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. 2014 Jan;95(1):83–93. doi: 10.1189/jlb.1112608

Figure 4. Knocking down PU.1 attenuates DAPK2 induction significantly during neutrophil differentiation.

Figure 4.

(A and B) NB4 cells were transduced with two independent shRNAs targeting PU.1 (shPU.1_1/_2) or with a nontargeting shRNA control (SHC002). NB4 control and PU.1 knockdown cells were differentiated toward neutrophils for 4 days using ATRA. (A) PU.1 mRNA levels were measured by qPCR. Values were normalized to the HMBS housekeeping gene. Results are shown as n-fold regulation compared with untreated NB4 SHC002 control cells. (B) DAPK2 mRNA levels analyzed as described for PU.1. (C) Western blot analysis of DAPK2 and PU.1 expression in NB4 PU.1 knockdown cells during neutrophil differentiation. Total protein was extracted and subjected to immunoblotting using anti-DAPK2 and anti-PU.1 antibodies. GAPDH is shown as a loading control. (D) HT93 APL cells were transduced with lentviral vectors expressing shRNAs targeting PU.1 as in A. DAPK2 mRNA levels in HT93 PU.1 knockdown or control cells upon neutrophil differentiation were determined as in A. (E) Western blot analysis of DAPK2 and PU.1 expression in PU.1 knockdown and control HT93 APL cells upon ATRA-induced neutrophil differentiation. Analysis as in C. (F and G) Knocking down Pu.1 in conditionally immortalized murine neutrophil progenitor cells (SCF-condHoxb8) impairs Dapk2 induction. SCF-condHoxb8 cells were transduced with a shRNA targeting Pu.1. Neutrophil differentiation was induced by removing 4-OHT from the cell culture medium. Six days later, cells were analyzed for DAPK2 and PU.1 expression. Pu.1 (F) and Dapk2 (G) mRNA levels were determined by qPCR. Values were normalized to the housekeeping gene Gapdh. Results are shown as n-fold regulation compared with undifferentiated SCF-condHoxb8 cells growing in the presence of 4-OHT. (H) Dapk2 and Pu.1 Western blot analysis of SCF-condHoxb8 cells treated as in G. (I and J) Attenuated neutrophil differentiation of SCF-condHoxb8 cells upon 4-OHT removal, as seen by reduced G-Csfr mRNA and Gr-1 cell-surface expression. ctrl, Control; SHC, SHC002. MWU: *P < 0.05; ***P < 0.001.