Fig. 2: siRNAs for mouse Fibcd1 induce mouse C2C12 myotube atrophy.
a Transmembrane (FL) and short (SH) versions of the mouse Fibcd1 protein (orthologous to Drosophila Fibcd1/CG8642) are encoded by the mouse genome. b Western blot of HEK293 cell lysates and cell culture supernatants 2 days after transfection with either an empty vector (EV) or with a vector encoding C-terminal Flag-tagged full length (FL) or short (SH) mouse Fibcd1. Expression of FL and SH is detected at similar levels in cell lysates. However, although neither FL nor SH are detected in the culture medium, a C-terminal ~38 kDa fragment of Fibcd1 derived from the proteolytic processing of FL Fibcd1 is detected in the cell culture supernatant. Coomassie blue staining is shown as loading control. A recombinant Fibcd1 protein (rFibcd1) that resembles the cleaved Fibcd1 FL fragment has been generated a. c NT or Fibcd1 siRNAs transfection into mouse C2C12 myotube-enriched cultures for 48 h, followed by treatment for further 24 h with rFibcd1 or a vehicle control. Representative images of myotubes stained for myosin heavy chain are shown. Scale bar, 100 μm. Measurement of myotube width indicates that Fibcd1 siRNAs induce atrophy and that myotube size is rescues by rFibcd1. Data are mean ± SD with the precise n indicated in the figure; ****P < 0.0001 and &&P < 0.01, compared to the indicated control; P values were determined by two-way ANOVA with Sidak’s multiple comparisons test. See also Supplementary Fig. 2. d Myotube-enriched cultures treated for 48 h with cachectic cytokines (IL-6 at 20 ng/mL, LIF at 20 ng/mL, and TNF-α at 100 ng/mL) and either rFibcd1 at 10 and 100 ng/mL or vehicle-alone control at the same time of the cytokine treatment. Representative images of myotubes stained for myosin heavy chain (green) are shown. Scale bar, 200 μm. Measurement of myotube width indicates that rFibcd1 rescues atrophy induced by cachectic cytokines. Data are mean ± SD; n = 101 myotubes/group. ***P < 0.001 compared to control; &P < 0.05, &&P < 0.01, and &&&&P < 0.0001 compared to control within each cytokine group. Statistical analysis was done by using two-way ANOVA with Sidak’s multiple comparisons test. Source data are provided in the Source Data file.
