Figure 6.

Role of p15Ink4b in the regulation of hematopoietic progenitor fate might be independent of its canonical function in the cell cycle. (a) Cell cycle analysis of EML and EMLp15Tuner (EMLp15) cells treated for 48 h with SH (100 nℳ) to induce expression of p15Ink4b. A representative experiment is shown (n=2). (b) Cell cycle analysis of Lin− bone marrow-derived cells of wild-type (WT) or Ink4bKO mice following re-expression of p15Ink4b. Ink4bKO cells were infected overnight with either the p15Ink4b-expressing vector (p15LentiX) or empty vector (LentiX). Following overnight infection, cells were transferred onto a S17 stromal cell layer and differentiated for 60 h in the presence of SH (100 nℳ) to induce p15Ink4b expression. Subsequently, the cells were fixed in ethanol and stained with propidium iodide (PI). ModFit LT software was used to analyze the data. A representative experiment is shown (n=2). (c) Number of BFU-E colonies obtained by plating 10 000 Lin− Ink4bKORbfl/fl bone marrow cells transduced with either empty vectors, MIG or pLVX-PTuner-Green (LentiX), or p15Ink4b-expressing vector (p15LentiX), or Cre recombinase (CreMIG)-expressing vector in M3436 medium. Following overnight infection, cells were transferred onto an S17 stromal cell layer and differentiated for 60 h in the presence of SH (100 nℳ) to induce p15Ink4b expression (n=4). To assess the deletion efficiency of pRb, a fraction of cells following overnight infection was transferred into the expansion medium for 3–5 days before harvesting them for western blot analysis (upper panel). The blot was quantitated using ImageJ and normalized to actin. (d) Number of myeloid colonies (CFU-GM, CFU-G and CFU-M) obtained by plating 1000 of the same cells as in (c) in M3534 medium (n=4). (e) Number of BFU-E colonies obtained by plating 1000 EML cells, pretreated for 24 h with Cdk4/6 IV inhibitor (CDK4/6i) or left untreated in M3436 medium.