Figure 1.Transwell culture with senescent myoblasts impairs myotube formation. (A) Representative phase contrast microscopy images of myoblasts stained for SA-β-gal at pH 6.0, captured 48h after dosing with vehicle (VEH) or bleomycin (BLEO). (B) Flow cytometry histograms of myoblasts stained with propidium iodide 48h after treatment. The fluorescent intensity of propidium iodide determines the cell cycle phase. (C) Relative mRNA expression of Cdkn1a (p21) following VEH or BLEO treatment. (D) Schematic diagram of myotubes exposed to SEN/NSEN myoblasts in a 0.4-μm transwell insert. This allows for intercellular communication exclusively through factors released into the cell culture media. Relative mRNA expression of senescence genes (E) Trp53 and (F) Cdkn1a in myotubes after 6 days of 0.4-μm transwell culture with NSEN or SEN myoblasts. (G) Representative immunofluorescence microscopy images of myotubes after co-culture with NSEN or SEN myoblasts. Myotubes are stained for MyHC (green) and nuclei (DAPI; blue). Graphical quantification of (H) myotube diameter, (I) myotube surface area, and (J) myonuclear index.
