Figure 5. Aire is critical for centrosome number sustenance and the integrity of mitotic spindle poles.
(A) Representative immunofluorescence SIM images of γ-tubulin (TUBG) and NEDD1 in control (Aire+/+;R26CreERT+/+ tamoxifen-/+and AireFL/FL;R26CreERT+/+ tamoxifen-) and Aire-/- (AireFL/FL;R26CreERT+/+ tamoxifen +) mES cells during S/G2 and M phases of the cell cycle. Scale bar: 0.7 μm. (B) Quantification of centrosome parameters in control and Aire-/- ES cells (groups same as in panel (A)). Centrosome number in 30 S/G2 cells (top panel) from three biological replicates were counted for each group, mean ± sd is shown. Each point represents one mitotic cell. p-values were calculated with Mann–Whitney–Wilcoxon test. γ-tubulin(TUBG) ring diameter in S/G2 cells (middle panel). Each point represents one centrosome. Mean ± sd was presented. p-values were calculated with Mann–Whitney–Wilcoxon test. γ-tubulin(TUBG) foci diameter in M phase cells (bottom panel). 30 spindle poles from three biological replicates were quantified for each group. Each point represents one spindle pole. Mean ± sd is presented. p-values were calculated with Mann–Whitney–Wilcoxon test. (C) Representative immunofluorescence SIM images of γ-tubulin (TUBG) and NEDD1 in control (Dox-) and AIRE or AIRE ΔLESLL-overexpressing mES cells (Dox+) during S/G2 and M phases of the cell cycle. Scale bar: S/G2 Figure 0.6 μm; M images in top three panels 0.5 μm, and bottom panel 0.9 μm. (D) Quantification of centrosome parameters in AIRE or AIRE ΔLESLL-overexpressing mES cells (groups same as in panel (C)) Centrosome number in S/G2 cells (top panel). Each point represents one mitotic cell. Mean ± sd is presented. p-values were calculated with Mann–Whitney–Wilcoxon test. γ-tubulin (TUBG) ring diameter in S/G2 cells (middle panel). Each point represents one centrosome. Mean ± sd was presented. p-values were calculated with Mann–Whitney–Wilcoxon test. γ-tubulin(TUBG) diameter in M phase cells(bottom panel). Each point represents one spindle pole. Mean ± sd is presented. p-values were calculated with Mann–Whitney–Wilcoxon test. (E) Representative immunofluorescence SIM images of spindles and MOTCs (γ -tubulin and α-tubulin) in control (Dox-) and AIRE or AIRE ΔLESLL-overexpressing mES cells (Dox+) during M phase. Scale bar: 1.3 μm. Magnified view of MTOC in AIRE ΔLESLL (Dox-) (top scale bar: 0.6 μm) and AIREΔLESLL (Dox+) (bottom scale bar: 0.4 μm).
Figure 5—figure supplement 1. Aire is critical for centrosome number sustenance and the integrity of mitotic spindle poles.
Figure 5—figure supplement 2. AIREΔLESLL overexpression caused defects in the pericentiole matter (PCM) around centrioles.
