The IRF8-KLF4 axis regulates the development of monocytes. (A) Induction of KLF4 expression by IRF8. Tot2 cells transduced with empty MSCV-puro, MSCV-IRF8-puro, or MSCV-KLF4-FLAG-puro were analyzed by qRT-PCR in triplicate (mean ± standard deviation) and western blotting analysis on day 4. RAW264.7 cells were also loaded for comparison. Data are representative of three independent experiments with similar results. (***) P < .001 (Student t test). (B) Wright-Giemsa stains of Tot2 cells transduced with indicated MSCV. Cells were classified into three categories (differentiated, intermediate, and undifferentiated) on day 4 (mean ± standard deviation). (C) Induction of monocyte-related genes. Transcript levels were analyzed by qRT-PCR on day 4 as in (A). (D) Surface marker analysis. Cells on day 4 were stained with the indicated antibodies or isotype-matched control antibodies and analyzed by flow cytometry. Forward scatter (FSC)hi SSChi cells were gated to analyze differentiated cells. Data are representative of three independent experiments. (E) Venn diagram for the relationship between genes that displayed more than a 2-fold change in expression 2 days after transduction with IRF8 or KLF4. Gene expression profiling by microarray was performed in biological duplicates as in Figure 1. P values by Fisher’s exact test are indicated.
