Phalloidin ifluor 594

For immunhistochemical staining of HAP1 cells, culture media was aspirated and cells were washed once with 1× PBS. Cells were fixed with ice-cold 4% paraformaldehyde in 1× PBS containing 15% sucrose for 20 min at room temperature (RT) and subsequently permeabilized with 0.1% Triton X-100 in 1× PBS for 3 min at 4 °C. For F-actin staining, Phalloidin-iFluor 594 (Abcam, Cambridge, United Kingdom) was diluted 1 : 1000 in 1% bovine serum albumin (Carl Roth, Karlsruhe, Germany) in 1× PBS and for staining of cell nuclei 0.5 μg mL⁻¹ DAPI (Carl Roth) was added. Cells were incubated for 1 h at RT, washed with 1× PBS and coverslips were mounted on microscope slides with Shandon Immu-Mount mounting media (Thermo Fisher Scientific).
Bright-field images of printed HAP1 and HEK293H cells were captured with a CKX53 inverted microscope with integrated Phase Contrast (iPC) and a XM10 monochrome camera (Olympus, Shinjuku, Japan). Fluorescence images were acquired with an IX83 inverted imaging system with a DP80 camera (Olympus) and a 4-channel high-specification LED System (Judges Scientific, London, United Kingdom). Olympus cellSense software was used with both microscopes, and adjustments of brightness and contrast were carried out with ImageJ (NIH, Bethesda, MD, USA).

PC-12 cells were transiently transfected with construct expressing GFP-Htt^ex1-polyQ74 or pEGFP vector. The cells were fixed in 4% PFA for 20 min in room temperature followed by washing in PBS twice. The cells were then incubated with permeable buffer for 2 min and stained with buffer containing phalloidin-iFluor 594 (1:1000 dilution in PBS; Abcam) [50 (link)]. Nuclei were stained with DAPI (blue). The signals were visualized and captured using the confocal microscope (R1, Nikon, Japan).

Cells were fixed with 4% paraformaldehyde (PFA), incubated with triton X 0.2% and blocked with 10% goat serum 10%. Human cells were stained with Phalloidin iFluor 488 (titer 1:100, A12379, Thermo Scientific), and mouse cells were stained with Phalloidin iFluor 594 (titer 1:100, ab176757, Abcam) at room temperature for one hour. Nuclei were stained with 1μg/ml 4’-6-diamino-2-phenylindole (DAPI, D9542, Sigma Aldrich). Staining was then evaluated by immunofluorescence microscopy using a Zeiss LSM 710 confocal microscope. RD and Rh30 human RMS cells were cultured and stained in uncoated 96-well-dishes. Kras;CDKN2A^null mouse sarcoma cells were cultured and stained on 10% matrigel (354234, Corning).

The rabbit antibody against HSPB5 was bought from Abcam (Cambridge, USA). The rabbit antibody against cleaved caspase 3 was from Cell Signaling Technology (Danvers, USA). The mouse antibodies against GFP and β-actin were from Proteintech (Wuhan, China). Phalloidin-iFluor 594 was from Abcam. Alexa Fluor 594 goat anti-rabbit IgG was from Thermo Fisher Scientific (Waltham, USA). Citrate synthase, DAPI and 302 serum-free medium were from Sigma (St. Louis, USA). PNGase F was from NEB (Beverly, USA). Caspase-Glo 3/7 assay kit was from Promega (Madison, USA). pEGFP-Htt^ex1-Q74 plasmid was from Addgene (Cambridge, USA). HiTrap Protein A HP column was from GE Healthcare (Little Chalfont, UK).

Coverslips were coated with poly-L-lysine overnight at room temperature. Cells were rinsed with PBS, fixed in 4% paraformaldehyde for 10 minutes at room temperature, and permeabilized with PBS containing 0.25% Triton X-100. Cells were blocked in PBS-Tween (PBST) containing 10% serum from the species in which the secondary antibody was raised for 30 minutes and incubated with the indicated primary antibodies in PBST in a humidified chamber at 4°C overnight. Cells were then incubated with Alexa Fluor 488- or Alexa Fluor 594-labeled secondary antibodies (Abcam, Cambridge, MA, USA) for 1 hour at room temperature in the dark. Phalloidin-iFluor 594 (Abcam) was used to detect F-actin. After the slides were washed and sealed, the cells were imaged using inverted microscopy (TE2000-U; Nikon).

For the fusion assay of freshly isolated SCs, sorted SCs were cultured for 10 days to induce spontaneous differentiation (Stuelsatz et al., 2015 (link)). For the fusion assay of MPCs, pharyngeal and gastrocnemius MPCs were seeded at low density (5 × 10³ cells/cm²) on Collagen I-coated plates to prevent cell-cell contact and differentiated for 2 days. Then, we counted and seeded cells at high density (7.5 × 10⁴ cells/cm²) to initiate prompt fusion and further differentiated them for additional 2 days. At the end of differentiation, cells were fixed in 2% formaldehyde in PBS for 10 min at room temperature and stained with Phalloidin-iFluor 594 (ab176757; Abcam, United Kingdom) for 30 min at room temperature. Nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI), and cells were mounted with Vectashield (Vector Labs, Burlingame, CA). Myoblast fusion was quantified by counting the number of myonuclei in myotubes, which were defined as containing two or more nuclei. Fusion index was calculated as the percentage of myonuclear number relative to the total number of nuclei in the images. Diameters of each myotube were measured at three points (1/4, 2/4, and 3/4 of the length) of a myotube and averaged for each myotube. We collected 10 images from random fields of view for each line.