Figure 4.
Inhibition of HDAC6 regulates the clustering of AChR complexes by organizing the MT network.(A) Schematic representation of the experimental time course for B and C. (B) 4-d-old C2C12 myotubes were pretreated for 24 h with HDAC6 inhibitors HPOB (5 µM) and TubA (5 µM) or with DMSO (CTL; 1 µl). AChR clusters were labeled with α-BTX–A488 (in green). (C) Quantifications of AChR cluster areas of three independent experiments (n = number of AChR clusters counted, between 65 and 124). (D) Schematic representation of the experimental time lapse imaging for E–H. (E and F) AChR clusters of 4-d-old C2C12 myotubes were labeled with α-BTX–A488 (in green). Myotubes were treated with either DMSO (CTL; 1 µl, curve in black), TubA (5 µM, curve in orange), TSA (0.1 µM, curve in red), nocodazole (10 µM, curve in blue), or taxol (10 µM, curve in purple) and imaged over 12 h (Video 1 and Video 2). Representative images are shown every 3 h; arrowheads point to representative AChR clusters. (F) Quantification of three independent experiments showing the disappearance of AChR clusters at the surface of myotubes between 3 and 12 h (n = total number of AChR clusters counted, between 162 and 286). *, P < 0.05; **, P < 0.01; n.s, not significant; two-way ANOVA. (G) 4-d-old myotubes were labeled with α-BTX–A488 (in green), then treated with drugs for 24 h, and finally labeled with α-BTX–A594 (in red) at day 5. Insertions of new AChR clusters are shown in preexisting AChR clusters (asterisks) or in new localizations (arrowheads). (H) Quantification showing the distribution of new AChR clusters at day 5 (three independent experiments; n = total number of AChR clusters counted, between 266 and 621). (C and H) Means ± SEM. *, P < 0.05; ***, P < 0.001; Mann-Whitney U test. (B, E, and G) Bars: 100 µm. noco, nocodazole; n.s, not significant; T, time.

Inhibition of HDAC6 regulates the clustering of AChR complexes by organizing the MT network. (A) Schematic representation of the experimental time course for B and C. (B) 4-d-old C2C12 myotubes were pretreated for 24 h with HDAC6 inhibitors HPOB (5 µM) and TubA (5 µM) or with DMSO (CTL; 1 µl). AChR clusters were labeled with α-BTX–A488 (in green). (C) Quantifications of AChR cluster areas of three independent experiments (n = number of AChR clusters counted, between 65 and 124). (D) Schematic representation of the experimental time lapse imaging for E–H. (E and F) AChR clusters of 4-d-old C2C12 myotubes were labeled with α-BTX–A488 (in green). Myotubes were treated with either DMSO (CTL; 1 µl, curve in black), TubA (5 µM, curve in orange), TSA (0.1 µM, curve in red), nocodazole (10 µM, curve in blue), or taxol (10 µM, curve in purple) and imaged over 12 h (Video 1 and Video 2). Representative images are shown every 3 h; arrowheads point to representative AChR clusters. (F) Quantification of three independent experiments showing the disappearance of AChR clusters at the surface of myotubes between 3 and 12 h (n = total number of AChR clusters counted, between 162 and 286). *, P < 0.05; **, P < 0.01; n.s, not significant; two-way ANOVA. (G) 4-d-old myotubes were labeled with α-BTX–A488 (in green), then treated with drugs for 24 h, and finally labeled with α-BTX–A594 (in red) at day 5. Insertions of new AChR clusters are shown in preexisting AChR clusters (asterisks) or in new localizations (arrowheads). (H) Quantification showing the distribution of new AChR clusters at day 5 (three independent experiments; n = total number of AChR clusters counted, between 266 and 621). (C and H) Means ± SEM. *, P < 0.05; ***, P < 0.001; Mann-Whitney U test. (B, E, and G) Bars: 100 µm. noco, nocodazole; n.s, not significant; T, time.

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