Extended Data Fig. 7: EB1 crotonylation regulates astral microtubule stability in cells.

(a) EB1-WT-GFP or EB1-K66TAG-GFP and pCMV-CrKRS/ acKRS were co-transfected into HeLa cells depleted of endogenous EB1. CRISPR/Cas9-mediated endogenous EB1 knockout was achieved by addition of Doxycycline (Dox, 1 μg/mL) into CRISPR/Cas9-edited HeLa cells. Cells were fixed by precooling methanol and stained with anti-γ-tubulin antibody. Scale bar, 10 μm. (b) Endogenous EB1-depleted HeLa cells expressing EB1-WT-GFP, AcK66-EB1-GFP and CrK66-EB1-GFP with or without FLAG-EB1CH-TOG2, and the acylation level and FLAG-EB1CH-TOG2 expression level were analyzed by indicated antibodies. (c) HeLa cells expressing NDP52 siRNA were transfected with or without FLAG-EB1CH-TOG2. Cells were synchronized to metaphase with MG132 treatment, fixed in pre-warmed paraformaldehyde and stained with α-tubulin antibody. The boxed areas in the α-tubulin channel are magnified in the right panels to show details of the astral microtubule region. Scale bar, 10 μm. (d) HeLa cells were transfected with NDP52 siRNA for 48 h. The knockdown efficiency of NDP52 siRNA and FLAG-EB1CH-TOG2 expression level were assessed by Western blotting. (e-f) Statistical analysis of astral microtubule number (e) and intensity (f) in c. For astral microtubule number, only microtubule length above 2 μm was included in statistics, for astral microtubule intensity, the average intensity was achieved by total intensity dividing the astral microtubule area. Control siRNA, n = 20; NDP52 siRNA, n = 20; NDP52 siRNA + TOG2, n = 20. Data represent mean ± s.e.m. from three independent experiments. Ordinary one-way ANOVA followed by Tukey’s post hoc test was used to determine statistical significance. NS (not significant) indicates p > 0.05. Related to Fig. 4.

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