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The role of nutrients and metabolism in cellular differentiation is poorly understood. Using RNAi screening, metabolic profiling and small-molecule probes, we discovered that the knockdown of three metabolic enzymes-phosphoglycerate kinase (Pgk1), hexose-6-phosphate dehydrogenase (H6pd) and ATP citrate lyase (Acl)-induces differentiation of mouse C2C12 myoblasts even in the presence of mitogens. These enzymes and the pathways they regulate provide new targets for the control of myogenic differentiation in myoblasts and rhabdomyosarcoma cells.

Carbon metabolism–mediated myogenic differentiation