Fig. 6: Discovery and functional characterization of previously unknown NMJ marker genes.

a Representative images from smRNA-FISH for Ufsp1, Lfrn5, Vav3, and Ano4 on skeletal muscle sections showing co-localization with the canonical NMJ protein, acetylcholine receptor (AChR) (n = 3). AChR was visualized through α-bungarotoxin labeling. b Quantitative real-time PCR (qPCR) analysis for the indicated genes not previously associated with the NMJ from normal (n = 4) and denervated (n = 5) muscle. c Schematic for a siRNA screen in C2C12 myoblasts designed to test the function of candidate NMJ genes. siRNA was transfected two days after differentiation and three days later α-bungarotoxin was used to analyze AChR clustering as a surrogate for NMJ formation. d qPCR analysis for the genes targeted with siRNA. A scrambled siRNA was used as a control. e Representative images of C2C12 myotube cultures after treatment with various siRNAs and staining with α-bungarotoxin. Cells were also stained with phalloidin and DAPI. f Quantification of AChR clusters per field of view from e. g qPCR analysis for genes associated with myogenesis and NMJ formation. Scale bars: a 50 μm (top left panel), 10 μm (top right and bottom panels), e 50 μm. Data in d, f, and g are from three independent experiments. All data are represented as mean ± standard deviation. An unpaired two-sided t-test was used to determine statistical significance, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.