Figure S4.

Inclusion of Itga7-X1 (exon 5) is sufficient to induce loss of MuSC polarity and reduction of myogenic progenitors. (A) Quantification of proportion of Dmd-positive muscle stem cells (MuSCs) in QKI-ctrl, QKI-cKO, and ASO-Q1-transfected myofibers cultured for 36 h from Fig 4D (error bars represent mean ± SEM, n = 3 biological replicates per group. ns, not significant, unpaired t test). (B) Biotinylated RNAs were used for RNA pull-down assays. The mutated sequence disrupting the core of the QKI response element is indicated in red. Immunoblotting of QKI-5 after RNA pull-down assay using C2C12 total cell lysates. Increasing NaCl concentrations in the wash buffer was used to define the high affinity RNA binding of QKI-5. (C) RT-qPCR of exon 23 of Itga7 in antisense oligonucleotide-treated MuSCs normalized to mock-transfected MuSCs (error bars represent mean ± SEM, n = 3 independent replicates, ns, not significant, unpaired t test). (D) RT–PCR validation of ASO-Q1 off-target alternative splicing events in proliferating MuSCs control (Ctrl) transfected or transfected with ASO-Q1. PCR products were separated on acrylamide gels and stained with ethidium bromide. The migration of DNA markers in base pairs (bp) is shown on the left.