FIG. 2.

Reduced levels of Mfrn1 or Mfrn2 affect iron incorporation into heme. (A) NIH 3T3 fibroblasts were treated for 48 h with nonspecific (N.S.) oligonucleotides or oligonucleotides specific for Mfrn1 (siRNA Mfrn1) and/or Mfrn2 (siRNA Mfrn2). The cells were lysed, and Western blotting was performed using anti-Mfrn1, anti-Mfrn2, or antitubulin. (B) NIH 3T3 fibroblasts grown in 1.2 mM ALA were treated as described for panel A with cells simultaneously transfected with pEGFP-Mfrn1 (CMV-Mfrn1) or pEGFP-Mfrn2 (CMV-Mfrn2). Iron (⁵⁹Fe) incorporation into heme was measured as described in Materials and Methods and expressed as the relative percentage of ⁵⁹Fe incorporation, where 100% represents cells treated with nonspecific oligonucleotides. All data were normalized for protein. (C) Protoporphyrin IX accumulation was measured in the 3T3 fibroblasts treated as described for panel B. The data are expressed as the fluorescence units/mg of protein. Error bars represent the standard deviations of the results from three independent experiments.