ZIPK positively regulates Wnt-mediated transcription in HeLa cells. A, 293T cells in a 24-well plate were transfected with TOPFLASH (100 ng) with or without ZIPK and/or NLK (+, 10 ng; ++, 100 ng) using jetPEI (Polyplus Transfection). At 36 h after transfection, cells were treated with LiCl (30 mm) for an additional 12 h. The cells were harvested and assayed for luciferase activity. The results are indicated as -fold induction of luciferase activity from triplicate experiments, and the error bars represent S.D. **, p < 0.01. TCL (1%) was blotted with an anti-FLAG or anti-Myc antibody (IB). B, 293T cells in a 24-well plate were transfected with TOPFLASH (100 ng) with or without ZIPK WT or KA mutant (10, 30, and 100 ng) using jetPEI. At 36 h after transfection, cells were treated with LiCl (30 mm) for an additional 12 h. The cells were harvested and assayed for luciferase activity as described the above. **, p < 0.01. TCL (1%) was blotted with an anti-FLAG antibody. C, HeLa cells in a 24-well plate were transfected with control, ZIPK-1, or ZIPK-2 siRNA (20 pmol) and then transfected with TOPFLASH or FOPFLASH (100 ng). At 36 h after transfection, cells were treated with LiCl (30 mm) for an additional 12 h. The cells were harvested and assayed for luciferase activity as described in A. **, p < 0.01. TCL (1%) was blotted with anti-ZIPK or anti-actin antibody. D, HeLa cells in a 24-well plate were transfected with control or ZIPK-1 siRNA (20 pmol) and then transfected with or without Myc-tagged mouse ZIPK (0.5 μg) together with TOPFLASH (100 ng). At 36 h after transfection, cells were treated with LiCl (30 mm) for an additional 12 h. The cells were harvested and assayed for luciferase activity as described in A. **, p < 0.01. TCL (1%) was blotted with anti-Myc or anti-actin. E, HeLa cells in a 24-well plate were transfected with control, ZIPK-1, or ZIPK-2 siRNA (20 pmol) and then transfected with TOPFLASH (100 ng). At 36 h after transfection, cells were treated with Wnt3a (culture supernatant of Wnt3a-transfected COS-7 cells) for an additional 8 h. The cells were harvested and assayed for luciferase activity as described in A. **, p < 0.01. F, HeLa cells in a 24-well plate were transfected with control, ZIPK-1, or ZIPK-2 siRNA (20 pmol). At 36 h after transfection, cells were treated with LiCl (30 mm) for the indicated periods. Cells were then lysed, and TCL (1%) was blotted with anti-β-catenin or anti-actin antibody. Densitometric quantification of the above results was also shown. The relative intensity of β-catenin was normalized to the actin protein of the same sample. G, HeLa cells were transfected in a 10-cm dish with control, ZIPK-1, or ZIPK-2 siRNA followed by transfection with TCF4 (10 μg). At 36 h after transfection, cells were treated with LiCl (30 mm) for an additional 2 h. A ChIP assay was then performed on siRNA-transfected cells using IgG or anti-β-catenin antibody. ChIP DNA was analyzed by RT-PCR with primers specific for the cyclin D1 promoter DNA.

ZIPK knockdown influences β-catenin/TCF-mediated gene expression in SW480 colon carcinoma cells. A, SW480 cells (3 × 10⁷) were lysed and immunoprecipitated with control or anti-NLK IgG, and blotted with anti-ZIPK antibody (IB). TCL (1%) was blotted with anti-NLK or anti-ZIPK antibody. B, SW480 cells were transfected in a 24-well plate with control, ZIPK-1, or ZIPK-2 siRNA. At 24 h after transfection, TCL (1%) was blotted with anti-ZIPK or anti-actin antibody. Total RNA samples isolated from these cells were also quantified by reverse transcription and quantitative real-time PCR analysis. Data represent the levels of these mRNA normalized to that of an actin internal control and are expressed relative to the value of control siRNA-treated samples. Shown is a representative experiment, which was repeated at least three times with similar results. C, SW480 cells were transfected in a 24-well plate with control, ZIPK-1, or ZIPK-2 siRNA (20 pmol) and then transfected with TOPFLASH or FOPFLASH (100 ng). At 36 h after transfection, cells were treated with LiCl (30 mm) for an additional 12 h. The cells were harvested and assayed for luciferase activity as described in Fig. 2A. *, p < 0.05. D, SW480 cells in a 24-well plate were transfected with control, ZIPK-1, or ZIPK-2 siRNA (20 pmol). Total RNA samples isolated from these cells were also subjected to RT-PCR analysis using cyclin D1, SURVIVIN, hZIPK, or G3PDH primers. These mRNA expression levels were also quantified by reverse transcription and quantitative real-time PCR analysis. Data represent the levels of these mRNA normalized to that of an actin internal control and are expressed relative to the value of control siRNA-treated samples. Results are representative of three independent experiments, and the error bars represent S.D. E, HeLa cells in a 24-well plate were transfected with control or ZIPK-1 siRNA (20 pmol) and then transfected with or without Myc-tagged mouse ZIPK (0.5 μg). Total RNA samples isolated from these cells were also subjected to RT-PCR analysis using cyclin D1, SURVIVIN, hZIPK, mZIPK, or G3PDH primers. These mRNA expression levels were also quantified by reverse transcription and quantitative real-time PCR analysis. Data represent the levels of these mRNA normalized to that of an actin internal control and are expressed relative to the value of control siRNA-treated samples. Shown is a representative experiment, which was repeated at least three times with similar results.

ZIPK interact physically with NLK in vivo. A, 293T cells (1 × 10⁷) were transfected with HA-tagged ZIPK (10 μg) and/or Myc-tagged NLK (10 μg). The cells were lysed 48 h later in lysis buffer, immunoprecipitated with anti-HA (IP), and blotted with anti-Myc or anti-HA antibody (IB). TCL (1%) was blotted with anti-Myc antibody. B, HeLa cells (3 × 10⁷) were lysed, immunoprecipitated with control or anti-ZIPK IgG, and blotted with anti-NLK antibody (upper panels). HeLa cells (3 × 10⁷) were also lysed, immunoprecipitated with control or anti-NLK IgG, and blotted with anti-ZIPK antibody (lower panels). TCL (1%) was blotted with anti-ZIPK or anti-NLK antibody. C, domain structure of ZIPK and its mutant fragments are shown schematically. D, 293T cells (1 × 10⁷ cells) were transfected with Myc-tagged ZIPK WT or its mutants (10 mg) together with or without FLAG-tagged NLK (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-Myc, and blotted with anti-FLAG or anti-Myc antibody. TCL (1%) was blotted with anti-FLAG antibody. E, domain structure of NLK and its mutant fragments are shown schematically. F, 293T cells (1 × 10⁷ cells) were transfected with FLAG-tagged NLK WT or its mutants (10 μg) together with or without Myc-tagged ZIPK (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-FLAG, and blotted with anti-Myc or anti-FLAG antibody. TCL (1%) was blotted with anti-Myc antibody. N, N-terminal domain; C, C-terminal domain.

ZIPK influences the interaction between NLK and TCF4. A, 293T cells (1 × 10⁷ cells) were transfected with FLAG-tagged TCF4 (10 μg) together with or without Myc-tagged ZIPK or FLAG-NLK (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-Myc or anti-TCF4 (IP), and blotted with anti-Myc or anti-FLAG antibody (IB). TCL (1%) was blotted with anti-FLAG antibody. HeLa cells (1 × 10⁷ cells) were transfected with FLAG-tagged TCF4 (10 μg) together with or without Myc-tagged ZIPK (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-Myc, and blotted with anti-FLAG or anti-Myc antibody. TCL (1%) was blotted with anti-FLAG antibody. B, 293T cells (1 × 10⁷ cells) were transfected with FLAG-tagged TCF4 together with or without Myc-tagged ZIPK FL, KD, or LZ (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-Myc, and blotted with anti-FLAG or anti-Myc antibody. TCL (1%) was blotted with anti-Myc antibody. C, 293T cells (1 × 10⁷ cells) were transfected with Myc-tagged NLK (10 μg) together with FLAG-tagged TCF4 and/or Myc-tagged ZIPK (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-FLAG, and blotted with anti-Myc or anti-FLAG antibody. TCL (1%) was blotted with anti-Myc antibody. D, 293T cells (1 × 10⁷ cells) were transfected with FLAG-tagged NLK (10 μg) together with Myc-tagged TCF4 (10 μg) and/or FLAG-tagged ZIPK WT or KA (10 μg). At 48 h after transfection, the cells were lysed, immunoprecipitated with anti-Myc, and blotted with anti-FLAG or anti-Myc antibody. TCL (1%) was blotted with anti-FLAG or anti-Myc antibody. E, 293T cells (1 × 10⁷ cells) were transfected with FLAG-tagged TCF4 (10 μg) with or without Myc-tagged NLK (10 μg) and/or Myc-tagged ZIPK (10 μg). The cell were lysed and immunoprecipitated with anti-FLAG antibody and assayed for in vitro kinase activity (top panel). The amounts of TCF4 proteins were shown to be similar by Western blotting with anti-FLAG antibody (second panel from top). TCL (1%) was also blotted with anti-FLAG or anti-Myc antibody (bottom three panels).

ZIPK knockdown influences cell growth in colon carcinoma cells. A, SW480 cells in a 6-well plate were transfected with control, ZIPK-1, or ZIPK-2 siRNA. At 24 h after transfection, siRNA-transfected cells were harvested and seeded into 96-well plates (5 × 10³/well). At the indicated time point, cell viability was determined by using Cell Counting Kit-8. Shown is a representative experiment, which was repeated at least three times with similar results. B and C, HCT116 (B) or Caco2 (C) cells in a 6-well plate were transfected with control, ZIPK-1, or ZIPK-2 siRNA. At 24 h after transfection, siRNA-transfected cells were harvested and seeded into 96-well plates (5 × 10³/well). An approximately 60% reduction of ZIPK mRNA in HCT116 cells and 50% reduction of ZIPK mRNA in Caco2 by ZIKP siRNAs was confirmed by quantitative real-time PCR analysis (data not shown). At the indicated time point, cell viability was determined by using Cell Counting Kit-8. Shown is a representative experiment, which was repeated at least three times with similar results.