Fig 1: Clks localize to the midbody in a mutually dependent manner.(a) Cells expressing Clk1:GFP, Clk2:GFP or Clk4:GFP proteins were analysed in cytokinesis with chromatin bridges. Green, GFP; red, CENP-A; blue, DNA. A minimum of 22 cells from three independent experiments were examined per treatment. (b,c) Localization of Clk2. Cells were transfected with negative siRNA (control), Clk1 siRNA (siClk1), Clk4 siRNA (siClk4) or treated with TG003 for 5 h. Green, Clk2; red, CENP-A; blue, DNA. (d) Mean midbody intensity of Clk2 in cells treated as in b,c. (e) Localization of phosphorylated Aurora B–S331 (pS331) in cells treated as in b. Green, pS331; red, CENP-A; blue, DNA. Insets show magnified midbodies. Scale bars, 5 µm. (f) Mean midbody intensity of pS331 in cells transfected with Clk2 siRNA (siClk2) or treated as in b,c. Data are from n cells from three independent experiments. Error bars show s.d. Values in control were set to 1. ***P<0.001 compared with control. The Mann–Whitney U-test was used.
Fig 2: Histological characterization of Clk4-cKO hearts with NEXN phosphorylation supplementation.a H&E-stained heart sections from AAV-WT, AAV-S437E, and AAV-S437A hearts. Scale bar: 1 mm. b, c Summary data for LV internal diameter (LVID) and interventricular septum (IVS) thickness. d, e WGA staining, and quantification of the cardiomyocyte cross-sectional area. Scale bar: 50 µm. f, g WGA staining, and quantification of the cardiomyocyte length. Scale bar: 25 µm. h, i Masson’s trichrome staining of heart sections and quantification of myocardial fibrosis. Scale bar: 50 µm. For all panels, n = 5 animal per group. All statistical analyses were performed using one-way ANOVA and Dunnett multiple comparisons test. Data are presented as the means ± S.E.M.; P values are shown in each graph. Source data are provided as a Source Data file.
Fig 3: Cardiac-specific Clk4 knockout (Clk4-cKO) leads to cardiac hypertrophy and dysfunction.a Schematic diagram of the gene targeting strategy. Clk4 exons 3–10 are flanked by two loxP sites. Clk4fl/fl mice were crossed with cardiomyocyte-restricted tamoxifen-inducible transgenic mice (αMHC-MerCreMer, MCM) to generate Clk4fl/fl × MCM mice. Then, tamoxifen was injected intraperitoneally to induce cardiomyocyte-specific deletion in the adult heart (Clk4-cKO). b qPCR detection of the expression of Clk4 mRNA in Clk4-cKO and MCM littermate control hearts. Normalized to Gapdh expression. n = 3 animals. c Western blot analysis of CLK4 expression in Clk4-cKO hearts. d Representative short-axis M-mode images of MCM control and Clk4-cKO left ventricles 3 weeks post tamoxifen initiation. e, f Summary data for the LV internal diameter, diastole (LVID, d) and LV ejection fraction (EF). n = 7 animals for MCM and n = 8 animals for Clk4-cKO. g Gross heart images of Clk4-cKO and MCM control mice. Scale bar: 2.5 mm. h Heart weight-to-body weight ratios (HW/BW). n = 5 animals per group. i Heart weight-to-tibial length ratios (HW/TL). n = 5 animals per group. j Lung weight-to-tibial length ratio (LW/TL). n = 5 animals per group. For b, statistical analysis was performed using one-way ANOVA and Dunnett multiple comparisons test; for e, f, h, i, and j, statistical analyses were performed using unpaired, two-tailed Student’s t test. Data are presented as the means ± S.E.M.; P values are shown in each graph. Source data are provided as a Source Data file.
Fig 4: CLK4 regulates cardiomyocyte hypertrophy, and its expression decreases with disease.a qPCR detection of the expression of Nppa and Nppb in NRVMs transfected with control or Clk1-4 siRNAs. b, c Representative image of NRVMs treated with control or Clk4 siRNA for 48 h with cell area quantification. Scale bar: 50 µm. d Western blot analysis of CLK4 protein expression in failed mouse hearts subjected to either transverse aortic constriction (upper) or ISO infusion (lower) and in corresponding control mouse hearts. GAPDH served as loading control. e Quantification of the western blots in d. Ctrl control, NRVMs neonatal rat ventricular myocytes, HF heart failure, TAC transverse aortic constriction, ISO isoproterenol. a n = 3 biologically independent samples per group; c, n = 4 biologically independent samples per group; e, n = 3 animals per group. a statistical analysis was performed using one-way ANOVA and Dunnett multiple comparisons test; for c and e, statistical analyses were performed using upaired, two-tailed Student’s t test. Data are presented as the means ± S.E.M.; P values are shown in each graph. Source data are provided as a Source Data file.
Fig 5: Phosphoproteomic screening of Clk4-cKO hearts identifies NEXN as a potential target.a Volcano plots showing changes in phosphopeptides in Clk4-cKO mice compared with MCM mice, as identified using mass spectrometry (MS). The red dots are peptides with fold-changes (Clk4-cKO/MCM ratios) > 1.2 (P < 0.05, t test), and the blue dots are those with fold-changes < 1/1.2 (P < 0.05, t test). In, increase; De, decrease. b Hierarchical clustering analysis of the specified phosphoproteome. A total of 366 phosphoforms met the aforementioned two criteria. c A table showing the top downregulated phosphopeptides and corresponding phosphoproteins obtained from differential gene expression analysis in Clk4-cKO hearts. d qPCR analysis of cardiac hypertrophic-related genes, including Nppa and Nppb, in NRVMs transfected with siRNAs targeting Pdha1, Snw1,Dusp27, Zc3hc1, Ndrg1, and Nexn; here, Clk4 siRNA was used as a positive control. e–g Coimmunoprecipitation of CLK4 with NEXN in NRVMs transfected with CLK4-flag and NEXN-myc (e, f), and in vivo myocardium infected with AAV NEXN-WT (g). The input represents 6% of the whole-cell lysate used for each immunoprecipitation. IgG was used as a negative control. h Representative western blots showing that CLK4 increased the serine phosphorylation level of NEXN in NRVMs. Samples incubated with calf intestinal phosphatase (CIP) served as a negative control. i Typical western blots illustrating that CLK4 phosphorylated the wild-type NEXN fragment (amino acids 295–671) but not the mutant type (S437A) in a cell-free system. j Western blot detection of the phosphorylated NEXN in Clk4-cKO mouse myocardia using a Phos-tagged acrylamide gel. For a, b, n = 3 animals per group; for d, n = 3 biologically independent samples per group. For a, statistical analysis was performed using unpaired, two-tailed Student’s t test; for d, statistical analysis was performed using one-way ANOVA and Dunnett multiple comparisons test. Data are presented as the means ± S.E.M.; P values are shown in the graph. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-CLK4 antibody