Figure 5 | Scientific Reports

Figure 5

From: Akap350 Recruits Eb1 to The Spindle Poles, Ensuring Proper Spindle Orientation and Lumen Formation in 3d Epithelial Cell Cultures

Figure 5

EB1 overexpression rescues AKAP350KD induced defective spindle orientation. MDCK cells with stable expression of EB1 fused to RFP (EB1OE) were generated and EB1OE with normal or decreased levels of AKAP350 (AKAP350KD-EB1OE) cells were prepared as described in Materials and Methods. (a) Merged images show the visualization of EB1 (green) and γ-tubulin (red) staining in metaphase control, AKAP350KD and AKAP350KD-EB1OE cells. Bars represent the percentage of EB1 fluorescence present at the centrosomes, defined at the γ-tubulin channel, and at astral microtubules in mitotic cells. Results are representative of at least 10 cells for each group analyzed in 3 independent experiments. Scale bar, 5 μm. (b) Cells were grown in Matrigel for 72 h and stained as indicated. Images were obtained by confocal microscopy, and spindle orientation in mitotic cells analyzed as described in Materials and Methods. Images show the projection of the central sections for γ-tubulin and DAPI staining corresponding to control, AKAP350KD cysts and AKAP350KD-EB1OE cysts, where the spindle angle of anaphase cells has been delineated, as explained for Fig. 2. Scale bar, 10 μm. The circular section histogram graphs show the population distribution of mitotic spindle angles in control, AKAP350KD and AKAP350KD-EB1OE cysts indicated in red. Results are representative of three independent experiments. *p < 0.05.

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