Figure 3.
CBA-1 Interacts with KDM3A
(A) Synthesis of CBA-B2, a biological active biotinylated version of CBA-1; Legend: a, 4-fluorobenzaldehyde, ethyl piperidine-4-carboxylate, K2CO3, dimethyl sulfoxide, 120°C, 3 h (63% yield); b, 2 M NaOH, 48 h, 25°C (100% yield); c, N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-yl)pentanamide (1 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.5 eq), 1-hydroxybenzotriazole (1.5 eq), triethylamine (1.5 eq), 12 h, 25°C (57% yield); d, 5-chloroquinolin-8-ol (1 eq), butyramide (6 eq), 150°C, 5 h (57% yield).
(B) CBA-B2 inhibited Wnt signaling (∗p < 0.0001; n = 3).
(C) KDM3A was pulled down from HEK293T cell lysates by CBA-B2 and streptavidin beads.
(D) Dose-responses of CBA-1 on the enzymatic activities of KDM3A.
(E) Knocking down of KDM3A reduced Wnt target genes expression in LS174T CRC cells. Densitometry was analyzed using three western blot images (∗p < 0.05; n = 3).
(F) Knocking down of KDM3A decreased Wnt-induced TOPFlash reporter activity (∗p < 0.01; #p < 0.05; n = 3).
(G) Knocking down of KDM3A inhibited LS174T CRC cell proliferation (∗p < 0.0001; n = 3).