Extended Data Fig. 5: Dynamic formation of de novo pyrimidine biosynthetic complexes.
From: De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence
a, The chromatography elution profiles of the fractions were shown. b, Co-fractionation of pyrimidinosome by fast protein liquid chromatography with Superose 6 columns. The size-exclusion chromatography of whole cell lysates from HEK293T/shSramble and HEK293T/shCAD cells was analyzed by immunoblotting. The dashed box indicates pyrimidinosome-containing fractions. The elution positions of a series of protein standards with known molecular masses were indicated. c, Sucrose density gradient centrifugation analysis of the pyrimidinosome using the whole cell lysates from HEK293T cells. d,e, HEK293T cells expressing CAD were cultured under pyrimidine-depleted medium with or without uridine supplementation for 12 h and subjected to IP analysis. f,g, Immunofluorescence analysis of the co-localization of Flag-CAD and GFP-GOT1 (f) or mCherry/mCherry-UMPS and VDAC3-Flag (g) proteins in HeLa cells growing in pyrimidine-rich or pyrimidine-deprived medium. Flag-CAD was labeled in red (f) and VDAC3-Flag (g) was labeled in green. Scale bar, 10 μm. h,i, Co-localization of GFP-fusion proteins and mCherry-fusion proteins in HeLa cells cultured in pyrimidine-deprived medium. GFP, DHODH-GFP, or GFP-GOT1 was co-transfected with mCherry-UMPS, and DHODH-mCherry was co-transfected with GFP-GOT1 (i). Scale bar, 10 μm. Pearson’s correlation coefficients for co-localization of the indicated proteins under pyrimidine-deprived conditions in i (h). The clusters from different cells in each group were analyzed using the co-localization analysis tools in ZEN software. Data are shown as the mean ± SD, n = 41 (GFP/mCherry-UMPS), n = 36 (GOT1/mCherry-UMPS), n = 60 (DHODH/mCherry-UMPS) and n = 45 (GOT1/DHODH-mCherry) biologically independent clusters. P values were obtained using one-way ANOVA with Sidak test. j, Immunofluorescence analysis of the co-localization of DHODH-GFP and MitoTracker Deep Red in HeLa cells growing in pyrimidine-rich or pyrimidine-deprived medium. Scale bar, 10 μm. (b-g, i, j) Data are verified in three replicates with similar results.
