Fig 2: UPF1 OE reduces C9orf72 HRE-mediated neurodegenerative phenotypes(A) Representative images of Drosophila eyes expressing the noted UPF1 expression modifiers in photoreceptors using a GMR driver. Top images are from flies expressing the noted alleles only, and bottom images are from flies expressing the noted alleles in the context of 30 GGGGCC repeats under the same driver. (B) Violin plots of eye degeneration scores of 30R flies expressing the noted UPF1 alleles. See Method details for details on scoring method. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ****p < 0.0001. From left to right, n = 108, 54, 101, 15, 60, 15. Data are represented as violin plots with indicated quartiles. (C) Representative fields of view of propidium-iodide-positive iPSNs in the absence (top row) or presence (bottom row) of 10 μM glutamate. Scale bar, 100 μm. (D) Quantification of the relative proportion of propidium-iodide-positive (dead) cells to total cells from images in (C), represented as “% Cell Death.” n = 3 pairs of age- and sex-matched control and C9orf72 iPSNs, 2 replicates per pair. Six fields of view were analyzed for each data point. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. *p < 0.05, ****p < 0.0001.Data are indicated as mean ± SD. See also Figure S3.

Fig 3: NMD targets the cellular UPR pathway to restrict transcription of ORF50.a Quantification of sXBP1 expression by RT-qPCR in latent (Dox−) and lytic (Dox+) TREx-BCBL1-RTA cells treated with the indicated siRNAs. b Western blot of cell lysates prepared from latent (Dox−) and lytic (Dox+) TREx-BCBL1-RTA cells. sXBP1, spliced XBP1; uXBP1, unspliced XBP1. c Quantification of IRE1α expression by RT-qPCR in latent (Dox−) and lytic (Dox+) TREx-BCBL1-RTA cells treated with the indicated siRNAs. d, e Quantification of XBP1 occupancy at the ORF50 promoter in latent (d) and lytic (e) TREx-BCBL1-RTA cells by ChIP-qPCR. Signals were normalized to input. f, g RNA was isolated from the indicated TREx-BCBL1-RTA cells and the expression of UPF1 (f) and sXBP1 (g) was quantified by RT-qPCR. h Western blot of cell lysates prepared from of latent (Dox−) and lytic (Dox+) TREx-BCBL1-RTA cells in the condition of UPF1 depletion and KIRA6 treatment. i Quantification of ORF50 expression in the indicated TREx-BCBL1-RTA cells. j Quantification of PAN RNA expressing cells by PAN FISH-FLOW in latent (Dox−) and lytic (Dox+) TREx-BCBL1-RTA cells following UPF1 depletion and KIRA6 treatment. k The model depicting how NMD regulates the expression of ORF50 at both transcriptional and post-transcriptional level. Data are presented as mean values ± SD (n = 3 biologically independent samples). p-values were determined by the two-tailed Student’s t-test. Source data are provided in Source Data file.

Fig 4: C9orf72 HRE sense RNA is in a complex with UPF1 and is stabilized by NMD inhibition(A) Relative abundance of sense repeat RNA following treatment with 0.5 μM SMG1i for the indicated periods of time (24 h 0.1% DMSO treatment used for normalization). n = 4 C9-ALS iPSN lines. Ordinary one-way ANOVA was used to calculate statistical significance. **p < 0.01. (B) Western blot against total UPF1 protein in samples following anti-UPF1 IP from C9-ALS iPSN lysates. Short and long exposures are shown on the top and bottom, respectively. (C) Fold enrichment of sense repeat RNA and ATF4 mRNA relative to GAPDH in IP fraction following anti-UPF1 pulldown as measured by qRT-PCR (input used for normalization). n = 5 C9-ALS iPSN lines. Ordinary one-way ANOVA was used to calculate statistical significance. *p < 0.05. Data are indicated as mean ± SD.See also Figure S1.

Fig 5: Examination of dnUPF1 protein expression in transgenic mouse tissues. (A) Representative western blot of dnUPF1 (green; antibody to HA epitope tag), total UPF1 (red; antibody to both human UPF1 and mouse Upf1), and tubulin (green; for normalization) in transgenic mice either vehicle alone or treated with 500 mg/ml doxycycline (dox) administration for 7 days. (B) Quantitation of total UPF1 protein by western blotting. Protein levels are expressed as the mean fold change (±s.d.) in protein levels from dox-treated mice relative to vehicle-treated mice (indicated by dashed line) and performed in triplicate from at least three mice per cohort. Exact P-values were calculated using an unpaired, two-tailed t-test and are shown in the figure. Note that multiple UPF1 bands are observed in some tissues, which is consistent with the presence of different isoforms due to alternative splicing. Yellow asterisks indicate the bands representing dnUPF1 in tissues with multiple isoforms.