Funding for this research was provided by:
Grantová Agentura, Univerzita Karlova (GA22-06405S, GA22-06405S, GA22-06405S, GA22-06405S)
Grantová agentura. Masaryk University (MUNI/J/0004/2021, MUNI/J/0004/2021, MUNI/J/0004/2021, MUNI/J/0004/2021)
Masaryk University
Article History
Received: 23 March 2023
Accepted: 9 June 2023
First Online: 26 June 2023
Declarations
:
: The authors declare that they have no competing interests.
: Not applicable.
: Not applicable.
: For Fig. InternalRef removedd, sequences of 23 Prickle paralogs were collected in the Uniprot database: Q96MT3, Q7Z3G6, O43900, and Q2TBC4 for human PRICKLE1-4; A0A2I3TH70, A0A2I3RJX6, K7DBC5, and H2R2U9 for <i>Pan</i> Prickle1-4; Q3U5C7, Q80Y24, Q80VL3, and D3Z6Q6 for mouse Prickle1-4; Q90Z06, A0A8J0V3T3, A8WH69, and D3Z6Q6 for <i>Xenopus laevis</i> Prickle1-4; A0A6I8QKU1, F6U3C9, A0A803K7N2, and B0JZ01 for <i>Xenopus tropicalis</i> Prickle1-4; and Q6WGK5, Q7ZZC4, and F1Q568 for <i>Danio rerio</i> Prickle1-3. Multiple sequence alignments were performed using the ClustalW algorithm, implemented in the BioEdit editor 7.2 [CitationRef removed]. Prickle phylogenetic tree was generated from a set of 23 Prickle sequences above plus Q9NDQ9 for <i>Ciona intestinalis</i> Prickle, A1Z6W3 for <i>Drosophila melanogaster</i> Prickle, and A0A260ZPH8 for <i>Caenorhabditis elegans</i>. Specifically, the sequences were first aligned using ClustalW algorithm, implemented in BioEdit editor 7.2 [CitationRef removed]. The total sum of amino acids and gaps (i.e. regions of insertion/deletion) in the multiple sequence alignment was 1170, and has been visualized in JalView2 desktop version with standardized Clustal X Colour Scheme (Suppl. Figure 1). Next, the best fitting amino acid substitution model and evolution parameters of evolution were estimated using SMS: Smart Model Selection in PhyML [CitationRef removed], with Akaike information criterion (available online on ExternalRef removed). The JTT model [CitationRef removed] was estimated as the best fitting model, in variant JTT + G + I + F (parameter gamma = 1.032, proportion of invariable sites estimated 0.043, and number of substitution rate categories 4). Based on the multiple sequence alignment and JTT substitution model, the (unrooted) phylogenetic tree was calculated using PhyML3.0 [CitationRef removed]. The <i>Ciona</i>, <i>Caenorhabditis</i> and <i>Drosophila</i> Prickle sequences were used here as an outgroup for a tree rooting. Finally, the phylogenetic tree was visualized using PRESTO (a Phylogenetic tReE viSualisaTiOn; implemented in PhyML 3.0 [CitationRef removed]), and manually edited in the Affinity Designer software.In Fig. InternalRef removede, 1f and Suppl. Figure 2, aligned sequences together with their conservation score (see below) were manually graphically edited in the Affinity Designer software. Conservation is visualized on the alignment or a sequence group as a histogram giving the score for each column. Pairwise Sequence alignment and Conservancy score were calculated via Emboss needle tool (ExternalRef removed) and the Jalview2 desktop version, respectively.Figure InternalRef removedg was created using PondrFit tool available on ExternalRef removed, and manually edited in the Affinity Designer software.Figures InternalRef removeda, 2b, and InternalRef removed were created with BioRender.com, the online app used to create, edit, and collaborate on scientific diagrams, and illustrations. Data for Fig. InternalRef removedC were adopted from the gnomAD database v2.1.1, available on ExternalRef removed on February 10, 2023.Data for Fig. InternalRef removed, including Suppl. Figures 3, 4, 5, and 6 were prepared using GEPIA2 and cBioPortal databases, and manually edited in the Affinity Designer software.GEPIA2 database analysis: the datasets analysed for this study can be accessed through the following website: ExternalRef removed. TCGA and GTEx data were accessed on January 9, 2023. The gene expression profile and box plot function were used to compare cancer versus normal <i>PRICKLE1-4</i> tissue expression, by comparing cancer TCGA with normal TCGA and GTEx data. The expression data were log2 (TPM + 1) transformed, with the log2FC = 1 cut-off and q-value cut-off = 0.01. Statistical significance between cancer and normal samples was analysed by ANOVA. The impact of <i>PRICKLE1-4</i> expression on Overall survival of TCGA cancer patients was visualized with the help of the survival heat map, using the median for cut-off, with no p-value adjustment. Log-rank significance level was p = 0.05, estimated using the Mantel–Cox test. For overall survival (OS) representation, Kaplan–Meier curves were generated, and the median was selected as the threshold for separating the high- and low-expression groups in TCGA data. The hazard ratio (HR) was calculated based on the Cox PH model; a 95% Confidence Interval was added (dotted line). Graphs were manually edited in Affinity Designer.cBioPortal analysis: The datasets used for this study can be accessed through the following website: ExternalRef removed. TCGA data were used for further analysis, accessed on January 9, 2023, and analysed using software-derived tools. Pie charts representing derived mutational data were generated with the help of the GraphPad Prism 8.0 software. Graphs were manually edited in the Affinity Designer software.All figures in this research paper were designed with colourblind-friendly colours to ensure maximum accessibility.