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. 2024 Dec 5;8(2):e202402757. doi: 10.26508/lsa.202402757

Figure 1. RuvBL overexpression reduces C9orf72-associated dipeptide repeat (DPR) proteins in vitro.

Figure 1.

(A, B, C, D) HeLa cells transfected with empty vector control (Ctrl), FLAG-RuvBL1, or HA-RuvBL2 (A, B, C, D) were co-transfected with empty vector (A) or AUG-driven synthetic codon-optimised V5-tagged 100 repeat poly(GA) (B), poly(GR) (C), or poly(PR) (D) DPR expressing constructs. RuvBL overexpression was confirmed via immunoblot with GAPDH indicating equal loading of samples. Levels of V5-tagged DPRs were determined via dot-blot. DPR levels were normalised to GAPDH and plotted relative to empty vector transfected control (mean ± SEM; one-way ANOVA with Tukey post-test: *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001; N = 3 independent experiments). (E) HeLa cells transfected with empty vector control (ev), FLAG-RuvBL1, or HA-RuvBL2 were co-transfected with empty vector or with 45 uninterrupted sense GGGGCC repeats (45xG4C2) with V5-tags in all three reading frames. RuvBL overexpression was confirmed via immunoblot with GAPDH indicating equal loading of samples. Levels of repeat-associated non-AUG translated V5-DPRs were determined via dot-blot. DPR levels were normalised to GAPDH and plotted relative to empty vector transfected control (mean ± SEM; one-way ANOVA with Tukey post-test: *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; N = 4 independent experiments).