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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

Fig. 1

SMN-depleted muscle cells secrete reduced levels of CTRP3 protein. Quantitative secretome analysis of metabolically labelled SMN-depleted muscle cells. a H-2Kb-tsA58 cells were differentiated to multinucleated myotubes within 3 to 5 days (10x, 20x). b Representative Western blots and quantification showing knockdown efficiency. Negative control siRNA (Control) or siRNA against Smn (SmnKD) was transfected, and cells were differentiated for 72 h. GAPDH was used as a loading control (n = 4). c Schematic drawing of quantitative secretome analysis. Simultaneous siRNA transfection and combined pulsed labelling of differentiating H-2Kb-tsA58 muscle cells with azidohomoalanine (AHA) and stable isotope-labelled amino acids. Newly synthesized proteins were isolated by click chemistry and analyzed by mass spectrometry. d Volcano plot of secretome analysis; statistical significance (−log10, p-value) against fold change (FC, log2, Control/SmnKD). Normalized SILAC ratios of 4 replicates were used to calculate the log2 ratio between SmnKD (heavy) and control (intermediate) conditions, and p-values were determined using an unpaired two-sided t-test. Proteins with a p-value < 0.05 are highlighted in green/red and proteins with a FC > 50% are additionally labelled with name. e Representative Western blots and quantification of C2C12 cells transfected with negative control siRNA (Control) or siRNA against Smn (SmnKD), and differentiated for 5 days. ACTB was used as a loding control. f Representative Western blots and quantification of serum-free conditioned media of control and Smn KD C2C12 cells, differentiated for 5 days. CTRP3 levels were normalized to the total protein amount (stainfree gel). Dot graphs represent data from independent experiments and the bar graphs depict the mean ± s.d. Two-tailed unpaired student’s t-test was used to determine statistical signaficance. ***p < 0.001; **p < 0.01

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