Figure 3.

Alteration of Metabolism in Response to Galactose

(A) Incorporation of [¹⁴C]galactose into laminin β2. HEK293 cells were transfected with mock or a C-terminal His-tagged laminin β2 expression construct, and were then cultured in medium with or without 0.3 mM [¹⁴C]galactose for 24 hr. Immunoprecipitates with His antibody were analyzed by silver staining, western blot, and autoradiography.

(B) The response of ECAR (upper panel) and OCR (lower panel). HEK293 cells were stimulated with glucose (3 mM), mannose (3 mM), or galactose (3 mM), and subsequently subjected to the mitochondria inhibitor rotenone (3 μM) and the glycolysis inhibitor 2-DG (100 mM) at the indicated times. Both datasets are from the same experiment, and each data point represents the mean ± SEM, n = 4.

(C) Metabolomic analysis of HEK293 cells treated with sugar-free (white), glucose (3 mM, red), and galactose (3 mM, green) conditions for 6 hr. The relative quantities of the annotated metabolites are represented as bar graphs.

(D) Representative immunoblot of PFKP in response to the indicated treatments.

(E) HEK293 cells were treated with the indicated conditions for 6 hr, and PFK1 activity was analyzed by measuring NADH consumption.

(F) HEK293 cells were sugar-deprived (SD) for 6 hr, followed by restoration with the indicated sugars (3 mM each) for 1 hr.

(G) HEK293 cells were cultured in medium containing glucose (3 mM) for 6 hr, and PFK1 activity was analyzed in a reaction mixture that included galactose (Gal) (3 mM), galactose-1-phosphate (Gal-1P) (3 mM), or UDP galactose (UDP-Gal) (3 mM). The data are the mean ± SEM. ***p < 0.005 by Dunnett's test. Cells were cultured under serum-free conditions.

See also Figure S7.