Fig 1: Acetylation of WISP2 prevents NEDD4-mediated ubiquitination. A, HL-60 cells were treated with or without RGFP966 for 24 h, and MG132 was added 6 h before harvesting cells. Proteins were co-IP with anti-WISP2 antibody, followed by immunoblotting with anti-ubiquitin or anti-WISP2 antibodies. B, HDAC3 lentivirus was infected into HL-60 cells for 72 h, and MG132 was added 6 h before harvesting cells. The ubiquitination of WISP2 was detected by co-IP with an anti-WISP2 antibody, followed by immunoblotting with the indicated antibodies. C, HL-60 cells were infected with HDAC3 lentivirus. After 72 h, the cells were treated with MG132 for 6 h. Total lysates were analyzed by Western blotting using an anti-WISP2 antibody. D, prediction of Ubibrowser indicated that NEDD4 was the top E3 ubiquitin ligase that probably targets WISP2. E and F, endogenous interaction between WISP2 and NEDD4 in HL-60 cells and Kasumi-1 cells was examined by co-IP assay. G and H, the protein levels of NEDD4 and WISP2 were confirmed by Western blotting in NEDD4-overexpressing AML cells. I, co-IP assay to assess WISP2 ubiquitination in HL-60 cells infected with NEDD4 lentivirus. J, co-IP assay to assess WISP2 ubiquitination in HL-60 cells transfected with NEDD4 overexpression plasmid and wt-WISP2-flag or WISP2_K6R-flag. K, HL-60 cells were infected with LV-oeHDAC3 and LV-shNEDD4 as indicated. Western blot analysis was performed to determine WISP2 expression. L, schematic diagram for the antileukemic effect of WISP2: HDAC3-induced deacetylation disrupts WISP2 stability by augmenting ubiquitination mediated by NEDD4, which contributes to the increase in tumor cell proliferation and the suppression of apoptosis. The blockade of histone deacetylation by HDAC inhibitors restores WISP2 expression in AML cells, thereby allowing WISP2 to exert its antileukemia effect. AML, acute myeloid leukemia; co-IP, coimmunoprecipitation; LV, lentivirus; ub, ubiquitin; WISP2, Wnt-1-induced signaling protein-2.