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. 2021 Feb 24;12:1265. doi: 10.1038/s41467-021-21021-w

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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. 3 — a Analysis of the total FMR1 mRNA and pre-mRNA levels in fibroblasts treated with ASOs. Fibroblasts were transfected with siRNA against RNase H1 or control siRNA and with ASOs (9 or 11 nt, 200 nM). Graphs present RT-qPCR results for N = 5 biologically independent samples. b Increase in the efficiency of in vitro transcription in the presence of ASO–CCG or RNase H. rCGG₁₀₀ signal was measured for N = 3 independent samples. c Correlation between R-loop formation and efficiency of transcription of RNA containing CGG₁₀₀. Graphs present quantification of the fluorescent signal coming from fluorescently labeled ASO–CCG-Cy3 bound to either rCGG₁₀₀ or R-loop structure for N = 3 independent samples. The area of the gel marked with a red box was exposed to higher laser power strength and the result is presented below the main gel. DNA template was the same as in b, but the observed signal came only from partially single-stranded DNA in the region of CGG/CCG repeats or R-loop bound with ASO–CCG-Cy3. d Cellular distribution of FMR1 mRNA in fibroblasts treated with ASOs (9 nt, 200 nM). Graphs present RT-qPCR results for the cytoplasmic and nuclear fractions for N = 3 biologically independent samples. Results were normalized to GAPDH. e Changes in the steady-state level of FMRP after treatment with ASO–CCG. Fibroblasts were transfected with 200 nM ASOs (9 or 11 nt). The graph presents western blot results for N = 4 biologically independent samples (CGG^norm/- (1) and CGG^exp/-) and N = 3 (other cell lines). a–e Graphs present means of indicated N with the SDs. a, b, d, e Red bars, ASO-ctrl; blue bars, ASO–CCG. a, d, e The used cell lines contained one allele with a normal CGG repeat length (CGG^norm/-; lines (1), 20 CGG repeats; (2), 31 CGG repeats; (3) and (4)); one allele with a normal CGG repeat length and one allele with CGG^exp (CGG^norm/CGG^exp); two alleles with CGG^exp (CGG^exp/CGG^exp) and one allele with CGG^exp (CGG^exp/-). b, c, d Gels were cropped. a–e Two-sided, unpaired Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, non-significant. Source data are provided as a Source Data file.