Simultaneous Runx1 and Pcgf1 depletion strongly increases the self-renewal of Lin⁻ BM cells. Lin⁻ cells isolated from Runx1^Δ/Δ (dark gray) or Runx1^fl/fl (light gray) control mice transduced with 2 different shRNAs targeting Pcgf1 (shPcgf1 #1 and shPcgf1 #2) or a scrambled shRNA (shCtrl) are shown. (A) Pcgf1 knockdown levels as determined by isolating total mRNA and performing qRT-PCR using primers specific for Pcgf1 are shown. RNA levels were normalized to β-actin. Mean values of 3 independent experiments are shown. The significance of knock-down levels was determined by the Student t test. *P < .05. Alternatively, protein lysates were analyzed by immunoblotting using an Ab specific for Pcgf1 or α-tubulin as a loading control. (B) Replating capacity of Lin⁻ BM cells transduced with the indicated shRNAs. Mean values and SDs of 3 independent experiments are shown. Significance was determined by the Student t test. *P < .05; **P < .01.