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. 2014 Apr 21;9(4):e95784. doi: 10.1371/journal.pone.0095784

Figure 6. 32Dcl3 cells with partial Cebpa knockdown retain capacity for nearly complete granulocytic maturation.

Figure 6

A) Parental 32Dcl3 cells, pools of 32Dcl3 cells stably transduced with the pLKO.1 vector (Vec) or Cebpa B9 or B11 shRNAs, or subclones of the B9 or B11 pools were subjected to Western blotting for C/EBPα and β-actin. B) Indicated 32Dcl3 lines were subjected to FACS analysis for GCSFR using biotin-G-CSF in the absence (heavy line) or presence (light line) of excess unbiotinylated G-CSF; x-axis is log scale (top). RNAs prepared from indicated 32Dcl3 lines cultured in IL-3 or after one or four days in G-CSF (G1, G4) were subjected to quantitative RT-PCR for Mpo (bottom, n = 3). C) Vector, B11-1, or B9-2 cells cultured in GCSF were subjected to Wright-Giemsa staining on the days of maximal differentiation, G6, G8, and G4 respectively (top row). Vector/GR, B11-1/GR, or B9-2/GR, G14, G14, and G6, respectively, were analyzed similarly (bottom row). D) Pools of vector, B11-1, or B9-2 lines transduced with pBabeNeo or pBabeNeo-GCSFR were subjected to FACS analysis for GCSFR (top). RNAs prepared from these lines in IL-3 or G-CSF were analyzed for Mpo expression (bottom, n = 3).