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. 2018 Oct 8;14(10):e1007682. doi: 10.1371/journal.pgen.1007682

Fig 2. C. elegans single-copy/knock-in sod-1 alleles increase SOD1 inclusions in motor neurons.

Fig 2

(A) Top: Cartoon illustration of the motor neurons examined to score hSOD1WT-YFP inclusion formation. Bottom: representative images of sod-1WTM and sod-1G85RM animals expressing hSOD1WT-YFP fusion protein [13] in ventral nerve cord motor neurons. sod-1G85RM animals had small neuronal inclusions that were largely absent in sod-1WTM wild type controls. Paraquat-induced oxidative stress (PQ or ox.) increased the number and size of the inclusions in sod-1G85RM animals. Inclusions in the middle image and right images were defined as small and medium size inclusions, respectively. Arrows point to inclusions. For oxidative stress trials, animals were treated with 2.5 mM paraquat for 3 hours. hSOD1WT-YFP was expressed under the pan-neuronal snb-1 promoter. Scale Bar = 10 um. (B) Single-copy ALS sod-1 mutations generated by MosSCI increased hSOD1WT-YFP inclusions compared to sod-1WTM controls. Three independent trials. All inclusions were small in size. N > 30 for every genotype in every panel. Chi-square test: * P < 0.05; ** P < 0.01; *** P < 0.001 in all panels. Unless otherwise indicated, comparisons were made to sod-1WTM animals. (C) sod-1G85RC and sod-1G93AC knock-in ALS model animals generated by CRISPR/Cas9 had increased hSOD1WT-YFP inclusions compared to sod-1WTC wild type controls. Neither sod-1L84VC nor loss of sod-1 altered inclusions compared to sod-1WTC and sod-1(+) controls, respectively. sod-1(+) designates the unedited wild type gene at the endogenous locus; this is the same allele present in standard N2 strain. All inclusions were small in size.(D) Paraquat-induced oxidative stress increased inclusions in sod-1H71YM and sod-1G85RM model animals compared to sod-1WTM wild type controls. Inclusions were scored manually as small, medium or large. If their diameters were more than four times larger than the small inclusions shown in Panel 2A, they were counted as large. (E) Paraquat-induced oxidative stress increased the percentage of animals with medium-size inclusions in sod-1A4VM, sod-1H71YM and sod-1G85RM animals. Likewise, emptyM control animals had larger inclusions compared to sod-1WTM controls. Inclusions were scored manually as small, medium or large, as described above.