Figure 9.
SNX10 regulates mitochondrial homeostasis and cell death in vivo. (A) Schematic diagram of human SNX10, zebrafish Snx10a, and Snx10b proteins. The percentage identity of the orthologues amongst each other and to the human counterpart is indicated. Also, the percentage identity of the zebrafish PX domains in comparison with the PX domain of human SNX10 is shown. (B) Temporal expression pattern of snx10a and snx10b. The graph shows the fold change in transcript levels relative to β-actin in whole zebrafish embryos from 1 to 5 dpf. Error bars indicate mean ± SEM. Data are collected from three individual experiments using 30 larvae for each experiment. (C) Dorsal and lateral view of the spatial expression pattern of snx10a and snx10b at 3 dpf as demonstrated by WM-ISH using an internal probe. Scale bars: 200 μm. Images are representative from three experiments. (D) Representative immunoblots of Snx10 and β-tubulin on whole embryo lysates from control (scrambled guide), single snx10a KO, and snx10ab DKO animals. β-tubulin served as a loading control. (E) Representative immunoblots of Snx10, Samm50, Cox-IV, and actin on whole embryo lysates from control (scrambled guide) and snx10ab DKO treated with 100 µM DMOG or DMSO control for 24 h at 2 dpf. (F) Quantification of the Cox-IV signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. (G) Quantification of the Samm50 signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. Data distribution was assumed to be normal but was not formally tested. (H) Representative images of TUNEL assay on control (scrambled sgRNA) and snx10ab DKO larvae treated with 100 µM DMOG or DMSO control for 24 h at 3 dpf. Orientation lateral. Scale bar: 500 μm. (I) Quantification of the mean fluorescent intensity from demarcated brain regions of images in H. A total of 45 control larvae (scrambled sgRNA) and 41 snx10ab DKO larvae were used for quantification, respectively. Values were normalized to control DMSO values. Control larvae were treated with DMOG as a comparison to snx10ab DKO larvae. n = 2 independent experiments. Plots demonstrate data distribution and median value (red line). Significance was determined by two-way ANOVA followed by Tukey’s post hoc test to compare all groups. Data distribution was assumed to be normal but was not formally tested. (J) Quantification of ROS levels obtained via FACS analysis of control (scrambled sgRNA), snx10ab DKO, and positive control larvae at 3 dpf incubated with MitoSOX. The values were presented as relative values after normalizing to control. Error bars indicate mean ± SEM. Quantification was from at least two independent experiments. Data distribution was assumed to be normal but was not formally tested. (K) Representative whole mount images shown as maximum intensity projection from z-stack of TUNEL assay performed on control (scrambled sgRNA) and snx10ab DKO larvae treated with or without 100 µM NAC at 3 dpf. Orientation lateral. Scale bar: 500 µm. (L) Quantification of the number of white puncta (dots) from the demarcated whole brain region shown in K. A total of >20 control larvae (scrambled gRNA) and >20 snx10ab DKO larvae treated or not with 100 µM NAC were used for quantification. Values were normalized to control values. Data are collected from three individual experiments. Plots show data distribution and median value (red line). Significance was determined by one-way Brown–Forsythe and Welch’s ANOVA tests to compare all groups. Data distribution was assumed to be normal but was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. NAC; N-acetyl cysteine. Source data are available for this figure: SourceData F9.

SNX10 regulates mitochondrial homeostasis and cell death in vivo. (A) Schematic diagram of human SNX10, zebrafish Snx10a, and Snx10b proteins. The percentage identity of the orthologues amongst each other and to the human counterpart is indicated. Also, the percentage identity of the zebrafish PX domains in comparison with the PX domain of human SNX10 is shown. (B) Temporal expression pattern of snx10a and snx10b. The graph shows the fold change in transcript levels relative to β-actin in whole zebrafish embryos from 1 to 5 dpf. Error bars indicate mean ± SEM. Data are collected from three individual experiments using 30 larvae for each experiment. (C) Dorsal and lateral view of the spatial expression pattern of snx10a and snx10b at 3 dpf as demonstrated by WM-ISH using an internal probe. Scale bars: 200 μm. Images are representative from three experiments. (D) Representative immunoblots of Snx10 and β-tubulin on whole embryo lysates from control (scrambled guide), single snx10a KO, and snx10ab DKO animals. β-tubulin served as a loading control. (E) Representative immunoblots of Snx10, Samm50, Cox-IV, and actin on whole embryo lysates from control (scrambled guide) and snx10ab DKO treated with 100 µM DMOG or DMSO control for 24 h at 2 dpf. (F) Quantification of the Cox-IV signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. (G) Quantification of the Samm50 signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. Data distribution was assumed to be normal but was not formally tested. (H) Representative images of TUNEL assay on control (scrambled sgRNA) and snx10ab DKO larvae treated with 100 µM DMOG or DMSO control for 24 h at 3 dpf. Orientation lateral. Scale bar: 500 μm. (I) Quantification of the mean fluorescent intensity from demarcated brain regions of images in H. A total of 45 control larvae (scrambled sgRNA) and 41 snx10ab DKO larvae were used for quantification, respectively. Values were normalized to control DMSO values. Control larvae were treated with DMOG as a comparison to snx10ab DKO larvae. n = 2 independent experiments. Plots demonstrate data distribution and median value (red line). Significance was determined by two-way ANOVA followed by Tukey’s post hoc test to compare all groups. Data distribution was assumed to be normal but was not formally tested. (J) Quantification of ROS levels obtained via FACS analysis of control (scrambled sgRNA), snx10ab DKO, and positive control larvae at 3 dpf incubated with MitoSOX. The values were presented as relative values after normalizing to control. Error bars indicate mean ± SEM. Quantification was from at least two independent experiments. Data distribution was assumed to be normal but was not formally tested. (K) Representative whole mount images shown as maximum intensity projection from z-stack of TUNEL assay performed on control (scrambled sgRNA) and snx10ab DKO larvae treated with or without 100 µM NAC at 3 dpf. Orientation lateral. Scale bar: 500 µm. (L) Quantification of the number of white puncta (dots) from the demarcated whole brain region shown in K. A total of >20 control larvae (scrambled gRNA) and >20 snx10ab DKO larvae treated or not with 100 µM NAC were used for quantification. Values were normalized to control values. Data are collected from three individual experiments. Plots show data distribution and median value (red line). Significance was determined by one-way Brown–Forsythe and Welch’s ANOVA tests to compare all groups. Data distribution was assumed to be normal but was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. NAC; N-acetyl cysteine. Source data are available for this figure: SourceData F9.

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