IMAGE

Fig. 3

ID
ZDB-IMAGE-180803-14
Genes
Antibodies
Source
Figures for Takayama et al., 2018
Image
Figure Caption

Fig. 3

Requirement of lysosomal acidification in the S6K activation. (A) Experimental scheme. NH4Cl or chloroquine (CQ) was treated from −12 to 3 or 24 hpa (B–G). LysoTracker solution was injected into the amputated fins 15 min before fixation (H–K). (B–E) Longitudinal ray sections and quantification of p-S6K fluorescence intensities per area that consist of the whole regenerates and 500 μm below the amputation plane in control, NH4Cl-, or CQ-treated fin stumps at 3 hpa; p-S6K and nuclei were visualized by immunohistochemical staining and DAPI staining, respectively (n = 5). Representative images (BD’) used for quantification are shown in (E). White dashed lines indicate the amputation planes. Scale bars: 50 μm (B–D) and 25 μm (B’D’). ***p < 0.001 by Student’s t test. Error bars represent the standard error. (F) Western blotting analysis of γTubulin, S6K, and p-S6K in the DMSO-, NH4Cl-, or CQ-treated fin stumps at 3 hpa. γTubulin serves as a loading control. Numbers below each lane show the level of p-S6K in NH4Cl-, or CQ-treated fin stumps relative to that in DMSO-treated fin stumps at 3 hpa normalized to loading control. (G) The relative expression of growth factor-related genes in NH4Cl- or CQ-treated fin stumps by qPCR at 24 hpa. ***p < 0.001, **p < 0.05 by Student’s t test. Error bars represent the standard error. (HK) Longitudinal ray sections and quantification of p-S6K+ and LysoTracker fluorescence+ cells in area that consist of the whole regenerates and 500 μm below the amputation plane at 3 hpa; p-S6K (green), nuclei (grayscale), and lysosomal acidification (red) were visualized by immunohistochemical staining, DAPI, and LysoTracker (Lyso.), respectively (n = 8). White boxed areas in (H) are enlarged in (IJ’), respectively. Representative images (HJ’) used for quantification are shown in (K). Arrowheads in (I’ and J’) indicate LysoTracker fluorescence and p-S6K double positive cells. Scale bars: 50 μm (H) and 25 μm (IJ’). A pie chart shows that 55.4% of p-S6K+ cells are the LysoTracker fluorescence positive cells (K).

Figure Data
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Sci. Rep.