Abnormal Thyroid Hormone Metabolism in sbp2-deficient Zebrafish

Descriprion excel file named "Supplementary_IJMS_Special Issue.Final.v1.xlsx" This dataset supports the study investigating the role of sbp2 in zebrafish thyroid development and function. The study analyzes the effects of knockdown and loss-of-function mutation in the sbp2 gene during zebrafish thyroid development, and post developmental functions. In particular, since the role of sbp2 is the insertion of Selenocysteine in the active centre of selenoproteins, including deiodinases, we explore TH metabolism in our zebrafish models. This dataset provides all raw numerical data corresponding to the graphs presented in the main text figures and supplementary materials of this study. Each subfolder is named according to the corresponding figure, as follows: FIGURE 1H: WISH signals of pax2a and nkx2.4b of CTRL, sbp2KD and sbp2MUT at 1 day-post fertilization (dpf), quantified using Fiji/ImageJ by defining a region of interest (ROI) encompassing the specific expression domain. Signal intensity was assessed by measuring the integrated density, calculated as the product of ROI area and mean pixel intensity. Data are presented as Mean +/- SEM of at least 15 embryos derived from three different biological replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 1I: WISH signals of tg, slc5a5, trh and tshba of CTRL, sbp2KD and sbp2MUT at 2 dpf quantified using Fiji/ImageJ by defining a region of interest (ROI) encompassing the specific expression domain. Signal intensity was assessed by measuring the integrated density, calculated as the product of ROI area and mean pixel intensity. Data are presented as Mean +/- SEM of at least 15 embryos derived from three different biological replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2E: Normalized WISH area of tshba and tg of CTRL, sbp2KD and sbp2MUT at 5 dpf. WISH-positive signal areas (um2) were normalized to the corresponding head area of each larva to account for potential differences in head size among experimental groups. Normalized areas were calculated as the ratio of the WISH-positive signal area to the total head area (signal area/head area) and expressed as dimensionless values. Consistent criteria for ROI selection, head area delineation, and image analysis were applied across all experimental groups within each comparison. Data are presented as Mean +/- SEM of 15 larvae derived from three different biological replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2F: WISH signal intensity of tshba and tg of CTRL, sbp2KD and sbp2MUT at 5 dpf, assessed by measuring the integrated density, calculated as the product of ROI area and mean pixel intensity. Data are presented as Mean +/- SEM of at 15 larvae derived from three different biological replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2G: Number of T4-producing follicles of CTRL, sbp2KD and sbp2MUT at 5 dpf, analyzed by immunofluorescence of T4 followed of manual counting of positive cells. Data are presented as Mean +/- SEM of at 15 larvae derived from three different biological replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2H: Corrected integrated densitiy of T4-producing follicles of CTRL, sbp2KD and sbp2MUT at 5 dpf. Fluorescence intensity was quantified using corrected integrated density, calculated according to the following formula: Corrected integrated density = Integrated density of the ROI − (ROI area × Mean background fluorescence intensity) Consistent criteria for ROI definition and background selection were applied across all experimental groups. At least 15 larvae per experimental condition, collected across a minimum of three independent experiments, were analyzed.Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2I: Corrected integrated densitiy of T3 in the gastrointestinal region of CTRL, sbp2KD and sbp2MUT at 5 dpf, as described in Figure 2H. At least 15 larvae per experimental condition, collected across a minimum of three independent experiments, were analyzed.Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2J: Total T4 (TT4, ng/g) analyzed by ELISA kit. Total concentration was normalized on wet weigth (ww) and larval number. Data are presented as Mean +/- SEM of three independent replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2J: Total T3 (TT3, ng/g) analyzed by ELISA kit. Total concentration was normalized on wet weigth (ww) and larval number. Data are presented as Mean +/- SEM of three independent replicates. Stastistics was calculated using ordinary one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparisons test. FIGURE 2L: Total T4/T3 ratio of CTRL, sbp2KD and sbp2MUT at 5 dpf. FIGURE 3B: Total T3 (TT3, ng/g) concentration of CTRL, sbp2KD and sbp2MUT at 5 dpf, in basal condition (1.2%DMSO) or after exposure of 100 nM levothyroxine (LT4). Data are presented as Mean +/- SEM of three independent replicates. Stastistics was calculated using ordinary ordinary two-way ANOVA and Šídák's multiple-comparisons test as post hoc analyses. FIGURE 3C: RT-qPCR of dio2 expression in CTRL, sbp2KD and sbp2MUT at 5 dpf, in basal condition (1.2%DMSO) or after exposure of 200 nM db-cAMP. Analysis were performed using rpl8 as endogenous control and the ddCT method. Data are presented as Mean +/- SEM of three independent replicates. Stastistics was calculated using ordinary ordinary two-way ANOVA and Šídák's multiple-comparisons test as post hoc analyses. FIGURE 3D: Total T3 (TT3, ng/g) concentration of CTRL, sbp2KD and sbp2MUT at 5 dpf, in basal condition (1.2%DMSO) or after exposure of 200 nM db-cAMP. Data are presented as Mean +/- SEM of three independent replicates. Stastistics was calculated using ordinary two-way ANOVA and Šídák's multiple-comparisons test as post hoc analyses. FIGURE A1: RT-qPCR of foxe1 and hhex in CTRL and sbp2KD at 1 dpf or mct8, oatp1c1, dio2 and dio3b in CTRL and sbp2KD at 2 dpf. Experiments were performed usign ddCT method and rpl8 as housekeeping gene. Data are presented as Mean +/- SEM of three independent replicates, using pools of 30 embryos each. Statistics was calculated by Unpaired student t-test. FIGURE A2: RT-qPCR of tshba in CTRL, sbp2KD and sbp2MUT at 5 dpf exposed to increasing doses of LT4 (0, 25, 50 or 100 nM) or T3 (0, 5, 10, 20 nM). Data are presented as Mean +/- SEM of three independent replicates, using pools of 20 larvae each. Stastistics was calculated using ordinary two-way ANOVA and Šídák's multiple-comparisons test as post hoc analyses. Detailed statistical results, including mean differences, 95% confidence intervals, and adjusted p-values where applicable, are provided in the accompanying Statistical Analysis Data File.