Figure 2.

Fibronectin Secreted by Myofibroblasts Is Required for Peripheral Nuclear Positioning

(A) Representative immunofluorescence image of 10-day myofibers treated with vehicle, mitomycin, or mitomycin supplemented with fibronectin, and stained for F-actin (myofibrils, green) and DAPI (nucleus, blue). Scale bar, 10 μm.

(B) Quantification of peripheral nuclei in 10-day myofibers treated with vehicle, mitomycin, or mitomycin supplemented with fibronectin or Pax7 sorted cells (satellite cells) supplemented or not with fibronectin. Data from three independent experiments were combined and error bars represent SEM from indicated number of nuclei (n) for each cohort. Unpaired t test was used to determine statistical significance, where ^∗p < 0.05, ^∗∗∗p < 0.001.

(C) Representative immunofluorescence image of 10-day myofibers knocked down for scrambled or fibronectin and stained for F-actin (myofibrils, green), fibronectin (magenta), and DAPI (nucleus, blue). Scale bar, 10 μm.

(D) Quantification of peripheral nuclei in 10-day myofibers from a culture knocked down for scrambled or fibronectin. Data from three independent experiments were combined and error bars represent SEM from indicated number of nuclei (n) for each cohort. Unpaired t test was used to determine statistical significance, where ^∗∗∗p < 0.001.

(E) Time-lapse (hr:min) transmitted light images of myofibroblasts transfected with GFP-fibronectin (green) locally depositing fibronectin on myofiber surface. Right-hand panels depict myofibroblast (blue), myofiber (dashed red), and fibronectin (black) interaction as outlines from the panels on the left. Scale bar, 10 μm.

(F) Representative immunofluorescence z-projection images of a 5-day myofiber and myofibroblast stained for F-actin (green), fibronectin (magenta), and DAPI (nucleus, blue). Yellow boxes represent 3× magnifications to highlight nuclei positioned at the periphery of the myofiber (1. nucleus is on the left; 2, nucleus is on the top). Scale bar, 10 μm.