Figure 2.

G-actin incorporation into filaments in dysferlin-deficient myoblasts. Fluorescently tagged G-actin incorporation into filaments was assayed in myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg²⁺, and 10 µM free Ca²⁺ during 6 min at 37 °C. After permeabilization, cells were fixed and nuclei were stained with DAPI. Confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. (a–c) Fluorescent actin filaments in control C25 and dysferlinopathy DYSF2, DYSF3, AB320, and ER myoblasts (a) and RCMH myoblasts non-transfected (N-T) or stably transfected with shRNA for dysferlin (c). Scale bar = 20 µm. Insets show digital zooms of the boxed areas; brightness and contrast were increased to appreciate better the pattern of fluorescent actin. (b–d) Data are means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of analyzed cells from five different cultures is indicated in parentheses. * p < 0.05 compared to C25 (b) or N-T RCMH myoblasts (c) (t-test).