EMBO/soda-nel huggingface dataset:
CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer (2018)
Figure 4

Fig 4: Overexpression of CEP55 rescue cell death during perturbed mitosis.

(A) Box and whiskers plot showing average time spent in mitosis and (B) mitotic outcomes in control and CEP55 knockdown MDA-MB-231 cells following treatment with the BI2536 (5 nM). Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis, characterized by membrane blebbing. Graph represents the mean±SEM of two independent experiments. For each experiments n=50 mitotic cells were counted per condition using Olympus Xcellence IX81 time-lapse microscopy.

(C, D) Cells were pre-treated for two hours with 50 µM of the pan Caspase inhibitor Z-VAD-FMK, followed by treatment with B12536 (5 nM), before assessment of cell fate (C) and an average time spent in mitosis (D) as described in panel A and B. Graph represents the mean±SEM of two independent experiments.

(E) Cells were synchronized using double thymidine then released into nocodazole (0.5 µM), and protein lysates were collected at the indicated time points. Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of Cdk1 activation/mitotic entry. COX-IV served as a loading control.

(F) Schematic representation of the mCerulean-CDK1-FRET biosensor. CDK1 activity is low when FRET signaling is low (high lifetime reading) vs. CDK1 activity is high when the FRET signaling is high (low lifetime reading). Adapted from (46), reprinted with permission from AAAS.

(G) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole (0.5 µM) treatment at indicated time points. Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment.

(H) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G. Blue: 1 ns; red: 3 ns. Graph represents the mean±SEM of three independent experiments.

(I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments.

(J) Control and CEP55 knockdown MDA-MB-231 cells were transfected with MAD2 siRNA (5 nM) for 48 hours followed by nocodazole (0.5 µM) treatment for an additional 12 hours. Cells were collected, fixed and sub-G1 analysis was performed as described in methods. Graph represents the mean±SEM of two independent experiments.
CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer (2018)
Figure 6

Fig 6: Combined MEK1/2-PLK1 inhibition specially kills basal-like breast cancer lines.

(A) Heat map showing relative cell viability of a panel of human breast cancer lines treated single or in combination with AZD6244 (1 µM) and BI2536 (2.5 nM) inhibitors and cell viability was determined after 6 days. DMSO treated control was used to calculate percentage of cells affected by individual or combination treatment. NN: near normal. Heat map represents data derived from three independent experiments.

(B) Immunoblot analysis was performed on MDA-MB-157 and SUM159PT cells treated with single or in combination with AZD6244 and BI2536 inhibitors after 96 hours and cleaved PARP and Caspase-3 were determined along with CEP55, MYC and both phosphorylated and total ERK1/2. COX-IV as a loading control (left panels). Percentage of sub-G1 population identified using propidium iodide staining and quantified by FACS following single and combination treatment with AZD6244 and BI2536 inhibitors after 96h. Graph represents the mean±SEM of two independent experiments (middle panels). Representative images of colony forming capacity at 14 days determined using crystal violet staining in cells treated with single and combination inhibitors (middle panels). Combination index (CI) values calculated through CompuSyn software (right panels) where 1 indicative of additive (no interaction), >1 indicative of antagonistic, and <1 indicative of synergistic.

(C) Percentage of mitotic outcomes determined using time-lapse microscopy as described in Figure 4A of MDA-MB-231 cells treated with single or in combination drugs of BI2536 (2.5 nM) and AZD6244 (1 µM). Graph represents the mean±SEM of two independent experiments. For each experiments n=50 cells were counted per condition.

(D) Immunoblot analysis was performed to determine the expression and activity of mitotic and apoptotic regulators upon single and combination treatment with AZD6244 and BI2536 inhibitors as indicated time points in both MDA-MB-231 and SUM159PT cells. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of CDK1 activation/mitotic entry. COX-IV as a loading control.
CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer (2018)
Figure 7

Fig 7: Co-blockade of MEK1/2 and PLK inhibits basal-like tumor outgrowth in vivo

(A) Left, Six week-old female BALB/c cohorts of mice were injected in the 4th inguinal mammary fat pad with the Cep55-ovexpressing mammary carcinoma cell line 4T1.2. Tumor size (area, mm2) was measured using a digital calliper and mean tumor size of each cohort is presented. Mice were treated with vehicle, AZD6244 (12.5mg/kg BID), BI6727 (12.5mg/kg thrice weekly), or combined AZD6244 and BI2536 treatment. Right, representative excised tumor images are shown. Graph represents the mean tumor area±SEM from six mice/group.

(B) Immunoblot analysis was performed on tumor lysates to determine target inhibition following single and combined treatments as indicated in panel A. Cleaved Caspase-3 was probed to determine apoptosis. GAPDH as a loading control.

(C) Six week-old female BALB/c cohorts of mice were pre-treated with single and combination BI6727 and AZD6244 inhibitors as indicated in panel A for 4 days, followed by 4T1.2 cells were injected in the 4th inguinal mammary fat pad. Survival of the mice was then monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P=0.0005); n=6 mice/group.

(D) Growth rate (mean tumor size, area, mm2) of MDA-MB-231 xenografts in BALB/c nude mice treated with vehicle, AZD6244, BI6727, or combined AZD6244 and BI6727 treatment as indicated in panel A and (E) survival of mice was monitored over the indicated period of time and the significant of data was analyzed by log-rank test (P<0.0001); n=6 mice/group.

(F) Schematic overview of MAPK pathway in controlling CEP55 level through c-MYC. Co-blockade of PLK1 (BI6727), a mitotic blocker with MEK1/2 (AZD6244) represents a potential therapeutic strategy to treat MYC-CEP55-dependent aggressive basal-like, triple-negative breast cancers.

EV Figure Legends:
Adaptation to high rates of chromosomal instability and aneuploidy through multiple pathways in budding yeast (2022)
Figure 3

Figure 3. Suppressor mutations in the Dam1 complex result in elevated spindle assembly checkpoint activity and minichromosome missegregation.

(A) Serial dilutions on YPAD media at the permissive (25˚C) or restrictive (35˚C) temperatures for the ipl1-321 mutation. The suppressor mutations from the Dam1c (blue) were the only mutants that rescued viability at the restrictive temperature.

(B) Top: representative images showing Bub3-mNeonGreen localization to kinetochores (Nuf2-mRuby3), indicating spindle assembly checkpoint activation in wild-type yeast. Scale bar is 2µm. Bottom: quantification of the proportion of prometaphase cells with Bub3-mNeonGreen foci. Only the mutations in Dam1 complex (blue) show a significant increase in SAC activity. Data are from at least 5 independent experiments. Means and standard deviations are shown.

(C) Proportion of colonies that grow on plates with restrictive (lacking uracil) versus permissive (YPAD) media after 24 hours growth under permissive conditions with a URA3-containing plasmid. The suppressor mutations in the Dam1c (blue) show a significant reduction of minichromosome segregation fidelity. Data are from 3 independent experiments. Means and standard deviations are shown.

Data information: (ns) non-signifcant; (*) P<0.05; (**) P<0.01; (***) P<0.001; (****) P<0.0001; unpaired t-test.
Adaptation to high rates of chromosomal instability and aneuploidy through multiple pathways in budding yeast (2022)
Figure 4

Figure 4. Suppressor mutations in the Dam1 complex have phenotypes similar to the dam1(S20D) phosphomimic mutation.

(A) A schematic showing the relative locations of the Ipl1 phosphosites in Dam1 that are mutated to aspartic acid in the phosphomimics.

(B) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. dam1(S20D) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1.

(C) Quantification of the proportion of prometaphase cells with Bub3-mNeonGreen foci localized to kinetochores (Nuf2-mRuby3), indicating SAC activation in all three mutant strains. Data are from at least 5 independent experiments. Means and standard deviations are shown.

(D) Degree of cell cycle delay as measured by percentage of large budded cells over time after release from G1 arrest. Wild-type (WT) cells treated with nocodazole and benomyl were used as a positive control for mitotic arrest.

(E) Proportion of colonies that grow on plates with restrictive (lacking uracil) versus permissive (YPAD) media after 24 hours growth under permissive conditions with a URA3-containing plasmid. Minichromosome segregation fidelity is reduced in the dam1(S20D) strain, similar to the duo1(P17L) strain. Data are from 3 independent experiments. Means and standard deviations are shown.

Data information: (ns) non-signifcant; (***) P<0.001; (****) P<0.0001; unpaired t-test.
Adaptation to high rates of chromosomal instability and aneuploidy through multiple pathways in budding yeast (2022)
Figure 5

Figure 5. Suppressor mutations in the Dam1 complex reduce spindle localization.

(A) View of the C. thermophilum Dam1c-ring, with focus on two adjacent protomers (labeled decamers 1 and 2). Interfaces 1 and 2 between adjacent decamers are shown with the blue dashed boxes as previously identified by Jenni and Harrison (Jenni & Harrison, 2018). Mutations in the S. cerevisiae proteins have been labeled based on sequence homology with C. thermophilum. Mutations within the structure are depicted as solid circles. Those that are located outside of the structured domains are shown as open-circles. Suppressor mutations identified in this study are colored magenta, and the residues mutated in the Dam1 phosphomimics are colored green. The marked residues are the closest approximations of the residues mutated in S. cerevisiae. N43 of Dad1 is conserved in C. thermophilum. The Dad2 residue K11 in S. cerevisiae is homologous to K35 in C. thermophilum. D119 of Spc34 maps to a short helix that contains H117 in C. thermophilum. The bottom right shows the location of the Dad1 residue N43 on the surface of interface 2. Images were made using ChimeraX (Pettersen et al, 2021).

(B) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in prometaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at kinetochores/spindles in prometaphase cells. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.

(C) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in anaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at the spindle in anaphase cells. The intensity was measured in the middle of the spindle using a perpendicular line-scan. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.

(D) Quantification of the intensity of Dad3-mNeonGreen at kinetochores in anaphase cells. Kinetochore position was based on Nuf2-mRuby3 localization. The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle (Fig 5C). Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments. The mean (red line) is shown.

Data information: (ns) non-signifcant; (*) P<0.05; (**) P<0.01; (***) P<0.001; (****) P<0.0001; unpaired t-test.
Adaptation to high rates of chromosomal instability and aneuploidy through multiple pathways in budding yeast (2022)
Figure 6

Figure 6. Cdc4 limits CPC localization in prometaphase independently of Sgo1.

(A) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. The met30-6 and cdc4-1 alleles are temperature sensitive. Two independent clones of each of these alleles are shown. The serial dilutions were performed at the permissive temperature (20˚C).

(B) The amount of mNeonGreen-Sli15 (CPC member) located at kinetochores (Nuf2-mCherry) was measured in prometaphase. Sgo1 was depleted by placing it under a galactose-inducible promoter and switching to glucose-containing media (YPAD). Representative images are on the left. Scale bar is 2µm. Averages from 3 independent experiments are shown.

(C) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores. Inner centromeres were identified as the regions directly between two centromeres (ACA staining). Images from an example WT cell are shown to the right. Scale bar is 5µm long. Each data point is an individual chromosome. More than 15 cells for each cell line were measured across two independent experiments. The mean (red line) is shown.

(D) Timeline of adaptation to high levels of CIN through aneuploidy and point mutations.

Data information: (ns) non-signifcant; (*) P<0.05; (***) P<0.001; (****) P<0.0001; unpaired t-test.

Expanded View figure legends:
ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function (2016)
Figure 1

Figure 1. PALB2 phosphorylation is induced upon DNA damage in an ATM/ATR dependent manner

A) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS-PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin. (NT, no treatment). The lysate from the 24 hours time point was treated with phosphatase (Ppase).

B) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies.

C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies.

D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies.

E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.
ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function (2016)
Figure 2

Figure 2. Extensive PALB2 phosphorylation after DNA damage requires three N-terminal ATM/ATR consensus target sites

A) Schematic figure of the human PALB2 protein. The N-terminus of PALB2 contains a coiled-coil region (CC) that mediates the interaction between PALB2 and BRCA1. PALB2 interacts with BRCA2 via the C-terminal WD40 domain, which also harbors interaction sites for RAD51. An additional region for RAD51 interaction is located in the N-terminus at amino acid residues 101-184. PALB2 has seven potential S/Q phosphorylation sites of which serines S59, S157, and S376 (depicted in red) were mutated either to alanine (triple mutant S to A, TMA) to create a phospho-deficient mutant, or aspartic acid (triple mutant S to D, TMD) for a phospho-mimicking mutant. GST-PALB2-N (amino acids 1-560) contains the N-terminal serines.

B) Characterization of the Doxycycline-inducible cell lines expressing siRNA resistant wild type (WT) or mutant (TMA or TMD) FLAG/HA-tagged PALB2. The cell lines were transfected with UNC (Negative Control) or PALB2 siRNA for 24 hours prior to induction with Doxycycline. Immunoblots were performed with indicated antibodies.

C) Cell line characterization of FLAG/HA-tagged PALB2 localization 2 hours following exposure to 15 Gy of IR. Z-stack max intensity image projection and background subtraction was done using ImageJ/Fiji. Scale bar = 5μm.

D) Kinase assay was performed as described in Figure 1E except that the SDS-PAGE gel was Coomassie stained following electrophoresis. WT was used at two different concentrations (1x and 0.75x).

E) Doxycycline-inducible stable U2OS cell lines were left untreated or treated with Doxycycline and/or IR (15 Gy, 2h recovery). Following protein extraction the lysates were immunoprecipitated (IP) with IgG or antibody recognizing phosphorylated S/Q sites (pS/Q) and analyzed by SDS-PAGE. Immunoblots were performed with indicated antibodies.
ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function (2016)
Figure 3

Figure 3. PALB2 phosphorylation promotes RAD51 foci formation

A) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later. Z-stack images were acquired with a 60X oil objective (Deltavision). Z stack max intensity projection, background subtraction, and foci count was done using ImageJ/Fiji. (* p<0.0001, unpaired Student’s t-test). Scale bar = 5μm.

B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student’s t-test). Scale bar=5μm.

C) HeLa DR-GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I-SceI together with empty vector (EV), WT-, TMA-, or TMD-PALB2 the next day. 72 hours later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars=SEM).

D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student’s t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.
ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function (2016)
Figure 4

Figure 4. PALB2 phosphorylation promotes genome maintenance

A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student’s t-test, error bars=SEM).

B) The relative intensity of phosphorylated H2AX (pS139) was examined in the total population of WT- and TMA-PALB2 expressing cell lines 18 hours post exposure to IR (5 Gy). Cell lines were prepared as in above, fixed at the indicated time point, and stained for pH2AX. Cells were imaged with a 20X air objective on a Scan^R workstation (Olympus), mean relative pH2AX intensity was calculated from background subtracted images using the Scan^R analysis software. (* p<0.0001, unpaired Student’s t-test).

C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.