Fig. 3

Fig. 3 VPS18 deficiency reduces CORVET and HOPS core complex subunit levels and leads to a disturbed vesicle homeostasis during neutrophil maturation.

A Schematic of CORVET and HOPS core (gray) and unique (green) subunits. B Expression levels of VPS11, VPS16 and VPS33A in indicated cell lines before (day 0) and after removal (day 2) of estrogen analyzed by mass spectrometry. C, E Expression levels of autophagy markers LC3B and p62 analyzed by mass spectrometry. D Representative Western blot of LC3B expression (left panel) and quantitative analysis (right panel) of LC3B-II expression in cell lysates from indicated cell lines during differentiation (day 0-4). Ratios of LC3B-II/GAPDH are presented as relative protein amount. n = 7. F Expression of p62 during differentiation (day 0-4) in indicated cell lines using flow cytometry. Relative p62 levels are shown (normalized to CTRL cells, 100%). n = 9. G Representative transmission electron microscopy images of CTRL, clone 1 and clone 2 dHoxb8 cells (left panel, scale bar, 2 µm). Squares, zoom-in areas shown in (a) (middle panel, scale bar, 500 nm) and (b) (right panel). Arrows, irregularly shaped vesicular structures. Arrowheads, autophagosomes. N, nucleus. H Quantification of horizontal (H) and perpendicular (P) size of vesicular structures and autophagosomes from (G). n = 1 independent experiment with n = 5 cells (CTRL & clone 1) and n = 3 cells (clone 2). I Expression levels of lysosomal marker LAMP1 analyzed by mass spectrometry. J Quantification of LysoTracker-stained lysosomes in indicated cell lines at day 4. n ≥ 13. Mean ± SD. **P < 0.01, ****P < 0.0001 compared to CTRL. ^####P < 0.0001 compared to Clone 1. One-way ANOVA, Tukey’s multiple comparisons test. B, C, E, I Mean ± SEM. n ≥ 2. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to CTRL. One-way ANOVA, Dunnett’s multiple comparison test. ns, not significantly different. D, F Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to CTRL. Two-way ANOVA, Tukey’s multiple comparisons test.