Fig 2: SSO treatment attenuates insulin receptor signaling and reduces vessel formation in SCID mice.A Mice were injected with 106 Rh30 cells either transfected with NS or SSO55. IGF1-R antibody was administered on days 1 and 3 post-injection. The matrigel plugs were extracted after 7 days for CD34 (endothelial cell marker) staining. The plugs were formalin-fixed and paraffin-embedded, and slides were stained for CD34. Quantification of the CD34 staining from NS and SSO55-treated grafts was done in a blinded manner, n = 10 mice in each condition. The representative IHC pictures for CD34 staining are depicted. The RT-PCR shows the splicing changes in the injected cells in the presence of SSO55. B Model depicting how insulin and IGF-2 bind to IR-A in tumor cells leading to proliferative signaling while in the presence of an IGF-1R antibody and an SSO that shifts the splicing to IR-B, the proliferative signaling (AKT) can be mitigated. Results are shown as the standard error of the mean (±SEM).

Fig 3: Splice switching oligonucleotide (SSO) treatment restores IR splicing to IR-B.A, B Representation of the insulin receptor minigene. The CUG-BP1 binding site is shown on the IR pre-mRNA. The blue lines represent the SSOs used for the macro-walk with the new SSO starting at the “bold” letter in the sequence. The red and black lines represent the SSOs used for the micro-walk. RT-PCRs depicting the IR isoforms in the presence of control SSOs or SSOs that target either the CUG-BP1 binding site are shown below the sequences. The capital letters in the sequence represent the binding site for SSO55. The percent exon 11 inclusion is shown as PSI, percent spliced in. C Representation of exon 11 SSOs (green lines). RT-PCR using SSOs on the second CUG-BP1 binding site in exon 11 is shown below. D Schematic to represent the region amplified and used as a probe for the RNA electrophoretic shift assay from the IR intron 10. The complex formed by CUG-BP1 and the I10 probe is depicted as * and the unbound I10 probe is depicted as **. Experiments were performed in triplicate and quantitated in the graph on the right. The bound fraction was calculated as (bound complex intensity)/(bound complex intensity + unbound probe intensity). Results are shown as the standard error of the mean (±SEM).

Fig 4: Hypoxic stress increases the expression of IR-A isoform.A The Gene expression data for HIF-1a expression were extracted from Schafer-Welle (Scafer-Welle-56-MAS5.0-u133a) dataset using R2: Genomics Analysis and Visualization platform for 26 normal muscle samples and 30 RMS samples35. The data distribution is represented by a box plot, the line inside the box represents the median and the whiskers represent minimum and maximum expression. p = 1.5e-28. B RT-PCR depicting the IR alternative splicing in HeLa S3 cells after 48 h of normoxia or hypoxia (1% O2). Western blot analysis of the Hif-1a induction by hypoxia treatment is shown with ß-actin as control. C Quantification of IR-B expression in control and hypoxic conditions; n = 6, p = 0.0018. D RT-PCR analysis for the INSR alternative splicing of the siRNA-mediated knockdown of Hif-1a followed by treatment of normoxia or hypoxia (1% O2). Western blot analysis for Hif-1a expression and ß-tubulin as a loading control. E Quantification of IR-B expression in control and hypoxic conditions of the Hif-1a knockdown experiments. n = 6, p = 0.0013. F Depiction of the wild-type insulin receptor (IR) minigene or the CUG-BP1 binding-site mutated minigene. RT-PCR depicting both INSR minigenes alternative splicing. G Quantification of the 10.11.12 isoform expression is shown n = 4, p = 0.405. Results are shown as the standard error of the mean (±SEM).

Fig 5: RMS cell lines and patient samples express increased levels of IR-A.A Schematic depicting the splicing of insulin receptor gene. B RT-PCR of control muscle samples and RMS cell lines depicting the two insulin receptor (IR) isoforms IR-A and IR-B. Cells were cultured in the required medium, RNA was extracted, and PCR was performed using the primers depicted by black arrows in (A). The PCR products were run on a 2% agarose gel and bands were quantified. RT-PCR was performed on (C). Embryonal (e) and D Alveolar (a) RMS samples (depicted with different numbers) using the primers for IR depicted in (A) (black arrows). Samples from normal adjacent tissue (for one sample) are shown as control (n). The fusion status (fp = fusion positive, fn = fusion negative) status is denoted on the alveolar tumor samples. All embryonal samples were also tested and found fusion negative. The percentages for IR-B are shown as PSI (percent spliced in). GAPDH is shown as a loading control. Results are shown as the standard error of the mean (±SEM).