v-ATPase activity is required for the S6K activation. (A) Experimental scheme. DMSO, ConcA, or BafA1 solution was injected into the amputated fins at 1 hpa. The red two-headed-arrow indicates LysoTracker treatment, which was applied 15 min before observation. (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 DMSO-, ConcA-, or BafA1-treated fin stumps at 3 hpa; p-S6K and nuclei were visualized by immunohistochemical staining and DAPI staining, respectively (n = 5). Representative images (B–D’) 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-, ConcA-, or BafA1-treated fin stumps at 3 hpa (n = 6). γTubulin serves as a loading control. Numbers below each lane show the level of p-S6K in ConcA-, or BafA1-treated fin stumps relative to that in DMSO-treated fin stumps at 3 hpa normalized to loading control. (G–I’) Images of bright-field and fluorescence (red) microscopy, and quantification of the LysoTracker fluorescence intensity (see Materials and Methods) of DMSO-, ConcA-, or BafA1-treated fin stumps (n = 8). Black boxed areas in (G–I) are enlarged in (G’–I’), respectively. The LysoTracker fluorescence intensities in red boxed areas were measured (G–I’). Representative images (G’–I’) used for quantification are shown in (J). White dashed lines indicate the amputation planes. Scale bars: 1 mm (G–H) and 500 μm (G’–H’). ***p < 0.001 by Student’s t test. Error bars represent the standard error.
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