Fig. 6. Ser556 phosphorylation in ULK1 facilitates DAPK3-mediated autophagy and tumor suppression in GC cells.

a HA-ULK1 WT or S556A mutant were transfected into DAPK3-overexpressed MKN45 cells with endogenous ULK1 depletion by shRNA. Cell lysates were immunoblotted as indicated. b DAPK3-silenced MGC803 cells transfected with Flag-DAPK3-WT or Flag-DAPK3-K42A were placed in EBSS starvation media or full media for 4h and immunoblotted as indicated. Cell growth rate, migration, and invasion ability of MKN45-DAPK3 cells were measured by XTT assay (c) and transwell assay (d) (***P < 0.001, student t test) (original magnification, 200×, scale bars:100 μm). Cell growth rate, migration, and invasion were examined by XTT assay (e) and transwell assay (f) in MGC803-shDAPK3 cells (***P < 0.001, student t test, original magnification, 200×, scale bars: 100 μm). g 1×10⁷ DAPK3-overexpressed and empty vector-transfected MKN45 cells expressing wild type ULK1 or ULK1-S556A mutant were subcutaneously injected into nude mice. Representative images of the xenograft tumors formed in the nude mice. Growth curves of tumors derived from the indicated cell lines were shown (***P < 0.001, Student’s t test).