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. 2020 Dec 17;296:100182. doi: 10.1074/jbc.RA120.013908

Figure 6.

Figure 6

BODIPY–PUT is taken up via endocytosis.A, cells were treated with 5-μM BODIPY–PUT for 90 min at 37 °C with or without 30 min pretreatment of 1-mM BV or endocytosis inhibitors (100-μM Dynasore, 50-μM genistein, and 50-μM Pitstop-2). Uptake was measured in terms of the mean fluorescence intensity (MFI) up to 1 × 104 events (debris free) per condition on the flow cytometer (n = 3–4). B, cells were treated with BV or endocytosis inhibitors, while being starved for 30 min. Afterward, they were incubated at 4 °C for 15 min, treated with 50 μg/ml Alexa647-transferrin for 20 min, and incubated at 37 °C, 5% CO2 for 15 min. Uptake was measured in terms of the mean fluorescence intensity (MFI) up to 1 × 104 events (debris-free) per condition on the flow cytometer (n = 5). C and E, cells were treated with BODIPY–PUT for 90 min at 37 °C with or without 30 min pretreatment of 1-mM BV and incubated with primary antibodies for EEA1, RAB11, RAB7, and LAMP1 (1:200) followed by staining with Alexa Fluor 594 goat anti-rabbit antibody (1:1000) for 60 min and the nuclear stain DAPI (200 ng/ml) for 15 min (n=3–5). Confocal microscopy images (scale bar = 20 μm; boxed areas are enlarged in the inset with scale bar = 5 μm) of BODIPY–PUT in CHO-WT ± BV and CHO-MG (C), and BODIPY–PUT colocalization with endosomal markers in CHO-MG (E) are shown. D, BODIPY–PUT, represented in panel C, was quantified by measuring the mean fluorescence intensities (MFI) of BODIPY normalized to that of DAPI (CHO-WT + BODIPY–PUT = 29 images; CHO-WT BODIPY–PUT + BV = 39 images; CHO-MG BODIPY–PUT = 25 images). F, colocalization of BODIPY–PUT with endosomal markers, demonstrated in panel E, was analyzed in terms of Pearson’s coefficient of BODIPY–PUT with EEA1 (49 images), RAB11 (30 images), RAB7 (29 images), and LAMP1 (30 images). Data represent the mean ± SD, and individual data points (representing replicates) are overlaid on bar graph plots (∗∗p < 0.01, ∗∗∗∗p < 0.0001), generated with two-way ANOVA and Bonferroni post hoc corrections. A3-DN, overexpression of ATP13A3 catalytically dead mutant D498N; A3-WT, overexpression of WT ATP13A3; BODIPY, boron dipyrromethene; BV, benzyl viologen; EEA1, early endosomal antigen 1; LAMP1, lysosomal-associated membrane protein 1; PUT, putrescine; RAB7/11, ras-associated binding protein 7/11.