Figure 2.

MAD1 regulates RNA synthesis, rDNA transcription and protein synthesis in granulocytes. Cultures of primary granulocytes from wild-type (WT; Mad1+/+) and Mad1-null (Mad1−/−) mice were stimulated with SCF and G-CSF. Cells were analyzed after 5 days of culture and equal cell numbers were used for all assays. (A) RNA synthesis as measured by incorporation of [³H]uridine into total cellular RNA (n=4 for each genotype). ^*P<0.05 compared to WT mice. (B) rDNA transcription was measured by nuclear run-on analysis of the 45S rRNA precursor (n=4 WT, 4 Mad1−/−). ^*P<0.05 compared to WT mice. (C) Cell volume of cultured granulocytes (n=8 WT, 7 Mad1−/−). ^*P<0.05 compared to WT mice. (D) Protein synthesis as measured by incorporation of [³⁵S]methionine into total cellular protein (n=4 WT, 5 Mad1−/−). ^*P<0.05 compared to WT mice. (E) Proliferation rate as determined by granulocyte concentration 5 days after wild-type and Mad1−/− cultures were seeded with 1 × 10⁵ cells/10 ml of culture medium (n=7 WT, 7 Mad1−/−).