Porphyrin and heme analysis of MEL cells treated with itaconate. Heme (A), PPIX (B), and total carboxyl porphyrins (C,D) were determined by high-performance liquid chromatography for wild-type MEL cells differentiated in dimethyl sulfoxide media for 72 hours. (E) Relative fluorescence of cyclic porphyrins in media from MEL cell cultures overexpressing Homo sapiens ALAS2 (hsALAS2) over 72 hours without dimethyl sulfoxide induction. Itaconate concentrations were 2.5 mM in all experiments shown here. Bars represent means ± 1 standard deviation (n = 3-4 biological replicates as indicated red dots). Multiple Student t tests combined with Bonferroni analysis produced P values <.01 for all phosphate-buffered saline (PBS) vs itaconate comparisons shown. RFU, relative fluorescence units.