Fig. 7: ZFYVE28 promoted the conversion of early endosomes to late endosomes.
a ZFYVE28 colocalized with early endosomes, whereas the colocalization disappeared with the FYVE domain deleted (ZFYVE28-ΔFYVE). Representative confocal images of four independent parallel experiments are shown, and the inset in the right panel shows a magnified view of the indicated area. Scale bar, 100 μm (left panel), 25 μm (right panel). b HepG2 cells overexpressing ZFYVE28 and ZFYVE28-ΔFYVE were fixed and stained at different time points after insulin stimulation at 10−6 M. ZFYVE28 promoted the formation of late endosomes (RAB7-labeled endosomes). Representative confocal images of four independent parallel experiments are shown. Scale bar, 50 μm. c, d After 90 min of insulin stimulation at 10−6 M, HepG2 cells were subjected to multiplex immunofluorescence staining using Opal™ 7 color kits (Akoya Biosciences). A large field of view was selected for confocal imaging. Representative confocal images of cells (c) and the quantification results of relative fluorescence density (d) of six independent parallel experiments are shown. Scale bar, 100 μm. e Representative western blot results of HepG2 cells overexpressing ZFYVE28 and ZFYVE28-ΔFYVE at different time points after insulin stimulation at 10−6 M. Independent experiments were repeated three times in parallel. f Representative western blot results of hepatic proteins in flox/flox mice and LKO mice after insulin injection (2 U/kg body weight) for 30, 60, 90, and 120 min. Independent experiments were repeated three times in parallel. g, i, j Representative results of immunohistochemical staining of liver sections from mice injected with AAV9-pTBG-GFP and AAV9-pTBG-Zfyve28 (g); independent experiments were repeated five times in parallel. The quantification results (i, j) are shown; n = 5 biologically independent samples per group. Scale bar, 100 μm. h, k, l Representative results of immunohistochemical staining of liver sections from flox/flox mice and LKO mice (h); independent experiments were repeated five times in parallel. The quantification results (k, l) are shown; n = 5 biologically independent samples per group. Scale bar, 100 μm. Data are shown as means ± SD, and P values are determined by unpaired two-tailed Student’s t-test (d, i–l). The exact P values are shown in the figure. Source data are provided as a Source Data file.
