Poly mount

Immunofluoresecent labeling was carried out as described previously (Luijsterburg et al., 2012 (link); Smeenk et al., 2013 (link)). Cells were either directly fixed or pre-extracted with 0.25% Triton-X100 (Serva) in cytoskeletal (CSK) buffer (10 mM Hepes-KOH, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl2, pH 7.4) for 2 min and subsequently fixed with 4% formaldehyde in PBS for 15 min. Cells were post-extracted with 0.5% Triton-X100 (Serva) in PBS, and treated with 100 mM glycine in PBS for 10 min to block unreacted aldehyde groups. Cells were rinsed with phosphate-buffered saline and equilibrated in wash buffer (WB; PBS containing 0.5% BSA, and 0.05% Tween 20 (Sigma-Aldrich)). Antibody steps and washes were in WB. The primary antibodies (see Supplementary file 1 for a list of antibodies) were incubated overnight at 4°C. Detection was done using goat anti-mouse or goat anti-rabbit Ig coupled to Alexa 488, 546 or 647 (1:1000; Invitrogen Molecular probes). Samples were incubated with 0.1 μg/ml DAPI and mounted in Polymount (Polysciences).

Cells were permeabilized with 0.5% triton X-100 (Sigma) in PBS for 10 min, followed by treatment with 100 mM glycine in PBS for 10 min to block unreacted aldehyde groups. Cells were rinsed with PBS and equilibrated in wash buffer (WB: PBS containing 0.5% BSA, and 0.05% Tween-20 (Sigma-Aldrich)) for 10 min. Antibody steps and washes were in WB. The primary antibody rabbit-p89 (1/100; Santa Cruz; SC-293; S19) was incubated for 2 h at RT. Detection was done using goat-rabbit Ig coupled to Alexa 488 (1:1000; Invitrogen). Cells were incubated with 0.1 μg/mL DAPI and mounted in Poly mount (Polysciences; 18606).

Samples of the mutant and WT plants were fixed with FAA (formaldehyde:glacial acetic acid:50% ethanol, 2:1:17) for 24 h at 4°C for histological analysis, or with PFA (4% [w/v] paraformaldehyde and 1% Triton X in 0.1 M sodium phosphate buffer) for 48 h at 4°C for in situ hybridization. They were then dehydrated in a graded ethanol series, after which the ethanol was substituted with 1-butanol, and the samples were embedded in Paraplast Plus (McCormick Scientific). The samples were sectioned at a thickness of 8 μm using a rotary microtome. The sections were stained with hematoxylin for histological analysis. After staining, the sections were mounted with Poly-Mount (Polysciences, Inc.) and observed under a light microscope.
For scanning electron microscopy, plant materials were fixed in PFA for 24 h and dehydrated in an ascending ethanol series, which was then gradually replaced with 3-methyl-butyl-acetate. Samples were critical-point dried, sputter-coated with platinum, and observed under a scanning electron microscope (S-4800, Hitachi) at an accelerating voltage of 10 kV.

For histological analysis of heart muscles paraffin blocks were cut at 5-μm sections. Samples were deparaffinized and subsequently stained with hematoxylin-eosin (Abcam, ab245880, Cambridge, MA, USA) and mounted (Poly-Mount, PolySciences Inc., Warrington, PA, USA) to analyze heart muscle structure. A BX51/IX70 Microscope (Olympus, Japan) was used for imaging.
For immunofluorescence analysis, OCT embedded frozen heart samples were cut with a cryotome (ThermoFischer, Waltham, MA, USA) at 5-μm cross-sections. Samples were fixed with ice-cold acetone for 8 min. Blocking was performed with 10% normal goat serum in 1% BSA for 60 min. Dystrophin was detected using primary rabbit anti-dystrophin antibody (1:50, ab15277, Abcam, Cambridge, MA, USA) and secondary goat anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:500, A11001, Life Technologies, USA). Nuclei were counterstained with DAPI (ab104139, Abcam, Cambridge, MA, USA). A Zeiss Meta confocal microscope with ZEN software (Carl Zeiss, Oberkochen, Germany) was used for fluorescence signal detection and ImageJ for analysis. The number of dystrophin-positive muscle fibers in five standardized regions of each cross-section were counted and normalized to total number of fibers; two non-serial cross-sections were quantified for each animal group (n > 2, mean ± SD, one-way ANOVA with post-hoc Tukey test) at day 90.

BAMs were fixed in 4% formaldehyde for 1 h and rinsed 3 × 5 min in PBS.
For tropomyosin staining, samples were permeabilized with methanol, incubated with mouse anti-tropomyosin antibody (Sigma #T9283) 1:100, followed by a secondary biotinylated anti-mouse IgG and then a preformed avidin and biotinylated horse radish peroxidase complex (Vector Laboratories, Burlingame, CA, United States). Development was by addition of DAB to produce a brown precipitate. Fixed BAMs were sent out for cross-sectional sectioning and hematoxylin and eosin staining (Mass Histology Service, Worcester, MA, United States). Slides were deparaffinized and then gradually rehydrated from 100% ethanol to H₂O and stained for collagen using Accustain Trichrome Stains (Masson) (Sigma-Aldrich). The Trichome protocol was adapted as follows. Slides were soaked overnight in Bouin’s solution (Sigma), stained in hematoxylin (Sigma), and then rinsed in running tap water. The slides were then placed in Biebrich scarlet acid fuchsin (Sigma), followed by 12 min in a working phosphotungstic/phosphomolybdic acid solution (Sigma), and then 6 min in an aniline blue Solution (Sigma). Sections were rinsed in double distilled water in between every staining step. The slides were then destained for 1 min using a 1% acetic acid solution (Sigma), dehydrated with ethanol, and mounted in Polymount (Polysciences Inc.).

After counting with trypan blue exclusion, 20,000–50,000 cells were loaded and cytospins were prepared at 1000 rpm for 5 min. Slides were air-dried and stained with StainRITE Wright-Giemsa Stain Solution (Polysciences) and mounted with Poly-mount (Polysciences). For flow cytometric analysis, K562, NB4 or MOLM13 cells with different treatments were collected and washed with chilled PBS, followed by staining with APC-CD61 (eBioscience, cat. no. 17-0619-42), PE-labelled anti-CD11b (BioLegend, cat. no. 101208) and APC-labelled anti-CD14 (eBioscience, cat. no. 17-0149-41) antibodies for 25 min. Cells were then fixed and analysed on a BD LSRFortessa or FACSAria III analyser (BD Biosciences).