PMC:5985359 / 30279-32685
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/5985359","sourcedb":"PMC","sourceid":"5985359","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5985359","text":"A series of six FECD CEC lines, with confirmed expanded CTG18.1 genotypes (Table S7), were selected for ASO treatment. Each independent line was transfected with 200 nM of either (CAG)7 or a control ASO of identical chemical structure and of comparable length but specific to a completely unrelated sequence. A final oligo concentration of 200 nM was selected for all experiments based on optimization data (Figure S1). All lines analyzed showed a striking reduction in foci number in response to (CAG)7 ASO treatment (Figures 5 and S5). Violin plots summarize the shift in distribution of RNA foci-positive nuclei observed comparing the control ASO-treated CECs versus (CAG)7 ASO treatment (n = 6) (chi square test, χ2 = 160.78, df = 4, p = 0.001; Figure 5B). The percentage of nuclei containing zero, one, two, three, and four or more foci was analyzed, and odds ratio (OR) test confirmed that the likelihood of finding zero foci was significantly increased in cells treated with the (CAG)7 ASO compared to cells treated with the control ASO (OR = 6.2024, 95% CI, p \u003c 0.0001).\nFigure 5 ASO-Mediated Treatment of Corneal Endothelial Cells (CECs) Significantly Reduces Foci Number and Rescues MBNL1 Nuclear Localization\n(A) Foci incidence for control and (CAG)7 antisense oligonucleotide (ASO)-treated FECD-affected subject-derived CECs. The graph shows percentages of nuclei that contain 0, 1, 2, 3, and 4 or more foci after treatment with the different ASOs. Mean ± SD are represented in each case (n = 6).\n(B) Violin plots representing the distribution of the frequencies of each group of nuclei (containing 0, 1, 2, 3, and 4 or more foci) in cells treated with control and (CAG)7 ASOs (n = 6).\n(C) Representative images of ASO treatment on foci incidence. Sense RNA foci detection using Cy3-(CAG)7 probe (red). Scale bars, 10 μm.\n(D) Number of MBNL1 puncta present per nucleus when cells were treated with either a control ASO or with the (CAG)7 ASO. The mean ± SD from 4 independent expansion-positive CEC lines, where a minimum of 95 nuclei were evaluated per line. p values were calculated using an unpaired two tailed t test; ∗∗∗∗p \u003c 0.0001.\n(E) Representative example illustrating the reduction of MBNL1 puncta and the changes in MBNL1 localization when cells were treated with control and (CAG)7 ASOs. In all cases CECs were treated with 200 nM (CAG)7 or control ASO for 24 hr. Scale bars, 25 μm.","divisions":[{"label":"label","span":{"begin":1079,"end":1087}},{"label":"p","span":{"begin":1088,"end":1219}},{"label":"p","span":{"begin":1220,"end":1508}},{"label":"p","span":{"begin":1509,"end":1697}},{"label":"p","span":{"begin":1698,"end":1833}},{"label":"p","span":{"begin":1834,"end":2149}}],"tracks":[]}