American Society of Hematology

Figure 5.

$Figure 5. The effect of MtFt on distribution and trafficking of intracellular 59Fe and on the availability of 59Fe for chelation by SIH. (A) Distribution of 59Fe-containing molecules. Induced and uninduced clone B9 cells were incubated with 59Fe-Tf for 1, 2, or 3 days and lysed with Triton X-100 lysis buffer (1.5% Triton X-100, 0.14 M NaCl, 0.1 M HEPES, pH 7.4, and proteinases cocktail). The lysates were centrifuged at 10 000g for 10 minutes and the soluble fractions (40 μg) were separated by native gradient gel electrophoresis, which was followed by 59Fe autoradiography. (B) Mobilization of iron from endogenous ferritin by MtFt overexpression. Uninduced clone B9 cells (80% confluence) were incubated with 1 μM 59Fe-Tf and after 24 hours some of the cells were processed immediately and the others were reseeded (50% confluence), and incubated for another 48 hours without (2a and 2b) or with (3a and 3b; a and b represent duplicates) tetracycline. After washing, lysing and centrifugation, the lysates were subjected to native PAGE, which was followed by 59Fe autoradiography. (C) 59Fe mobilization from wild-type and MtFt-expressing cells by the iron chelator SIH. Induced and uninduced clone B9 cells were cultured for 48 hours without (-) or with (+) tetracycline, and then for another 24 hours with 1 μM 59Fe-Tf. After washing 3 times with cold PBS, the cells were incubated without or with 200 μM SIH. After 5 hours of incubation, 59Fe in the culture medium and the cells was measured by a γ counter. Data shown are the means ± SD of triplicate determinations from a typical experiment that was performed 3 times. (D) 59Fe mobilization from cytosolic ferritin and MtFt by the iron chelator SIH. Cells were treated as described in panel C. After the pellets were lysed, the extracts were subjected to native PAGE, which was followed by 59Fe autoradiography. The intensities of the bands, corresponding to cytosolic ferritin, MtFt, and fraction Y, were quantified by densitometry. (Note: exposure time for fraction Y was 3-fold longer that that for ferritins.) For panel D, a representative of 5 experiments that gave similar results is shown.$

The effect of MtFt on distribution and trafficking of intracellular ⁵⁹Fe and on the availability of ⁵⁹Fe for chelation by SIH. (A) Distribution of ⁵⁹Fe-containing molecules. Induced and uninduced clone B9 cells were incubated with ⁵⁹Fe-Tf for 1, 2, or 3 days and lysed with Triton X-100 lysis buffer (1.5% Triton X-100, 0.14 M NaCl, 0.1 M HEPES, pH 7.4, and proteinases cocktail). The lysates were centrifuged at 10 000g for 10 minutes and the soluble fractions (40 μg) were separated by native gradient gel electrophoresis, which was followed by ⁵⁹Fe autoradiography. (B) Mobilization of iron from endogenous ferritin by MtFt overexpression. Uninduced clone B9 cells (80% confluence) were incubated with 1 μM ⁵⁹Fe-Tf and after 24 hours some of the cells were processed immediately and the others were reseeded (50% confluence), and incubated for another 48 hours without (2a and 2b) or with (3a and 3b; a and b represent duplicates) tetracycline. After washing, lysing and centrifugation, the lysates were subjected to native PAGE, which was followed by ⁵⁹Fe autoradiography. (C) ⁵⁹Fe mobilization from wild-type and MtFt-expressing cells by the iron chelator SIH. Induced and uninduced clone B9 cells were cultured for 48 hours without (-) or with (+) tetracycline, and then for another 24 hours with 1 μM ⁵⁹Fe-Tf. After washing 3 times with cold PBS, the cells were incubated without or with 200 μM SIH. After 5 hours of incubation, ⁵⁹Fe in the culture medium and the cells was measured by a γ counter. Data shown are the means ± SD of triplicate determinations from a typical experiment that was performed 3 times. (D) ⁵⁹Fe mobilization from cytosolic ferritin and MtFt by the iron chelator SIH. Cells were treated as described in panel C. After the pellets were lysed, the extracts were subjected to native PAGE, which was followed by ⁵⁹Fe autoradiography. The intensities of the bands, corresponding to cytosolic ferritin, MtFt, and fraction Y, were quantified by densitometry. (Note: exposure time for fraction Y was 3-fold longer that that for ferritins.) For panel D, a representative of 5 experiments that gave similar results is shown.

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