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. 2014 Dec 16;112(2):329–337. doi: 10.1038/bjc.2014.589

Figure 2.

Figure 2

ATRA induces differentiation and prevents the induction of apoptosis by VPA. (A) Flow-cytometry analysis of NB4 cells treated with 1 μM ATRA and/or 1.5 mM VPA for the indicated period of time. The diagram shows the subG1 fractions of PI-stained cells (mean±s.e.m., n⩾3). Statistics were done with the two-tailed unpaired Student's t-test, *P⩽0.05, **P⩽0.01, ***P⩽0.001, n.s. not significant P⩾0.05. (B) Whole-cell protein extracts were prepared from treated NB4 cells. Immunoblot was used to detect the protein expression of C/EBPɛ, acetylated histones H3 (acetyl-H3), cleaved caspase-3 (cl., cleaved form) which is the active form of the caspase, and α-tubulin as a loading control. All samples were analysed on one membrane. (C) Flow-cytometry analysis of NB4 cells treated with 1 μM ATRA and/or 1.5 mM VPA for the indicated period of time. Treated NB4 cells were stained with an antibody against CD11b and analysed by flow cytometry. All treated samples show more CD11b-positive cells than control cells, significance ***P⩽0.001. Statistics were done with the two-tailed unpaired Student's t-test. (D) To test maturation at the morphological level, we analysed NB4 cells exposed to 1.5 mM VPA, 1 μM ATRA for 72 h and to 100 nM LBH589 (LBH) for 24 h by May-Grünwald–Giemsa staining. This approach revealed that only ATRA evokes cellular maturation. An at most partial induction of differentiation by VPA was also found in other studies (Müller and Krämer, 2010). A 63-fold magnification was used.