Figure 3
Figure 3. The transcription factor MTF-1 is required for zinc-induced increase in FPN1 transcription. (A) NIH3T3 cells transfected with pcDNA-mouse-MTF-1-Flag were incubated with nonspecific (siNS) or MTF-1 specific (si MTF1) oligonucleotides. Seventy-two hours after silencing, cells were lysed and MTF-1 mRNA analyzed (left panel) or MTF-1-Flag and tubulin levels determined by Western blot analysis (right panel). The middle panel is a quantification of the Western blot. (B) Macrophages incubated with nonspecific (siNS) or MTF-1 specific (siMTF1) oligonucleotides as in panel A for 48 hours were exposed to 100μM iron (FAC) or 50μM zinc overnight at 37°C. RNA was harvested and semi quantitative RT-PCR was performed for MTF-1, MT-1 and actin mRNA. The data were quantified and normalized to actin with 100% FPN1 or MT-1 mRNA representing the mRNA levels in the non specific (siNS) samples without iron or zinc. The data represent the average of 3 independent experiments. Asterisks in the figures with lines identify columns being compared and show a P value < .05 as determined by Student t test. (C) NIH3T3 cells treated as in panel B were lysed and Fpn and tubulin levels determined by Western blot analysis. (D) NIH3T3 cells expressing either mouse or human MTF1-Flag (Mo and Hu, respectively) were transfected with non specific shRNA (shNS) or sh-mouse MTF1 (shMTF1) containing vectors. Ninety-six hours after shRNA transfection cells were harvested, lysed as described in “Immunofluorescence and microscopy,” and the levels of MTF-1-Flag and tubulin determined by Western blot. (E) NIH3T3 were transfected with shMTF1 or shNS and 72 hours later with either pcDNA-human-MTF-1-Flag (Hu-MTF1) or pcDNA-mouse-MTF-1-Flag (Mo-MTF1). Cells were exposed to 100μM iron (FAC) or 50μM zinc overnight. RNA was harvested and semi quantitative RT-PCR were performed for FPN1, MT-1 and actin mRNA. Histograms show gel quantification of PCR band intensity for 3 independent experiments. Asterisks represent P values less than .05 as determined by Student t test.

The transcription factor MTF-1 is required for zinc-induced increase in FPN1 transcription. (A) NIH3T3 cells transfected with pcDNA-mouse-MTF-1-Flag were incubated with nonspecific (siNS) or MTF-1 specific (si MTF1) oligonucleotides. Seventy-two hours after silencing, cells were lysed and MTF-1 mRNA analyzed (left panel) or MTF-1-Flag and tubulin levels determined by Western blot analysis (right panel). The middle panel is a quantification of the Western blot. (B) Macrophages incubated with nonspecific (siNS) or MTF-1 specific (siMTF1) oligonucleotides as in panel A for 48 hours were exposed to 100μM iron (FAC) or 50μM zinc overnight at 37°C. RNA was harvested and semi quantitative RT-PCR was performed for MTF-1, MT-1 and actin mRNA. The data were quantified and normalized to actin with 100% FPN1 or MT-1 mRNA representing the mRNA levels in the non specific (siNS) samples without iron or zinc. The data represent the average of 3 independent experiments. Asterisks in the figures with lines identify columns being compared and show a P value < .05 as determined by Student t test. (C) NIH3T3 cells treated as in panel B were lysed and Fpn and tubulin levels determined by Western blot analysis. (D) NIH3T3 cells expressing either mouse or human MTF1-Flag (Mo and Hu, respectively) were transfected with non specific shRNA (shNS) or sh-mouse MTF1 (shMTF1) containing vectors. Ninety-six hours after shRNA transfection cells were harvested, lysed as described in “Immunofluorescence and microscopy,” and the levels of MTF-1-Flag and tubulin determined by Western blot. (E) NIH3T3 were transfected with shMTF1 or shNS and 72 hours later with either pcDNA-human-MTF-1-Flag (Hu-MTF1) or pcDNA-mouse-MTF-1-Flag (Mo-MTF1). Cells were exposed to 100μM iron (FAC) or 50μM zinc overnight. RNA was harvested and semi quantitative RT-PCR were performed for FPN1, MT-1 and actin mRNA. Histograms show gel quantification of PCR band intensity for 3 independent experiments. Asterisks represent P values less than .05 as determined by Student t test.

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