Fig 2: Activation of β-catenin partially rescues zG morphology, but not function, in Wnt2b KO mice.a. Treatment protocol with 0.06% lithium chloride (LiCl) or water from birth to 6 weeks of age.b. Representative images and quantification from 6-week-old adrenals stained for DAB2 (gray, n=11 WT, n=7 KO, n=8 KO+LiCl), Gαq (magenta, n=10 WT, n=7 KO, n=8 KO+LiCl), β-catenin (β-cat, red, n=10 WT, n=6 KO, n=8 KO+LiCl) and CYP11B2 (green, n=6 WT, n=8 KO, n=6 KO+LiCl) mice. Positive cells were quantified and normalized to nuclei (DAPI, blue) in the cortex. Scale bars: 10μm. One-way ANOVA with Tukey’s post-test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Data are represented as mean ± SEM.c, d. Quantification of (c) aldosterone levels from (female, n=5 WT, n=6 KO, n=3 KO+LiCl; male, n=6 WT, n=4 KO, n=5 KO+LiCl) and (d) plasma renin (female, n=5 WT, n=6 KO, n=3 KO+LiCl; male, n=6 WT, n=4 KO, n=6 KO+LiCl) mice. Data are represented as mean ± SEM. One-way ANOVA with Tukey’s post-test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM.

Fig 3: RepID-CUL4A dissociates from the chromatin during megakaryocytic differentiation. A-C RepID WD40 domain is required for inhibition of DAB2 expression. Construction of RepID full-length (FL) and lacking WD40 fragments (ΔWD40) (A). Total protein was extracted from RepID-deficient K562 cells transfected with RepID fragments and K562 RepID WT cells as indicated, and immunoblot analysis was performed using DAB2 antibodies. Alpha-tubulin was used as a loading control (B). Quantification of DAB2 levels, normalized to its levels in RepID WT cells. All error bars represent the standard deviation from 3 independent experiments (*P-value < 0.05, ****P < 0.0001, n. s.; not significant) (C). D, E RepID WD40 domain is required for inhibition of megakaryocytic differentiation. Indicated cells were incubated with PMA (1 day) and polyploidy was measured by flow cytometry analysis (D). Percentages of polyploidy (> 4N) are indicated (dark blue) (E). F Stability of DAB2 protein is not altered regardless of RepID expression. K562 RepID WT or KO cells incubated with or without the protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132. Cell lysates were subjected to the immunoblot analysis and relative densitometry was indicated. G Differential changes of transcription level for selected CRL components (CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7, DDB1, RepID) in PMA-treated K562 cells (20 nM, 2 days) in published microarray dataset (GSE57734), or in published RNA-Sequencing database from MKs and normal HSCs in both species (human and mouse) (https://repository.uel.ac.uk/). H RepID and CUL4A dissociates from chromatin during megakaryocytic differentiation. K562 RepID WT or KO cells incubated with PMA were collected at the indicated times, and chromatin-bound fractions were extracted and immunoblot analysis was performed using anti-RepID, anti-CUL4A, anti-CUL4B and anti-DDB1 antibodies. Alpha-tubulin and histone H3 was used for loading as well as fractionation control. Quantification of RepID, CUL4A, CUL4B and DDB1 levels in whole cell lysates and chromatin-bound fractions after normalizing alpha-tubulin or histone H3, respectively. Quantification was presented under the blots

Fig 4: Transcript and protein levels of endocytic traffic proteins in cells cultured under static or OSS conditions. (A) RNA transcript expression (average raw reads) of the genes involved in endocytic traffic in cells cultured for 96 h under static (blue bars) or OSS (red line) conditions is plotted in descending order of expression. (B) Equivalent amounts of lysates from cells cultured for 96h under static (ST) or OSS conditions were blotted with antibodies against Dab2, Dynamin-2 (Dyn), Rab11a, and Caveolin1 (Cav1). The migration of molecular mass markers is shown on the left, and the arrowheads denote the predicted molecular mass of each protein. The asterisk denotes a band also observed in representative blots by the supplier that is presumed to represent a post-translational modification of Rab11a. (C) Band intensities in western blots were quantified and values for 96h OSS (black circles) were normalized to 96 h Static for each protein above (dashed line). Individual values and the mean ± SD of three independent experiments are plotted. *P = 0.029 vs. 96 h Static by one sample t-test.

Fig 5: RhoA-mediated myosin-II contractility suppresses Dab2 recruitment at integrin-beta3.(a,b) FRET-based biosensor reveals that the RhoA-GTP level on RGD-glass is higher than the level on RGD membranes. (c) Dab2-mCherry is found at integrin-β3-GFP clusters after treatment of ROCK inhibitor (Y27632, 10 μM), even when the cell adheres on fixed RGD-coated substrate. In the control cell, Dab2 do not localize at integrin-beta3. (d,e) Overexpression of RhoA-Q63L and MRLC T18D/S19D mutants suppresses Dab2 recruitment on RGD membranes. (f) Pearson's correlation analysis of Dab2 and RGD clusters. With overexpression of RhoA-Q63L and MRLC T18D/S19D, Dab2 recruitment to RGD clusters is suppressed. All data are the mean of three independent experiments (n>25 in each condition). P value by unpaired Kruskal–Wallis test is: ****P<0.0001. Error bars represent s.d., and all samples represent biological replicates. Detailed statistical test results are available in Supplementary Table 1. Scale bar, 10 μm.