FIGURE 2: Heme mediates the inhibitory effect of DHA on fermentable media.

(A) The heme biosynthesis pathway in Saccharomyces cerevisiae.

(B) Deletion of HEM1 essentially eradicates DHA inhibition of yeast on fermentable media. YPD medium supplemented with Tween-80 and cholesterol (YPD*) was used. HEM2-knockdown (HEM2-KD) strain is also resistant to DHA on fermentable media.

(C) RT-PCR analysis of RNA extracted from wild type and HEM2-KD strain. Semi-quantitative RT-PCR reveals a down-regulation at the mRNA level of HEM2 in the HEM2-KD strain. ACT1 is the control.

(D) Overexpression of HEM2&3 (HEM2&3 OE) sensitizes yeast to DHA in the presence of δ-aminolevulinic acid (ALA). The effect was not as dramatic and could not be detected with the plate assay and thus a liquid growth assay was adopted. A petite strain was used. The grey bar represents HEM2&3 OE strain and white bar is the control strain with an empty vector. ***, P < 0.001.

(E) The growth of the WT petite and HEM2-KD petite yeast in the presence and absence of ART in YPD liquid medium. HEM2-KD petite yeast is not more resistant than normal petite yeast to ART in fermentable media. Similar results were observed in 3 independent experiments.

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