(A) Acentrosomal cells exhibits prolonged mitosis. Measurement of mitotic duration of wild type RPE1 and PLK4as cells dividing in the presence or absence of 3MBPP1 with live-cell imaging. With …
Wild type RPE1 and PLK4as cell line treated with 3MBPP1 for seven days stained with antibodies against centrin-2 and γ-tub to mark centrosomes. Scale bar, 5 μm.
Positions of sgRNA target site within the ORF of the p53 gene is depicted in the map. Descriptions of mutant indels are depicted below. Green colored nucleotides are insertions. sgRNA target site is …
(A) Schematic representation of the loss-of-function screen for components required for centrosome loss induced G1 arrest using the human genome-scale CRISPR knockout library (GeCKO). (B) Binary …
Positions of sgRNA target sites within the ORF of each gene are depicted on maps. Descriptions of mutant indels are depicted below. Red colored bases represent deleted nucleotides. Green colored …
BrdU incorporation assay for Figure 2H.
(A) Domain organization of 53BP1. BRCT (BRCA1 carboxy-terminal), UDR (ubiquitylation-dependent recruitment). p53 and USP28 interact with 53BP1 through the tandem BRCT domain. (B) 53BP1ΔBRCT mutant …
Western blot of PLK4as; 53BP1-/- cells mildly expressing (10 ng/ml doxycycline) wild type and mutant 53BP1.
BrdU incorporation assay for Figure 3B.
53BP1TESE delocalizes from the DNA damage sites marked by γ-H2AX. Mutant proteins of 53BP1 were mildly expressed under the tetracycline inducible promoter in stable, clonal, centrosomal PLK4as; …
(A) Organization of the conserved catalytic domains in USP28. UCH-1 (Cys box, amino acids 162–196) and UCH-2 (His box, amino acids 580–649). (B) Catalytic-inactive USP28CI cannot rescue the G1 …
Western blot of PLK4as; USP28-/- cells mildly expressing (10 ng/ml doxycycline) USP28WT and USP28CI. USP28 was probed with anti-HA antibody.
BrdU incorporation assay for Figure 4B.
(A) 53BP1 normally disassociates from the kinetochores during anaphase, but can do so prematurely in prometaphase upon mitotic stress/delay. Immunofluorescence images of centrosomal (left) or …
(A and B) Cells arrested or delayed in prometaphase due to Eg5 inhibitor treatment or acentrosomal cell division stained with the antibodies indicated. Bright BubR1 or Mad2 signals indicate SAC …
(A) Assay for 53BP1 mitotic kinetochore localization for Figure 5B. (B) SAC reactivation assay for Figure 5C. (C) BrdU incorporation assay for Figure 5F.
Candidate genes enriched in the eight independent screens for genes involved in centrosome loss-induced G1 arrest.
Enrichment compared to p53 | Gene symbol | Gene name or description | Number of sgRNAs enriched out of total 6 |
---|---|---|---|
1.0000 | TP53 | Tumor Protein P53 | 5/6 |
0.9386 | TP53BP1 | Tumor Protein P53 Binding Protein 1 | 4/6 |
0.9039 | TRIM37 | Tripartite Motif Containing 37 | 3/6 |
0.8895 | USP28 | Ubiquitin Specific Peptidase 28 | 4/6 |
0.8858 | P21 | Cyclin-Dependent Kinase Inhibitor 1A (P21, Cip1) | 6/6 |
0.7559 | CHD8 | Chromodomain Helicase DNA Binding Protein 8 | 4/6 |
0.6945 | FBXO42 | F-Box Protein 42 | 3/6 |
0.6846 | RNF20 | Ring Finger Protein 20, E3 Ubiquitin Protein Ligase | 3/6 |
0.6665 | PCDHGC5 | Protocadherin Gamma Subfamily C, 5 | 2/6 |
0.6604 | TSPY8 | Testis Specific Protein, Y-Linked 8 | 3/6 |
0.6395 | RWDD3 | RWD Domain Containing 3 | 2/6 |
0.6364 | UGT1A9 | UDP Glucuronosyltransferase 1 Family, Polypeptide A9 | 2/6 |
0.6285 | PDSS1 | Prenyl (Decaprenyl) Diphosphate Synthase, Subunit 1 | 2/6 |
0.6275 | BPIFB4 | BPI Fold Containing Family B, Member 4 | 2/6 |
0.6240 | PCDHAC2 | Protocadherin Alpha Subfamily C, 2 | 3/6 |
0.6218 | CSNK2A1 | Casein Kinase 2, Alpha 1 Polypeptide | 2/6 |
0.6201 | GAGE7 | G Antigen 7 | 3/6 |
0.6119 | WTAP | Wilms Tumor 1 Associated Protein | 2/6 |
0.6093 | PRDM11 | PR Domain Containing 11 | 2/6 |
0.6086 | OR56A1 | Olfactory Receptor, Family 56, Subfamily A, Member 1 | 2/6 |
0.6059 | TBCEL | Tubulin Folding Cofactor E-Like | 2/6 |
0.5997 | DNAJC9 | DnaJ (Hsp40) Homolog, Subfamily C, Member 9 | 2/6 |
0.5991 | NPIPL3 | Nuclear Pore Complex Interacting Protein Family, Member B3 | 3/6 |
0.5987 | SYDE1 | Synapse Defective 1, Rho GTPase, Homolog 1 | 2/6 |
0.5959 | TECPR2 | Tectonin Beta-Propeller Repeat Containing 2 | 2/6 |
0.5683 | STOML3 | Stomatin (EPB72)-Like 3 | 2/6 |
0.5507 | DVL3 | Dishevelled Segment Polarity Protein 3 | 2/6 |
Log2 scaling of sgRNAs enrichment normalized to the top scoring hit p53. The sgRNAs for genes highlighted in red were validated in this study. For a gene to be scored as a candidate, at least two of its six sgRNAs were repeatedly enriched in independent screens, with the HiSeq reads at least 3 times higher than the average of the baseline read.