Figure 1. Iron content and ferroportin-1 expression in primary Nos2+/+ and Nos2−/− PE-MΦ. (A) Cellular iron content of primary Nos2+/+ and Nos2−/− PE-MΦ was measured by atomic absorption spectrometry. Results were normalized for protein content and compared by Student’s t test. Values are depicted as lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges and statistical significance between Nos2+/+ and Nos2−/− cells indicated (PE-MΦ isolated from n = 9 individual mice per group). (B) Quantitative RT-PCR (qRT-PCR) was used to analyze Fpn1 mRNA levels. Data, normalized for mRNA levels of the housekeeping gene Hprt, were compared and are depicted as in A (PE-MΦ isolated from n = 9 individual mice per group). (C–F) Wild-type PE-MΦ were stimulated with 50 µM of the NO donor Nor5 for different time periods, as indicated. (C) Fpn1 expression was determined by qRT-PCR. Data were compared and are depicted as mean and SEM (n = 4 independent experiments). (D) Western blotting was used to assess Fpn1 protein expression. Actin expression was assessed by reprobing membranes and is shown as loading control. One of four independent experiments is depicted. (E) Fpn1 and actin levels were quantified by densitometric scanning of membranes. Data were compared by ANOVA using Bonferroni’s correction (n = 4 independent experiments). (F) Hepcidin mRNA expression was assessed by means of qRT-PCR and data are presented as described for Fpn1 (n = 4 independent experiments). (G) Activation of the 8.4-kb full-length murine Fpn1 promoter Firefly reporter construct in response to Nor5 was measured in a dual luciferase assay (right) using co-transfection with the Renilla luciferase pRL-SV40 vector as reference. Cells transfected with the empty pGL3 reporter plasmid served as controls (left). Firefly luciferase activity was normalized to Renilla luciferase activity and is depicted as relative activity in arbitrary light units (ALU; n = 4 independent experiments). (H) Primary human CD14+ monocytes were exposed to solvent, Nor5 (50 µM), or FeSO4 (50 µM) for 6 h and FPN1 mRNA expression was quantified by means of qRT-PCR and normalized for levels of the housekeeping gene RPL27 (cells of n = 7 individual subjects). **, P < 0.01; ***, P < 0.001.
Figure 1.

Iron content and ferroportin-1 expression in primary Nos2+/+ and Nos2−/− PE-MΦ. (A) Cellular iron content of primary Nos2+/+ and Nos2−/− PE-MΦ was measured by atomic absorption spectrometry. Results were normalized for protein content and compared by Student’s t test. Values are depicted as lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges and statistical significance between Nos2+/+ and Nos2−/− cells indicated (PE-MΦ isolated from n = 9 individual mice per group). (B) Quantitative RT-PCR (qRT-PCR) was used to analyze Fpn1 mRNA levels. Data, normalized for mRNA levels of the housekeeping gene Hprt, were compared and are depicted as in A (PE-MΦ isolated from n = 9 individual mice per group). (C–F) Wild-type PE-MΦ were stimulated with 50 µM of the NO donor Nor5 for different time periods, as indicated. (C) Fpn1 expression was determined by qRT-PCR. Data were compared and are depicted as mean and SEM (n = 4 independent experiments). (D) Western blotting was used to assess Fpn1 protein expression. Actin expression was assessed by reprobing membranes and is shown as loading control. One of four independent experiments is depicted. (E) Fpn1 and actin levels were quantified by densitometric scanning of membranes. Data were compared by ANOVA using Bonferroni’s correction (n = 4 independent experiments). (F) Hepcidin mRNA expression was assessed by means of qRT-PCR and data are presented as described for Fpn1 (n = 4 independent experiments). (G) Activation of the 8.4-kb full-length murine Fpn1 promoter Firefly reporter construct in response to Nor5 was measured in a dual luciferase assay (right) using co-transfection with the Renilla luciferase pRL-SV40 vector as reference. Cells transfected with the empty pGL3 reporter plasmid served as controls (left). Firefly luciferase activity was normalized to Renilla luciferase activity and is depicted as relative activity in arbitrary light units (ALU; n = 4 independent experiments). (H) Primary human CD14+ monocytes were exposed to solvent, Nor5 (50 µM), or FeSO4 (50 µM) for 6 h and FPN1 mRNA expression was quantified by means of qRT-PCR and normalized for levels of the housekeeping gene RPL27 (cells of n = 7 individual subjects). **, P < 0.01; ***, P < 0.001.

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