Hoechst 33342 solution

Copper(II) chloride dihydrate
(CuCl₂·2H₂O), gadolinium(III) chloride
hexahydrate (GdCl₃·6H₂O), sodium hydroxide
(NaOH), bovine serum albumin, sodium sulfide pentahydrate (Na₂S·5H₂O), methylene blue, and Hoechst 33342
solution were purchased from Fujifilm Wako Pure Chemical Co. (Osaka,
Japan). Sulfo-NHS-Cy5.5 ester was obtained from Abcam (Cambridge,
UK). Cyclo(RGDfk) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA)
were obtained from Cayman Chemical (Michigan, USA). Sulfo-NHS-acetate
was purchased from BroadPharm, California (USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS),
Alexa Fluor 488 phalloidin, LysoTracker Green DND-26, calcein AM,
and propidium iodide (PI) were obtained from Thermo Fisher Scientific
(Massachusetts, USA). All other chemicals of analytical reagent grade
were obtained from qualified reagent suppliers.

Drug screening was performed on myocytes on a CellCarrier‐384 Ultra microplate. Small molecules in dimethyl sulfoxide (DMSO) were added at a concentration of 3 μM on day 7 and, 24 hours later, cells were fixed with 2% (wt/vol) paraformaldehyde (163‐20145; Wako) in phosphate‐buffered saline (PBS, 045‐29795; Wako) for 30 minutes. Cells were then blocked with 5% normal goat serum (16210064; Thermo Fisher Scientific) with 0.4% (vol/vol) Triton X‐100 in PBS for 1 hour before immunostaining. The following primary antibodies were used: anti‐dysferlin (1:800, ab124684; Abcam, Cambridge, U.K.) and anti‐myosin heavy chain (MHC; 1:800, MAB4470; R&D Systems, Minneapolis, MN). Secondary antibodies included Alexa Fluor 647 goat anti‐mouse (1:000, A21236) for MHC and Alexa Fluor 488 goat anti‐rabbit (1:1,000, A11034, both Thermo Fisher Scientific) for dysferlin. Nuclei were stained by Hoechst 33342 solution (1:2,000, 346‐07951; Wako). Stained cells were imaged using the Opera Phenix High Content Screening System (PerkinElmer) and the effect of small molecules was evaluated by the relative staining intensity of dysferlin to 0.1% DMSO control in each plate, as calculated by Harmony High Content Imaging and Analysis Software (PerkinElmer). All 622 small molecules tested in this study were from an external repositioning library of Takeda Pharmaceutical Company Ltd.

Cells were seeded on 6-well culture plate and incubated for 24 h. After treatment with VK3, VK3-OH, or CDDP for 24 h, cells were stained with Hoechst 33342 solution (Wako) and incubated for 15 min. The stained cell nuclei were visualized using a fluorescence microscope IX71 (Olympus, Tokyo, Japan).