Figure 6
- ID
- ZDB-FIG-230518-118
- Publication
- Parameswaran et al., 2023 - Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2α-dependent ISR in C9ORF72 FTD/ALS
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(A) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing (G4C2)75 or (G4C2)2 repeats. (B) Representative images of sense and antisense RNA foci in HEK293T expressing (G4C2)75 repeats together with either control antisense oligonucleotides (ASOs), ASOs targeting sense G4C2RNAs or ASOs targeting antisense C4G2 repeat expanded RNAs. Foci were detected by RNA FISH. Red, foci; blue, DAPI. (C) Immunoblotting and (D) quantification of p-PKR and p-eIF2α in HEK293T cells expressing (G4C2)75 or (G4C2)2 repeats together with either control ASO or ASOs targeting sense G4C2 repeat expanded RNAs. Phosphorylated PKR levels were detected using anti-p-PKR (phosphor T446) and normalized to total PKR. Phosphorylated eIF2α levels were detected using anti-peIF2α (Ser51) and normalized to total eIF2α. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using unpaired Student’s t-test. (E, F) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing (G4C2)75 or (G4C2)2 repeats together with either control ASO or ASOs targeting antisense G4C2 repeat expanded RNAs. P-PKR (T446) and p-eIF2α (Ser51) were normalized to total PKR and eIF2α, respectively. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. (G) Representative images and (H) quantification of FMRP and G3BP1 staining in HEK293T cells expressing (G4C2)75 or (G4C2)2 repeats in the presence and absence of antisense ASO2. Error bars represent SD (n = 150 cells/condition and three independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. Scale bars, 20 µm.
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