FIGURE 2.

Inositol synthesis is up-regulated in IP6K1-KO cells. A, mIno1 mRNA levels are increased in IP6K1-KO cells. WT and IP6K1-KO MEF cells were grown in DMEM with 10% FBS and harvested for mRNA extraction. mIno1 mRNA levels were determined by RT-PCR (two independent experiments with two replicates each) (*, p < 0.05). B, mINO1 protein levels were profoundly increased in IP6K1-KO cells (left). The expression of Ip6k1 in IP6K1-KO cells restored mINO1α and mINO1γ levels (right). MEF cells were grown in DMEM with 10% FBS in the presence or absence of 4 μg/ml blasticidin. Cells were harvested and lysed for protein extraction. 30 μg of total cell extract from each sample were subjected to Western blot analysis using 10% SDS-polyacrylamide gel. C, IP6K1-KO cells exhibited increased intracellular inositol levels. Intracellular inositol levels in MEF cells were determined as described under “Experimental Procedures” (three independent experiments with two replicates each) (*, p < 0.05). D, intracellular Glc-6-P levels were decreased in IP6K1-KO cells. Intracellular Glc-6-P levels in MEF cells were determined by enzyme-coupled fluorescence assay as described under “Experimental Procedures” (three independent experiments with two replicates each) (*, p < 0.05). Values are mean ± S.E. Statistical significance was determined by unpaired t test.