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. Author manuscript; available in PMC: 2021 Apr 26.
Published in final edited form as: Nat Cell Biol. 2020 Oct 26;22(11):1332–1345. doi: 10.1038/s41556-020-00595-5

Figure 5. H19 mimics and Nifenazone attenuate Ub-DMD.

Figure 5.

a, IP and IB detection of indicated proteins in hiPS-SkMCs derived from healthy donor (GM09503) or BMD patients (GM04981, GM02298, GM05089, GM04569). b, RIP assay using indicated antibodies in hiPS-SkMCs derived from healthy donor (GM09503) or BMD patients (GM04981, GM02298, GM05089, GM04569). Mean values±SD, n=4 independent experiments, two-way ANOVA. c, In vitro poly-Ub chain formation using His-tagged DMD (aa.3046–3685), UBA1, UB2G1, TRIM63, Ubiquitin, and indicated mimics or small molecular inhibitors. The Ub-DMD were subjected to Ni-NTA affinity resin pulldown and IB detection using indicated antibodies. d, Half-life determination of DMD protein in human iPSC-CMs introduced with H19 mimics or Nifenazone. Mean values±SD, n=3 independent experiments. e, IB detection of indicated proteins in iPS-SkMCs derived from healthy donor or BMD patient upon transfection of Scr mimic, H19 mimic or treatment Nifenazone (10 μM) for 72 hours. f, Representative images of IF using indicated antibody (top) and statistical analysis of DMD staining intensities (bottom) of hiPS-SkMCs derived from healthy donor or BMD patients, upon indicated treatments. Scale bars, 50 μm. Mean values±SD, n=5 independent experiments, two-way ANOVA. g, Representative images of IF using indicated antibodies (top) and statistical analysis of Ub-DMD (Lys3584) staining intensities (bottom) of hiPS-SkMCs derived from healthy donor or BMD patients, upon indicated treatments. Scale bars, 50μm. Mean values±SD, n=5 independent experiments, two-way ANOVA. No significance [n.s.], FDR > 0.05, *, FDR < 0.05, **, FDR < 0.01, ***, FDR < 0.001, and ****, FDR < 0.0001. Immunoblots are representative of two independent experiments. Statistical source data and unprocessed immunoblots are provided as Source Data Fig. 5.