Zito F , Bonaventura R , Costa C , Russo R .

	Figure 1. . Inhibition of CAN activity by AZ treatment on sea urchin embryo development. (a) Schematic drawing depicting the experimental procedure. Blue line, culture in the presence of AZ; red arrow, AZ addition; sampling, sampling times after AZ addition. AZ has been added to blastula embryos (16 h post-fertilization, hpf). Bl, blastula; emBl, early mesenchyme blastula; mBl, mesenchyme blastula; MG, middle gastrula; LG, late gastrula; ePl, early pluteus. (b) WB analysis of total proteins from control (Ctrl) and 24 h AZ-treated embryos, reacted with polyclonal antibodies against the recombinant Pl-CAN fusion protein. Molecular markers are indicated on the left. (c–e), (i–k) Control embryos cultured in the presence of DMSO for 24 h. (f–h), (l–n) Embryos cultured in the presence of 8 mM AZ for 24 h. (d–e), (g–h), (i–n) Indirect IF of embryos labelled with 1D5 (d,g), Endo1 (e,h), 295 (i,l), anti-acTub (j,k,m,n) antibodies. (j,k) and (m,n) are focal planes of the same control and AZ embryo, respectively. White arrow, pseudopodial cables formed by the PMCs. Scale bar, 20 µm.
	Figure 2. . Inhibition of CAN activity does not affect spatial expression of msp130 and p19 genes. (a) Schematic drawing depicting the PMCs distribution in late gastrula embryo. WMISH of control (Ctrl; (b–e, j–m)) and AZ-treated (AZ; (f–i, n–q)) embryos at 1 h, 3 h, 6 h and 9 h with msp130 (b–i) and p19 (j–q) RNA probes. Scale bar, 20 µm.
	Figure 3. . Inhibition of CAN activity affects the spatial expression of biomineralization-related genes. (a) Schematic drawing depicting the PMCs distribution and corresponding skeleton elements in Prism, early pluteus and pluteus (ventral and lateral views) embryos. WMISH of control (Ctrl; (b–g)) and AZ-treated (h–m) embryos at 24 h with msp130 (b,h), sm50 (c,i), sm30 (d,j), p19 (e,k), p16 (f,l), can (g,m) RNA probes. Scale bar, 20 µm.
	Figure 4. . Spatial and temporal expression of biomineralization- and pigment-related genes in AZ-treated embryos. (a) Comparative qPCR analyses of mRNA levels in embryos collected at 3 h (blue) and 24 h (green) after AZ addition (+AZ). The stages of the related controls (Ctrl) were mesenchyme blastula (mBl) and early pluteus (ePl), respectively. The morphologies of AZ-treated embryos are also indicated, i.e. mBl and prism-like (Pr-like). Relative mRNA levels are expressed in arbitrary units as mRNA fold change compared to Ctrl samples assumed as one in the histograms, using the endogenous gene Pl-Z12-1 for normalization. Each bar represents the mean of at least three independent qPCR analyses ± s.d., using cDNAs obtained by three independent AZ-treatment experiments. Mean values were significantly different according to the Student's t-test. The asterisks indicate statistically significant variations to the relative control, * (p < 0.05), ** (p < 0.01, *** (p < 0.001). (b–e) WMISH of control (Ctrl; (b,c)) and AZ-treated (d,e) embryos at 24 h with tbr (b,d) and jun (c,e) RNA probes. Scale bar, 20 µm.
	Figure 5. . Effects of CAN inhibition on gene expression evaluated by NanoString analysis. (a) Graphical representation of genes showing biologically significant fold changes in their expression levels in response to inhibition of CAN activity. Data are presented as the percentage of genes showing significant changes in expression in 3 h and 24 h AZ-treated embryos, compared to control embryos, grouped according to their function (i.e. transcriptional regulation, signalling, development, biomineralization and defence response). (b) Scatter graph showing of changes in gene expression levels of 127 genes grouped according to their function (i.e. biomineralization, transcriptional regulation, signalling, development, immune response and defence response). The grey box indicates the threshold for insignificant changes following AZ treatment, i.e. fold values between −2 and +2 at 3 h (blue triangle) and 24 h (red dot).
	Figure 6. . WB analysis of total proteins from control (C) and AZ-treated embryos (A), collected at 1, 3, 6 and 24 h after AZ addition, and probed with anti-p-p38, anti-p-ERK, anti-c-jun and anti-tubulin (tub) antibodies. Molecular weight markers are indicated on the left (kDa). Protein levels represented in the histogram below were calculated as fold increase/decrease of AZ samples compared to controls, normalized using tubulin (tub) detected on the same membranes. Each bar represents the mean value (±s.d.) of at least two independent experiments. Values included within the −2/+2 range (light grey) were not considered as significantly changed.
	Figure 7. . Phylogenetic analysis of sea urchin and human CAN proteins. The phylogenetics was conducted in MEGA X, using the neighbour-joining method. The percentages of replicate trees in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
	Figure 8. . Bioinformatics characterization of the CAN isoforms and DEGs interactome. Human orthologous of the P. lividus DEGs were identified and subjected to STRING analysis to reveal interactions among them. The graphic is displayed as nodes, representing proteins and lines, representing the predicted biological relationships between nodes. All the proteins were grouped as described in the text, i.e. biomineralization, transcriptional regulation, signalling (including growth factor and wnt signalling), development (including axes patterning, neural differentiation and adhesion) and defence response. Human CAs are listed close to each group of DEGs they interact with, according to the colour code shown in the legend.
	Figure 9. . Summary of the genes/proteins expression and localization in 3 h and 24 h AZ-embryos.
	Figure 10. . Interactomic model of CAN isoforms in P. lividus embryo. The model simulated connections of CANs to different proteins and to physiological processes/activities occurring from the blastula stage onwards, including both putative (dashed lines/arrows) and validated (solid lines/arrows) data from literature (blue), results from AZ-treatment (red, this study) and results from PI3K-treatment (green) [22]. The model does not contain information on the possible subcellular localization of the potential P. lividus CAN isoforms.

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