Extended Data Fig. 8: DMD K3584R resists TRIM63-dependent polyUb and protein degradation.
From: The lncRNA H19 alleviates muscular dystrophy by stabilizing dystrophin
a, Graphic illustration of the strategies used to generate knockin cell lines derived from hiPS cells. b, EGFP expression of hiPS cells upon electroporation. Scale bars, 50 µm. Data represent 3 independent experiments. c, DNA agarose gel detection of single-cell colonies derived from 04981 or 25313 using indicated genotyping primers. Green forward: Puro-GT-R(LJL); Green reverse: PM20003-A-R-GT-R; Red forward: PM20003-A-L-GT-F; Red reverse: Puro-F. d, DNA agarose gel detection of single-cell colonies derived from 04981 or 25313 respectively, following removal of resistance cassettes, using indicated genotyping primers. Blue forward: PM20003-A-L-GT-F; Blue reverse: PM20003-A-L-GT-R3. *: mixed colonies containing cells with resistance cassettes not removed. Yellow boxes: single cell colonies used for following studies. e, Sanger sequencing validation of DMD K3584R mutant. Data represent 3 independent experiments. f, IB detection using indicated antibodies of GM04981 parental (Par) or K3584R mutant cells in the presence of indicated treatment. MG132 was used as proteasome inhibitor. g-i, Concentration measurement of H19 mimic or AGR-H19 per milligram of tissue at indicated time points post i.p. administration of H19 mimics (g), SubQ administration of H19 mimics (h), or SubQ administration of AGR-H19 (i) (10 mg/kg, single dose). Mean values±SD, n = 5 animals per time point per experimental condition. j, Left, representative images of α-bungarotoxin staining (top) or Duolink PLA (proximity ligation assay) using antibodies targeting LPR4 and Biotin respectively (bottom) of hiPS-SkMCs with indicated treatment. rAgrin, recombinant Agrin (Z+ form, aa. 1260–2045). Scale bars, 50 µm. Right: Percentage of cells with AchR clustering (blue bars) or Duolink PLA (red bars) of hiPS-SkMCs with indicated treatment. Mean values±SD, n = 6 independent experiments, two-way ANOVA, ***, p < 0.001. DNA agarose gel and immunoblots are representative of two independent experiments. Statistical source data and unprocessed immunoblots are provided as Source Data Extended Data Fig. 8.
