(A) Northern blot analysis of induction of PU.1/Spi-1 gene expression in the PU.1-sense transfectants. Total RNA was extracted from the PU.1-sense clone 1 cells at the indicated time after the addition of 100 μmol/L ZnCl₂ to the culture and hybridized with ³²P-labeled probes. (B) Western blot analysis of overexpression of the PU.1 protein in the PU.1-sense transfectants. Total protein was extracted from the parental MEL-B8/3 cells (lane 1), PU.1-sense clone 1 cells (lane 2), and PU.1-sense clone 1 cells cultured for 8 hours with 100 μmol/L ZnCl₂ (lane 3) and was probed with antimouse PU.1 antibody. Total protein of PU.1-negative T-cell lymphoma EL4 was also loaded as a negative control (lane 4). (C) Western blot analysis of suppression of expression of the PU.1 protein in the PU.1-antisense transfectants. Total protein was extracted from the PU.1-sense clone 1 cells cultured for 8 hours with 100 μmol/L ZnCl₂ (lane 1), PU.1-antisense clone 1 cells (lane 2), and PU.1-antisense clone 1 cells cultured for 10 hours (lane 3), 24 hours (lane 4), and 48 hours (lane 5) with 100 μmol/L ZnCl₂ and was probed with antimouse PU.1 antibody.