Fig 1: Anti-sIL7R ASOs correct the enhanced exclusion of IL7R exon 6 in the presence of the MS risk “C” allele of rs6897932. Control (ASO-Ctrl) or lead anti-sIL7R morpholino ASOs IL7R-005 and IL7R-006 were transfected at 10 µM with Endo-Porter into HeLa cells stably expressing versions of the GFP-IL7R reporter containing either the protective “T” allele or the risk “C” allele of the MS SNP rs6897932 (T and C reporter, respectively). (A) Representative gel image of exon 6 splicing in transcripts from the GFP-IL7R reporter containing either the protective “T” allele or the risk “C” allele of the MS SNP rs6897932 (SNP ? T or C), and transfected with control (ASO-Ctrl) or lead anti-sIL7R (IL7R-005 and IL7R-006) morpholino ASOs. Percentage of exon 6 exclusion (mean ± S.D.) for each condition is plotted below the gel image. (B) GFP MFI (mean ± S.D.) from cells in panel A shown relative to the T reporter treated with ASO-Ctrl. In both panels, the bars are color-coded as follows: T reporter treated with ASO-Ctrl (black), C reporter treated with ASO-Ctrl (gold), and C reporter treated with anti-sIL7R ASOs IL7R-005 or IL7R-006 (white). The data are shown for one representative experiment in triplicate wells (n = 3 per condition) out of three independent experiments with similar results. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.
Fig 2: Screen of ASOs targeting functional cis-acting splicing elements within IL7R exon 6. (A) Schematics of the GFP-IL7R splicing reporter, wherein GFP is only expressed by exclusion of IL7R exon 6. (B) Schematics of approach comparing the effects of mutagenesis of functional cis-acting splicing elements to steric blocking of the corresponding elements with ASOs using the GFP-IL7R reporter. The blue dotted lines expand the exon 6 sequence (gray box), and the relevant cis-acting elements are color-coded: enhancer ESE2 (green), silencers ESS2 and ESS3 (red), and 5'ss (yellow). The SNP rs6897932 is indicated by the red C letter (nt 25). Mutations to ESE2, ESS3, and the 5'SS are shown underneath each element: ?ESE2 and ?ESS3 represent mutations to ESE2 and ESS3, respectively, and 5'Cons and 5'Mut represent consensus and inactivating mutations to the 5'ss, respectively. The sequence of ASOs targeting these cis-elements (IL7R-001–IL7R-005) is shown above the exon sequence: IL7R-001 blocks the 5'ss of exon 6, IL7R-002 blocks ESE2, IL7R-003 blocks ESS2 and ESS3, IL7R-004 blocks the sequence in between ESS2 and ESS3, and IL7R-005 blocks ESS3. (C,E) Percentage (%) of exon 6 exclusion (mean ± S.D.) in transcripts from the reporter in HeLa cells stably expressing wild-type or mutant versions of the reporter (C), or cells stably expressing the wild-type reporter and transfected with control (ASO-Ctrl) or experimental (IL7R-001–IL7R-005) morpholino ASOs (E) are shown under the gel images. The top band in the gels represents the exon 6 included product, whereas the lower band represents the exon 6 excluded product. (D,F) Mean fluorescence intensity (MFI) of GFP expression in the cells from panel C stably expressing the different versions of the reporter (D), or in cells from panel E transfected with ASOs (F). The left panels show representative GFP MFI histograms for selected mutants (wild-type, 5'Cons and 5'Mut) (D) and selected ASOs (ASO-Ctrl, IL7R-001, and IL7R-005) (F), with quantification shown on the right as log2(fold-change) [log2(FC)] relative to wild-type for all mutants (D) and relative to control for all ASOs (F). The data are shown for one representative experiment in triplicate wells (n = 3 per condition) out of three independent experiments with similar results. (***) P < 0.001.
Fig 3: Pro-sIL7R ASOs enhance exon 6 exclusion in the presence of the protective “T” allele of the SNP rs6897932. (A) Representative gel image of exon 6 splicing in transcripts from the GFP-IL7R reporter containing either the MS risk “C” allele or the protective “T” allele of the IL7R SNP rs6897932, and transfected with control (ASO-Ctrl) or lead pro-sIL7R (IL7R-001 and IL7R-004) morpholino ASOs. Percentage of exon 6 exclusion (mean ± S.D.) for each condition is plotted below the gel image. (B) GFP MFI (mean ± S.D.) from cells in panel A shown relative to the C reporter treated with ASO-Ctrl. In both panels, the bars are color-coded as follows: C reporter treated with ASO-Ctrl (gold), T reporter treated with ASO-Ctrl (black), and T reporter treated with pro-sIL7R ASOs IL7R-001 or IL7R-004 (gray). The data are shown for one representative experiment in triplicate wells (n = 3 per condition) out of three independent experiments with similar results. (***) P < 0.001.
Fig 4: Lead ASOs modulate expression of IL7R protein isoforms in human primary CD4+ T cells. (A) Representative gel image of exon 6 splicing in the endogenous IL7R transcripts from human primary CD4+ T cells transfected with control (ASO-Ctrl) or experimental (IL7R-001, IL7R-004, IL7R-005, and IL7R-006) morpholino ASOs. Percent (%) exon 6 exclusion (mean ± S.D.) is shown for each ASO under the gel image. (B) Levels of secreted sIL7R (mean ± S.D.) in supernatants from cells in panel A. Levels for IL7R-006 were below the limit of detection. (C) Relative MFI values (mean ± S.D.) of mIL7R cell surface expression in cells from panel A. The data are shown for one representative experiment in triplicate wells (n = 3 per condition) out of three independent experiments with similar results. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.
Fig 5: Concentration-dependent effect of lead ASOs on IL7R exon 6 splicing. (A) GFP expression in the wild-type reporter cell line transfected with increasing concentrations of control (ASO-Ctrl) or experimental (IL7R-001, IL7R-004, IL7R-005, and IL7R-006) morpholino ASOs. Overlapped representative histograms are shown on the left for each ASO at 0 µM (light gray), 1 µM (blue), 5 µM (green), and 10 µM (dark gray), with quantification of MFI plotted on the right as dose-response curves of log2(FC) relative to 0 µM for ASO-Ctrl (black), IL7R-001 (blue), IL7R-004 (green), IL7R-005 (red), and IL7R-006 (yellow). (B,C) Percentage (%) of exon 6 exclusion in transcripts from the GFP-IL7R reporter (B) or the endogenous IL7R gene (C). Representative gel images for each ASO are shown at the top with quantification of percent exon 6 exclusion (mean ± S.D.) plotted at the bottom as a function of ASO concentration (color-coded as the plot in A). The data are shown for one representative experiment in triplicate wells (n = 3 per condition) out of two independent experiments with similar results. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.
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