗∗∗p < 0.001).

(B) Graphic showing the neddylation and deneddylation cycles of cullins.

(C) Gene set enrichment analysis of the pre-ranked (based on differential expression score) transcriptomic dataset of individual 1- and 2-derived fibroblasts compared to 3 healthy control fibroblast lines. For this analysis, all genes were taken into account. The top 10 most significantly enriched (based on FDR (Q-value); blue, downregulated; red, upregulated) pathways are shown. Pathways in bold are associated with protein degradation and proteasome function. Pathways were aligned to the GO Ontology database (https://doi.org/10.5281/zenodo.4495804, released 2021-02-01).

(D) Cell death in fibroblasts derived from individuals 1 and 2, parents of individual 1, and healthy control subjects, after treatment with MG132 for 24 h at the indicated concentrations (in μM). Cell death was assessed by dividing the amount of PI-positive cells by the total amount of cells. Error bars represent SEM of 3 technical replicates of 2 donors for each group. two-way ANOVA. Post-hoc: Fisher’s least significance difference test (LSD) (∗∗∗∗p < 0.0001).

(E) Cell death in fibroblasts derived from individuals 1 and 2 (NAE1−/−) with and without a vector expressing WT NAE1 after treatment with MG132 for 24 h at the indicated concentrations (in μM). Cell death was assessed by dividing the amount of PI-positive cells by the total amount of cells. Errors bars represent SEM of 3 technical replicates of 2 donors for each group. Two-way ANOVA. Post-hoc: Fisher’s LSD (∗∗∗∗p < 0.0001).

(F) Cell death in fibroblasts derived from parents of individual 1 (NAE1+/−) with and without addition of NAE1 shRNA after treatment with MG132 for 24 h at the indicated concentrations (in μM). Cell death was assessed by dividing the amount of PI-positive cells by the total amount of cells. Error bars represent SEM of 3 technical replicates of 2 donors for each group. Two-way ANOVA. Post-hoc: Fisher’s LSD (∗∗p < 0.01).