Figure 4
Comparative phenotypic analyses of MT networks in wt and mutant muscles. (A) Fluorescence microscopy imaging of P1f, GLUT4, and tubulin in GFP-P1f/mCherry-GLUT4-double transfected myocyte cultures after seven days of in vitro differentiation. Of the two adjacent cells shown in the center, one is double-transfected, overexpressing full-length P1f (green) and GLUT4 (red), while the other is single transfected, expressing only GLUT4. Note reduced density of MT network (amber) in the P1f-expressing cell (especially in regions with high P1f expression) due to lower number of MTs. Blue, nuclei. Bar, 10 μm. (B) Quantification of MTs in differentiated myocytes with or without overexpressed P1f. Note reduction of MT-specific signal in myotubes expressing GFP-tagged P1f (P1f) to 62.7% relative to control cells (Ctrl, 100%) (n ≥ 12 per genotype; four different myotubes per genotype; ***P < 0.001). (C) Teased EDL muscle fibers (from wt and mutant mice) were immunolabeled using primary antibodies to α-tubulin. Arrowheads, positions of virtual cross-sections shown in small panels. Bars, 10 μm. Blue, nuclei. (D and E) Quantitative immunoblotting analysis of GC muscle lysates using antibodies to α-tubulin (D) (n = 4 per genotype, **P < 0.01 compared to wt), or antibodies to acetylated tubulin (E) (n = 4 per genotype), with corresponding Coomassie-stained gels. Values represent percentages relative to wt (100%); are shown. Note that in (E) no significant differences were observed between any of the samples. (F) Total length of MTs (per cell area) measured after nocodazole treatment of primary myofibers. MTs were visualized as in (C); (n ≥ 65 per genotype; *P < 0.05, **P < 0.01). Representative examples of cells subjected to the measurements are shown in Additional file 7: Figure S6. (G) Quantitative immunoblotting analysis of GC muscle lysates using antibodies to tau with corresponding Coomassie-stained gel. Note elevated levels of high molecular weight (HMW) isoforms of tau in dKO and cKO muscles compared to wt (100%) and mdx (109%) specimens (n = 4 per genotype; *P < 0.05, **P < 0.01). Positions of molecular mass marker (kDa) are indicated in immunoblots and corresponding Coomassie-stained gels (D, E, and G). Data in bar graphs (D-G) are presented as mean ± SEM.