Figure 5.

Differentiated Abcb10 shRNA MEL cells show reduced Alas2 mRNA levels. A, MEL cells stably expressing control shRNA or Abcb10-specific shRNA were treated with 1.5% DMSO for 3 days, and differentiated cells were shifted to growth in glycine-deficient medium (−glycine) and incubated with 1 μm Tf(Fe)₂ for 30 min, followed by the addition of [¹⁴C]glycine for 1 h at 37 °C. Heme and porphyrins were extracted, and radioactivity was measured as described under “Experimental procedures.” The data are expressed as cpm/mg of total cell protein. Error bars, S.E. B, mRNA from MEL cells stably expressing control shRNA or Abcb10-specific shRNA was treated with 1.5% DMSO for 3 days, and qPCR for Mfrn1, Abcb10, β-Globin, Alas2, and Fech was performed using the primers listed in Table 3. Error bars, S.E. Western blot analysis of Abcb10, Alas2, and Porin protein levels in control and shRNA Abcb10 undifferentiated and differentiated MEL cells is shown. C, control shRNA or Abcb10-specific shRNA cells were transduced with a lentivirus containing either a control vector or human Abcb10-FLAG, and hemoglobinization and qPCR were performed. A representative experiment is shown. Error bars, S.E. D, hemoglobinization was assessed in cells as in B plus or minus 1.2 mm ALA. E, cells as in D were harvested, mRNA was isolated, and qPCR was performed. *, p ≤ 0.05. Error bars, S.E.