Figure 4. Stable expression of murine ALAS2 variants in K562 cells results in PPIX accumulation when culture medium is supplemented with either glycine or deferoxamine.

(A) PPIX fluorescence 18 hours after supplementation of the culture medium with glycine to yield 10 mM and 100 mM final concentrations. Normalized fluorescence values were obtained by first dividing the mean PPIX fluorescence by the mean ZsGreen1 fluorescence for each cell population and secondly dividing by the mean PPIX fluorescence/ZsGreen1 fluorescence ratio of the pIRES2-ZsGreen1-transfected cells. (B) PPIX fluorescence 18 hours after supplementation of the culture medium with deferoxamine to a final concentration of 100 μM (+D). (C) PPIX fluorescence of K562 cell populations supplemented with glycine to a 100 mM final concentration. The blue area represents cells that do not express ZsGreen1, and the green area represents the population that expresses ZsGreen1. K562 cells that express ZsGreen1 are not affected by glycine supplementation of the culture medium (upper plots), while cells that express WT exhibit a significant increase in PPIX fluorescence when the culture medium is supplemented with glycine (lower plots). (D) PPIX fluorescence of K562 cell populations grown in culture media containing 100 μM deferoxamine. Cell growth in the presence of deferoxamine significantly increases the PPIX fluorescence in K562 cells expressing WT, but does not affect PPIX fluorescence in cells expressing only ZsGreen1 or HPVT, as indicated by the number of cells within the red dotted-line boxes.