Figure 1.

Luteolin functions as a potent inhibitor in vitro and in cells. A, schematic representation of the initiation of O-GalNAc glycosylation by ppGalNAc-Ts. B, chemical structure and IC₅₀ of luteolin against ppGalNAc-T2 detected by HPLC-based ppGalNAc-T assay. The data are expressed as percentages of the control (DMSO, 100%) and are shown as the mean ± S.D. (n = 3). C, effects of luteolin on the O-GalNAc glycosylation in CHO-ldlD cells detected by the metabolic labeling and click chemistry. CHO-ldlD cells were preincubated with 100 μm Ac₄GalNAz for 12 h and then treated with the indicated concentrations of luteolin for an additional 36 h. Total cell lysates were collected to react with biotin alkyne for 3 h before being analyzed by streptavidin blotting. D, effects of luteolin on glycans of proteins in HEK 293T cells. Cells were incubated with luteolin at 25 μm for 36 h, and total lysates were analyzed by lectin blot. Control, DMSO-treated cells. E, effects of luteolin on glycans in Jurkat cells detected by immunofluorescence. Cells were treated with 25 μm luteolin for 24 h and stained with different lectins. Green, HPA and ConA; blue, nuclei stained with DAPI. Scale bars, 50 μm. Control, DMSO-treated cells. F and G, effects of luteolin on the glycosylation of endogenous APP in HEK 293T cells (F) and A549 cells (G). Cell were incubated with 25 μm luteolin for 36 h and collected and analyzed by Western blotting. L, long-term exposure; S, short-term exposure. H and I, effects of luteolin on the glycosylation of transfected APP (H) and PDPN (I) in HEK 293T cells. FLAG-tagged APP and PDPN were transfected for 24 h and then incubated with 25 μm luteolin for another 24 h. APP was then immunoprecipitated from the cell lysates (Input) using an anti-FLAG M2 affinity resin. The mean density of APP-H or PDPN-H was quantified and normalized with APP-L or PDPN-L. Cells expressing APP or PDPN without luteolin treatment were considered 100%. IP, immunoprecipitation. 50 μg of total protein was used to analyze endogenous APP, and 10 μg was used for transfected APP and PDPN. The data shown represent the mean ± S.D. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. F–I, two-tailed Student's t test. β-Actin and α-tubulin were used as loading control. EV, empty vector.