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. 2021 Oct 15;12:6025. doi: 10.1038/s41467-021-26303-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic diagram showing ΔC9 ^ubi, C9 ^ubi, and UAG ^ubi constructs with the snb-1 promoter. The orange and green arrows show primer location to detect C9orf72 and nLuc mRNAs, respectively. nLuc: nanoluciferase; UTR: untranslated region. b The G4C2 repeat mRNA (C9orf72 intronic mRNA) and act-1 mRNA were assessed by RT-PCR on three different transgenic lines per genotype: ΔC9 ^ubi (kasEx154, kasEx259, kasEx260), C9 ^ubi (kasEx153, kasEx261, kasEx262), or UAG ^ubi (kasEx155, kasEx263, kasEx264) (mean ± s.e.m.). One-way ANOVA with Dunnett’s multiple comparisons test was performed. c Representative fluorescent images of the head of adult (day 1) ΔC9 ^ubi (kasIs9), C9 ^ubi (kasIs7) and UAG ^ubi (kasIs10) animals. N = 25. G4C2 RNA foci are visualized with a Quasar 670 probe. Nuclei are demarcated with DAPI (blue). Scale bar = 10 μm. G4C2 RNA foci are detected in head neurons and motor neurons of the retrovesicular ganglion (circled) both in C9 ^ubi and UAG ^ubi animals. The signal detected on the left of the region indicated “Head neurons” belongs to non-neuronal cells (no neurons reside that far anterior in the C. elegans head). d, e Western blotting with poly-GA and α-tubulin antibodies and quantification on three different transgenic lines per genotype: ΔC9 ^ubi (kasEx154, kasEx259, kasEx260), C9 ^ubi (kasEx153, kasEx261, kasEx262), or UAG ^ubi (kasEx155, kasEx263, kasEx264) (mean ± s.e.m.). One-way ANOVA with Dunnett’s multiple comparisons test was performed. f Luciferase assay was performed in worm lysates from the same three transgenic lines per genotype shown in panel e (mean ± s.e.m.). g, h ELISA for poly-GP and poly-GR in ΔC9 ^ubi (kasIs8), C9 ^ubi (kasIs7), and UAG ^ubi (kasIs10) animals (n = 4, mean ± s.e.m.). One-way ANOVA with Dunnett’s multiple comparisons test was performed. i Immunofluorescent staining for poly-GA (red) in ΔC9 ^ubi (kasIs9) and C9 ^ubi (kasIs10) adult (day 1) worms. Representative images of the head and ventral nerve cord region taken at ×40 magnification. N = 20. Anterior to the left. Motor neuron nuclei in blue (DAPI) shown with arrowheads. Scale bar = 20 μm.