Figure 3.

PKCβ may not be involved in enz-ATRA treatment-induced differentiation and apoptosis. (A) NB4-R1 (left panel) and NB4-R2 (right panel) cells were treated with 2 μM enzastaurin (ENZA), 1 μM ATRA (RA) alone and in combination (ENZA+RA) for 3 h. The activation of PKCβ was measured by Western-blotting analysis of phosphorylated PKCβ at serine 641. The same membrane incubated with anti-phospho-PKCβ (Ser 641) was stripped and followed by detection of PKCβ. The expression of β-actin was evaluated as internal control. The morphology of NB4-R1 (B) and NB4-R2 (C) cells treated with 200 nM PKCβ inhibitor and/or 1 μM ATRA (RA) for four days. Scale bar represents 5 μm and the magnification is 1,000. One representative experiment among three independent assays is shown. Differentiation was also assessed by flow-cytometric analysis of CD11b expression (D), and each value represents the mean ± SD of three independent measurements. ##P<0.01, versus ATRA treated cells. The representative histograms of flow-cytometric analysis of CD11b expression in NB4-R1 (E) and NB4-R2 (F) cells with the indicated treatment for four days are also shown. Apoptosis was evaluated by flow-cytometric analysis of Annexin-V in NB4-R1 (G) and NB4-R2 (H) cells with the indicated treatment for four days. The percentages of CD11b⁺ cells or Annexin-V⁺ cells are shown in the corresponding panels. Similar results were obtained in three independent experiments.