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. 2015 Nov 13;5:16523. doi: 10.1038/srep16523

Figure 4. Regeneration of cardiotoxin-injected muscle is impaired by AKAP6 shRNA lentivirus.

Figure 4

(A) Time table for the animal experiment a using GFP-expressing shRNA lentivirus for AKAP6 (shAKAP6-GFP) or control shRNA lentivirus (shMock-GFP) and CTX injection. (B) Transverse sections of muscle harvested 2 weeks after CTX injection. Muscle fibers infected by the GFP-expressing lentivirus show green fluorescence (n = 4 mice per group; magnification: ×400). (C) Knockdown of AKAP6 expression by the shAKAP6-GFP lentivirus was confirmed using proteins from lentivirus-infected tissues by western blotting. The quantification graph shows relative fold changes (n = 4, *p < 0.05 versus shMock-GFP). (D) Cross-section of TA muscle was immunostained for laminin α2 (red)/nuclei (blue) 4 weeks after CTX injection. Magnification: ×400. Muscle fibers infected with shMock-GFP or shAKAP6-GFP lentivirus fluoresced green. Skeletal muscle fibers were strongly stained by laminin α2 in the shMock-GFP lentivirus infection group (arrows). In the shAKAP6-GFP lentivirus group, muscle fibers stained with laminin α2 presented an irregular shape and were weakly detectable (asterisks; n = 4 per group). (E) The graph shows the percentage of central nuclei fibers divided by the number of fibers (n = 5 each, **p < 0.01). (F) Cross-sectional area (CSA) of the myofibers in the injured TA muscles 4 weeks after CTX injection. Myofiber CSA (μm2) was measured using the ImageJ software. Myofiber size in the shMock-GFP lentivirus infection group was larger than that in the shAKAP6-GFP lentivirus infection group. The shAKAP6-GFP lentivirus group showed a shift toward smaller fibers. Measurements of four different sections per mouse in four mice were used. Data are presented as mean (SEM). **p < 0.01 versus shMock-GFP (one-way ANOVA). (G) Rotarod test. One week before CTX injection, lentivirus was injected into the TA muscle (both legs) to induce virus amplification in vivo. We monitored muscle power in three groups 2 weeks after CTX injection: injury control with CTX injection (CTX only, n = 9), injury with shMock-GFP lentivirus infection (CTX+shMock, n = 17), injury with shAKAP6-GFP lentivirus infection (CTX+shAK6, n = 17). #p < 0.05 versus CTX after injury; *p < 0.05 versus CTX+shM after injury (one-way ANOVA).