American Society of Hematology

Fig. 4.

$Fig. 4. Microarray assessment of iron-mediated translational control. / Cytoplasmic extracts from hemin- and desferrioxamine-treated HeLa cells were sedimented through a 10%-40% sucrose gradient (see “Materials and methods”). The profile on the top denotes the A254 absorption profile. The positions of polysomes, monosomes (80S), and mRNPs are indicated. (A) Northern blot analysis was performed with total RNA extracted from the 6 individual fractions obtained from the sucrose gradient. The Northern blot was sequentially probed with radiolabeled probes for actin and L-ferritin. The signals obtained from the individual fractions were quantified on Fluoroimager (Molecular Dynamics) and the signals in the polysomes and the 80S and mRNP fractions were calculated as a percentage of the sum of signals in all lanes. The ratio of the 80S and mRNP fractions to the polysomal fractions (PS) is indicated. (B) The 3 fractions containing polysomal (PS) and the 3 fractions containing the monosomal and mRNP-derived RNA were pooled for each condition and analyzed on the IronChip. The regulatory ratio between mRNP and polysomal fractions is indicated for L-ferritin (L-fer), H-ferritin (H-fer), actin, and GAPDH.$

Microarray assessment of iron-mediated translational control.

Cytoplasmic extracts from hemin- and desferrioxamine-treated HeLa cells were sedimented through a 10%-40% sucrose gradient (see “Materials and methods”). The profile on the top denotes the A254 absorption profile. The positions of polysomes, monosomes (80S), and mRNPs are indicated. (A) Northern blot analysis was performed with total RNA extracted from the 6 individual fractions obtained from the sucrose gradient. The Northern blot was sequentially probed with radiolabeled probes for actin and L-ferritin. The signals obtained from the individual fractions were quantified on Fluoroimager (Molecular Dynamics) and the signals in the polysomes and the 80S and mRNP fractions were calculated as a percentage of the sum of signals in all lanes. The ratio of the 80S and mRNP fractions to the polysomal fractions (PS) is indicated. (B) The 3 fractions containing polysomal (PS) and the 3 fractions containing the monosomal and mRNP-derived RNA were pooled for each condition and analyzed on the IronChip. The regulatory ratio between mRNP and polysomal fractions is indicated for L-ferritin (L-fer), H-ferritin (H-fer), actin, and GAPDH.

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