Centrosome defects cause microcephaly by activating the 53BP1‐USP28‐TP53 mitotic surveillance pathway

A, B

Quantification of the number of (A) centrin foci and (B) γ‐tubulin foci in mitotic cells in the VZ of E14.5 cortices. WT n = 175 cells, N = 4 embryos; Usp28 ^−/− n = 137 cells, N = 3 embryos; Cep63^T/T;Usp28 ^−/− n = 150 cells, N = 3 embryos; Sas4^cKO;Usp28^cKO n = 150 cells, N = 3 embryos. #, chi‐square test, with * post hoc analysis, comparisons are made to WT. WT data are from Fig EV1F and G and shown alongside for comparison.

C

Ratio of mitotic (PH3⁺) to cycling (Ki67⁺) cells in E14.5 cortices. WT N = 5, Trp53bp1 ^−/− N = 3, Usp28 ^−/− N = 3, Trp53 ^−/− N = 3; one‐way ANOVA with post hoc analysis. WT data are from Fig 1H and shown alongside for comparison.

D

Percentage of proliferating, arrested, and apoptotic progeny in Sas4^cKO;Usp28^cKO dissociated NPCs calculated from data shown in Fig 5F. #, chi‐square test with *, post hoc analysis, comparisons are made to WT. WT and Sas4^cKO data are from Fig EV2A and shown alongside for comparison.

E

Genome‐wide copy number plots of single cells sequenced from NPCs dissociated from Sas4^cKO;Usp28^cKO E14.5 brains. Individual cells are represented in rows with copy number states indicated by colors; arrow on the zoomed in view (bottom) indicates an aneuploid cell. Sas4^cKO;Usp28^cKO n = 41 cells, N = 2 embryos.

F

Percentage of aneuploid cells from the single‐cell sequencing data shown in E.

G

Schematic of the prolonged mitosis assay.

H

Percentage of proliferating, arrested and apoptotic progeny in WT and Usp28^cKO dissociated NPCs with delayed mitosis (nocodazole delayed; WT n = 45 cells, N = 3 embryos; Usp28^cKO n = 56 cells, N = 3 embryos) and normal mitosis (After Washout; WT n = 90 cells, N = 3 embryos; Usp28^cKO n = 100 cells, N = 3 embryos) calculated from data shown in Fig EV5I–L; #, chi‐square test with *, post hoc analysis, comparisons are made to WT.

I, J

Graph showing the mitotic duration and fate of (I) WT and (J) Usp28^cKO E14.5 NPCs that were delayed in mitosis using nocodazole (0.08 μM). Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. WT n = 45 cells, N = 3 embryos; Usp28^cKO n = 56 cells, N = 3 embryos.

K, L

Graph showing the mitotic duration and fate of (K) WT and (L) Usp28^cKO NPCs from the same experiment in I and J that entered mitosis after nocodazole washout. WT n = 90 cells, N = 3 embryos; Usp28^cKO n = 100 cells, N = 3 embryos.

Figure EV5. Prolonging mitosis results in USP28‐dependent cell death in the progeny of NPC divisions. Related to Fig 5 .