Fig 1.
Fiber morphology of the quadriceps in WT and SMA mice at P10.
Panels A-B, Cox immunoreaction on quadriceps of WT (panel A) and SMA (panel B) mice, revealing the mitochondrial activity and the fiber types. Scale bar = 40μm. Panels C-D showed the same muscles (WT: panel C; SMA: panel D) stained by hematoxylin/eosin. Scale bar = 80μm. Panel E, quantification of fiber type I and II in quadriceps of WT and SMA mice, expressed as percentage of the total number of fibers. Panels F-H, respectively single fiber area, number of fibers and total area of quadriceps in WT and SMA mice. *p<0.05; ***p<0.001 by t-test analysis.
Fig 2.
Architecture of the NMJ of the quadriceps in WT and SMA mice at P10.
Panels A-B, Immunoreactions for α-bungarotoxin (red) and neurofilament (green) proteins in NMJs of quadriceps of WT (panel A) and SMA (panel B) mice. Inset C, isosurface module of Imaris software. Nuclei were labeled in blue with DAPI. The arrow in A points a WT NMJ correctly innervated, whereas arrowheads in B show the abnormal accumulations of neurofilament in SMA. Scale bar = 10μm. Panels D-F, morphology of NMJs at higher magnification: WT mice display endplates with a more mature morphology (panel D), compared to SMA ones (panel E). Additionally rare SMA NMJs appear fragmented, as indicated by arrowhead (panel F). Scale bar = 10μm. Panel G, quantification of multi-innervated, mono-innervated and denervated endplates in quadriceps of WT and SMA mice. Panel H, distribution of NMJ area in WT (continuous line) and SMA (dashed line) endplates expressed as percentage of total number of plaques. Panel I, mean value of NMJ area in WT and SMA quadriceps. *p<0.05 by t-test analysis.
Fig 3.
HDAC4 and FGFBP1 expression levels in C2C12 cells transiently transfected with miRNA-206.
Panels A-B, representative Western blots displaying HDAC4 protein levels in C2C12 cells transfected at 30% confluence (panel A) and at 60% confluence (panel B) with a mimic miRNA-206 (miRNA-206) and with a non targeting miRNA (NT miRNA) used as control. Graphs below each image report the quantification of HDAC4/GAPDH protein levels. Panel C, evaluation of FGFBP1 mRNA levels in C2C12 cells transfected at 30% and 60% confluence with miRNA-206 and NT miRNA. *p<0.05 vs respective control by t-test analysis.
Fig 4.
pri-miRNA-206, mature miRNA-206, HDAC4 and FGFBP1 expression levels in quadriceps of WT and SMA mice at P5 and P10/P12.
Panels A,B,D, evaluation of pri-miRNA-206 (panel A), mature miRNA-206 (panel B) and FGFBP1 (panel D) mRNA levels in WT and SMA mice, five (P5) and ten (P10/12) days after birth. #p<0.05 vs P10/12 WT, § p<0.05 vs all group by ANOVA followed by post hoc Newman-Keuls test. Panels C, representative Western blots displaying HDAC4 protein levels in WT and SMA mice, at P5 (left blot) and P10 (right blot). Graphs below each image report the quantification of HDAC4/GAPDH protein levels. *p<0.05 vs WT by t-test analysis.