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. 2019 Jan 24;116(8):2987–2995. doi: 10.1073/pnas.1820161116

Fig. 4.

Fig. 4.

Methotrexate inhibits Wnt signaling, as well as Wnt-induced endocytosis, lysosomal activity, and GSK3 and PRMT1 sequestration through the folate cycle. (A) One-carbon metabolism is composed of the methionine and folate cycles; methotrexate (MTX) inhibits DHF from entering the cycle. (B) Methotrexate (20 μM) inhibited Wnt signaling (12 h), as assessed by BAR-Renilla luciferase assays. Note that Wnt inhibition was rescued by the addition of SAM (lane 4). (C) Methotrexate induced significant cellular toxicity only at concentrations above 30 μM in HEK293 cells stably expressing Renilla (12-h treatment with MTX). (D–F) Methotrexate treatment inhibited Wnt-induced lysosomal activity (assessed by the fluorescence of endocytosed BSA-DQ after 30 min of Wnt3a treatment). (G) Quantification of the inhibition of Wnt-induced endolysosomal activity by MTX using ImageJ. (H–W) MTX (20 μM) inhibited the formation of vesicles by Wnt3a (by phase contrast microscopy) and decreased PRMT1 and GSK3 sequestration (by in situ proteinase K protection). Note that the addition of 1 mM SAM rescued the effects of MTX (arrowheads). (X) Quantification of the inhibition by MTX, and rescue by SAM, of Wnt-induced endolysosomal vesicle formation assessed by phase contrast microscopy. (Y) Quantification of GSK3 and PRMT1 colocalization in endolysosomes/MVBs after 30 min of Wnt3a treatment by Pearson’s correlation coefficient. (Scale bars: 10 μm.) *P < 0.05; **P < 0.01; n < 3.