Figure S5.

FAMIN Activity Affects Purine-Containing Cofactor Turnover, Related to Figure 4

(A) Fraction of ATP, NAD⁺, NADH, FAD, acetyl-CoA, HMG-CoA and succinyl-CoA labeled as the indicated isotopomer in M1 macrophages after a 3 h pulse with [¹⁵N₅] adenine. Fractions of different labeled states (averaged across Famin^p.254I, Famin^p.254V and Famin^p.284R genotypes) are depicted as pie charts. Asterisks indicate significantly altered isotopic labeling across Famin genotypes (n = 6 per genotype).

(B) Total, unlabelled and [¹³C₁¹⁵N₂] guanine and guanosine levels in M1 macrophages after a 3 h pulse with [¹³C₁¹⁵N₂] guanosine (n = 6).

(C) Differential metabolite levels in Famin^p.254I versus Famin^p.254V M1 macrophages. Data depicted as a volcano plot using p value and log₂ fold change. Grey dots are non-significant, while black dots correspond to LCMS features with significantly altered abundance following Benjamini-Hochberg correction for multiple testing. Differential metabolites shown in red were confirmed and identified as indicated (n = 6).

(D) Cytoplasmic pH measured using pHrodo in Famin^p.254I, Famin^p.254V, Famin^p.284R M0 macrophages (n = 18)

(E) Cytoplasmic pH measured using BCECF with dual excitation at 440nm and 490nm in Famin^p.254I, Famin^p.254V, Famin^p.284R M1 macrophages (n = 18). Reduced 490:440 ratio corresponds to lower (more acidic) pH_c.

(F) Cytoplasmic pH measured using pHrodo in control and FAMIN-silenced HepG2 cells 48 h after transfection (n = 9).

(G) Heatmap of metabolites in control and FAMIN-silenced HepG2 cells 24 h after transfection (n = 3).

Data are represented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (unpaired, two-tailed Student’s t test or one-way ANOVA).