Hoechest

Cells were grown at the desired confluence on a glass cover slide for 24 hours and treated with 1,000 ng/mL doxycycline for another 48 hours (for the corresponding experiment). Cells were washed with phosphate-buffered saline (PBS) three times and fixed for 10 minutes with 4% paraformaldehyde, washed with PBS three times, and permeabilized with PBS containing 0.2% Triton-X100. For immunofluorescence, cells were blocked for 30 minutes in 10% FCS in PBS, incubated overnight at 4°C with the primary antibody, washed three times with PBS-T (0.05% Tween-20 in PBS), incubated for 1 hour in the dark at room temperature with the secondary antibody, washed three times with PBS-T, and mounted on a slide with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting media. As colocalization staining, DDX1-mCherry or DDX1-mCherry-Δ269-295aa inducibly expressed KELLY cells were seeded on 8-well μ-slide (ibidi) for 48 hours in the presence or absence of doxycycline (1 μg/mL, Sigma-Aldrich). Then, 30 minutes before fixation, cells were incubated with MitoTracker (500 nmol/L, Cell Signaling Technology) and Hoechest (1 μg/mL, Thermo Fisher) at 37°C. After fixation, cells were washed 3 times with PBS and mounted with PBS. Cells were imaged using a Leica TCS SP5 II (Leica Microsystems) and quantified using ImageJ.

Tissues collected from patients were fixed in 4% Paraformaldehyde and washed with Phosphate buffered saline (PBS).Tissues were permeabilized with 0.5% triton-100 followed by PBS wash. The tissues were then blocked with 1%BSA prior to overnight incubation with primary Antibody. The detection was done using Cy3.5 secondary antibody and counterstained with Hoechest (Thermo fisher Cat No-33342).Further actin fibers of the tissues were stained with 100 nM of Phalloidin-488 (Cytoskeleton, INC. Cat No-PHDG1) after permeabilization. The tissues were incubated for 45 min in dark and Counter-stained with Hoechst. The tissues were imaged using Zeiss Microscope using 2 different channels.

10⁵ cells per well were seeded in 6-well plate and were first synchronized by serum starvation for 36 hrs and stained with hoechest (Molecular Probes, Eugene, OR, USA) for 10 min at room temperature at relevant time points for flow cytometry and analyzed using Modifit analysis (Beckman Coulter, Brea, CA, USA) software.

C2C12 cells were seed on Collagen coated glass and induce differentiation. Every indicated timepoint, the cells were fixed with 4% paraformaldehyde for 10 min and treated with 0.1% Triton X-100 in PBS for 10 min at room temperature, and then block with 3% BSA in PBS for 2 hr. Following incubation with the indicated antibody over night at 4 °C, cells were incubated with a conjugated secondary antibody at 37 °C for 1 hr. Finally, the nuclei were stained with Hoechest (Molecular Probes) for 5 min.