FIGURE 5:

GM130 and Golgi microtubule nucleation are dispensable for neutrophil migration. (A) Western blot of two CRISPR/Cas9 GM130 knockout clones compared with the CRISPR/Cas9 luciferase control. (B) Representative immunofluorescence images showing GM130 depletion in two GM130 knockout clones compared with a wild-type control. Cells were stained for GM130 (green) and the cis-Golgi marker giantin (magenta). Scale bar: 5 µm. (C) 2D mean track speed and chemotactic index of GM130 knockout clones compared with luciferase control ± centrinone (data displayed as mean + SEM; N = 3 repeats, n = 9 devices, 3720–5911 cells). Cells were imaged in a 10-µg/ml fibronectin-coated microfluidic device migrating toward an fMLP gradient every 30 s for 45 min. Significance was determined by mixed-effects REML regression with a Satterthwaite degrees of freedom approximation. (D) Representative track plots of GM130 knockout clones or luciferase control ± centrinone. (E) Representative immunofluorescence images of GM130 KO or luciferase control. Cells were seeded on fibronectin-coated coverslips and stimulated with 100 nM fMLP for 5 min. Cells were stained for microtubules (green) and nuclei (blue). Scale bar: 10 µm. n.s. = not significant; *, p < 0.05; **, p < 0.01.