Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 1. Low WISP2 expression predicts reduced survival of AML patients. A, correlation of WISP2 expression with the survival probability of AML patients via the UALCAN cancer database. B, analysis of WISP2 expression in AML patients with different subtypes via the UALCAN cancer database. C, Western blot analysis of WISP2 expression in bone marrow mononuclear cells from six healthy donors and 16 AML patients. D, Co-IP assay to detect the acetylation of WISP2 in bone marrow mononuclear cells from healthy donors and AML patients. E, the protein expression and acetylation level of WISP2 in normal bone marrow mononuclear cells and AML cells. Ac, acetylated lysine; AML, acute myeloid leukemia; Co-IP, co-immunoprecipitation; WISP2, Wnt-1- induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 2. WISP2 overexpression inhibits the proliferation and promotes the apoptosis of AML cells. A and B, WISP2 lentivirus or negative control (NC) was infected into HL-60 cells (A) or Kasumi-1 cells (B) for 24, 48, 72 h, 96 h, and 120 h, and cell viability was determined by CCK8 assay. Data represent the mean ± SD (n = 5 for each group). C and D, apoptosis of HL-60 cells (C) or Kasumi-1 cells (D) was measured by flow cytometry after 72 h of infection with LV-WISP2 or LV-NC. Data represent the mean ± SD of three independent experiments. E and F, FACS analysis of the mitochondrial membrane potential of HL-60 cells (E) or Kasumi-1 cells (F) after 72 h of infection with lentivirus. Data represent the mean ± SD of three independent experiments. G and H, Western blot for WISP2 and apoptosis-related markers in whole cell lysates, as well as cytochrome C in mitochondrial and cytoplasmic fractions of HL-60 cells (G) or Kasumi-1 cells (H). p values in Panels A and B were determined with two-way ANOVA followed by Tukey’s multiple comparisons test, and the p values in Panels C–F were determined with one-way ANOVA followed by Tukey’s test (**p < 0.01, ****p < 0.0001). AML, acute myeloid leukemia; cl cas-3, cleaved caspase-3; cl cas-9, cleaved caspase-9; cl PARP, cleaved PARP; cyt c, cytochrome C; cyto, cytoplasmic; LV, lentivirus; LV-WISP2, lentivirus plasmids containing full-length WISP2; mito, mitochondrial; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Over Expression, Negative Control, Infection, CCK-8 Assay, Cytometry, Membrane, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 3. WISP2 overexpression exerts antileukemic effects in the in vivo model of AML. A, percentage of CD45+ cells in the bone marrow of mice transplanted with HL-60 cells. B, Leishman–Wright–Giemsa staining of mouse bone marrow. Black arrows indicate ring-like differentiated cells. The p value in Panel A was determined with an unpaired t test (****p < 0.0001). AML, acute myeloid leukemia; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Over Expression, In Vivo, Staining

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 4. Pan-HDAC inhibitors induce apoptosis and efficiently prevent WISP2 degradation in AML cells. Two pan-HDAC inhibitors, VPA and TSA, were used to treat AML cells. A and B, HL-60 cells and Kasumi-1 cells were treated with VPA or TSA for 24 h and then subjected to CCK8 assay. Data represent the mean ± SD (n = 5 for each group). C and D, apoptosis of AML cells was measured by flow cytometry after 24 h of VPA or TSA treatment. Data represent the mean ± SD of three independent experiments. E and F, quantitative analysis of the percentage of apoptosis. G and H, Western blot for WISP2 in VPA- or TSA-treated AML cells. I and J, the acetylation of WISP2 in VPA- or TSA-treated AML cells was assessed by co-IP assay. K and L, AML cells were treated with VPA or TSA for 12 h and treated with CHX (25 μg/ml) for the indicated times, followed by Western blot analysis of WISP2 expression. p values were determined with one-way ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Ac, acetylated lysine; AML, acute myeloid leukemia; CHX, cycloheximide; co-IP, co-immunoprecipitation; HDAC, histone deacetylase; TSA, trichostatin A; VPA, valproic acid; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: CCK-8 Assay, Cytometry, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Histone Deacetylase Assay

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 5. HDAC3 interacts with and deacetylates WISP2 in AML cells. A and B, co-IP was performed with anti-WISP2 antibody and IgG as a control, followed by immunoblotting with anti-HDAC1, anti-HDAC3, anti-HDAC6, and anti-HDAC7 antibodies in AML cells. C, the correlation between HDAC3 or HDAC1 and WISP2 expression levels from 151 AML samples analyzed by StarBase. D, Western blot analysis of HDAC3 expression in bone marrow mononuclear cells from healthy donors and AML patients. E and F, the protein and acetylation levels of WISP2 in RGFP966-treated AML cells. G and H, the protein and acetylation levels of WISP2 and the protein expression of HDAC3 in AML cells infected with HDAC3 lentivirus. Ac, acetylated lysine; AML, acute myeloid leukemia; co-IP, co-immunoprecipitation; HDAC, histone deacetylase; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Expressing, Infection, Immunoprecipitation, Histone Deacetylase Assay

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 6. The lysine K6 residue is essential for WISP2 acetylation. A, prediction of acetylation on internal lysine residues indicated two potential acetylated lysine (K) residues, K6 and K20, within WISP2. B, HEK293 cells were transfected with wildtype WISP2 (wt-WISP2-flag), mutant WISP2_K6R (WISP2_K6R-flag), or mutant WISP2_K20R (WISP2_K20R-flag) for 48 h, and the transfection efficiency was determined by immunoblotting with an anti-Flag antibody. C and D, HEK293 cells were transfected with wt-WISP2-flag, WISP2_K6R-flag, or WISP2_K20R-flag for 48 h and then treated with VPA or RGFP966 for 24 h. Whole-cell lysates were subjected to detection with anti-acetylated-lysine antibody. Ac, acetylated lysine; VPA, valproic acid; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Residue, Transfection, Mutagenesis, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 7. 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 in- crease 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.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Western Blot, Infection, Transfection, Over Expression, Plasmid Preparation, Expressing