Loss of Myc leads to spontaneous differentiation and upregulation of differentiation-associated genes. (A) Confirmation of c-Myc or N-Myc knockdown by quantitative real-time (qRT)-PCR PCR. Levels of Myc RNAs measured by qRT-PCR were normalized to the levels of loading control peptidylprolyl isomerase A (PPIA; cyclophilin A). Error bars are standard deviations. N = 3. (B) Alkaline phosphatase staining of vector alone or Myc small hairpin (sh)RNA human embryonic stem (ES) cell colonies. Images were taken at either 4 × (top panels) or 10 × magnifications (bottom panels). (C) Gene ontology (GO) analysis of genes that are differentially expressed upon Myc knockdown with indicated P values.