Induction of expression of myelomonocyte-specific and B-cell–-specific genes in MEL cells after overexpression of PU.1.
(A) Total RNA was extracted from parental MEL-B8/3 cells (lanes 1-4), mock cells (lanes 5-8), and PU.1-1 cells (lanes 9-12) cultured for 48 hours without reagents (lanes 1, 5, and 9), with DMSO (lanes 2, 6, and 10), with ZnCl2 (lanes 3, 7, and 11), or with DMSO and ZnCl2 (lanes 4, 8, and 12). Expression of myelomonocyte-specific and B-cell–specific genes was examined by Northern blot (the C/EBP genes) or RT-PCR analysis. (B) The intensity of the bands obtained in the RT-PCR analysis was quantified, and ratios of the values for parental MEL-B8/3 (■), mock (░), and PU.1-1 (▨) cells treated with ZnCl2 are shown in the graphs. The calculated ratio of the value for PU.1 cells to the average value for parental and mock cells is shown in each graph. (C) Total RNA was extracted from PU.1-2 cells (lanes 1 and 2) and PU.1-3 cells (lanes 3 and 4) cultured for 48 hours without reagents (lanes 1 and 3) or with ZnCl2 (lanes 2 and 4). Expression of myelomonocyte-specific and B-cell–specific genes was examined by RT-PCR analysis. (D) Total protein was extracted from PU.1-1 cells (lanes 1 and 2), PU.1-2 cells (lanes 3 and 4), and PU.1-3 cells (lanes 5 and 6) cultured for 8 hours without reagents (lanes 1, 3, and 5) or with ZnCl2 (lanes 2, 4, and 6). Expression of PU.1 protein was examined by Western blot analysis.
