EMBO/soda-nel huggingface dataset:
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 7

Figure 7. T172 phosphorylation is selectively enriched in tumours, and its expression is correlated with cancer patient prognosis.

A Immunohistochemical staining of p-T172-BCL9 expression in a human HCC tissue. MN: matched normal tissues, T: tumour tissues. Scale bar represents1000 μm.

B IHC staining and the expression scored in a tissue array of 75 pairs of human lung cancer patient tissues. Scale bar represents 1000 μm. Significance was measured by two-tailed unpaired t-test,***p < 0.001, n=75, all data are the mean ± SD.

C A representative figure of p-T172-BCL9 IHC staining in HCC to indicate the expression in mitotic or in interphase cells. Scale bar represents 100 μm.

D-E Representative figures of p-T172-BCL9 IHC staining in interphase cells of breast cancer (D) or lung cancer (E) patient tissue. Scale bars represent 100 μm.

F Immunoprecipitation analysis of the interaction in 5 pairs of human HCC patient tissues by a p-T172-BCL9 antibody or a BCL9 antibody. CLTC relative binding with BCL9 was determined.

G-H Log-rank (Mantel-Cox) survival analysis based on high and low p-T172-BCL9 expression scored by immunohistochemical staining in two tissue arrays with (G) 75 lung cancer samples or (H) 145 breast cancer samples.

See also Fig EV7, Table EV5.
Attenuated CSF-1R signalling drives cerebrovascular pathology (2020)
Figure 2

Figure 2. The ΔA781_N783 and P824R Variants in CSF-1R Are Loss of Function and Have Conserved Cellular Processing

A) Structure-sequence analysis and context of the CSF-1R ΔA781_N783 and P824R variants. Sequences used in the alignment correspond to Uniprot sequences for CSF-1R as follows: Human P07333, Cynomolgus Monkey A0A2K5WG91, Mouse P09581, Bovine A7Z067, Cat P13369, Zebrafish Q9I8N6.

B) Western blot for CSF-1R and Actin in HEK293 cells transfected with native and variant CSF-1R as indicated.

C) Densitometry (top) and qPCR analysis of native and variant CSF-1R expression in HEK293 cells relative to native-transfected at 24 and 48 h (**** p < 0.0001, *** p < 0.0005, , ** p < 0.005 one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, error bars indicate SEM).

D-G) Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM).

H) Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).

I) Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).

J) Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).
Attenuated CSF-1R signalling drives cerebrovascular pathology (2020)
Figure 4

Figure 4. CSF-1R expression in endothelial cells and macrophages affects response to amyloid injection

A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm.

B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).

C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red).

D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).
Attenuated CSF-1R signalling drives cerebrovascular pathology (2020)
Figure 5

Figure 5. Endothelial - macrophage crosstalk regulates CSF-1R expression and TJs

A) Schematic for experimental design.

B) Western blot for CSF-1R in b.End3 cells treated with different BV2 conditioned media or PLX3397, with corresponding densitometry (right). (n = 3 independent BV2 media conditionings and b.End3 treatments, one-way ANOVA with Dunnett's post-test for multiple comparisons, *p < 0.05, ***p < 0.0005, error bars indicate SEM))

C) Western blot for tight junction proteins in b.End3 cells treated with unconditioned, microglia conditioned media or PLX3397.

D) Densitometry of tight junction protein changes (top) and transcriptional changes in Tjp1, Ocln and Cld5 (bottom) following conditioned media treatments or 20 μM PLX3397. (n = 3 independent BV2 media conditionings and b.End3 treatments, one-way ANOVA with Dunnett's post-test for multiple comparisons, *p < 0.05, **p < 0.009, error bars indicate SEM))

E) Schematic for experimental design.

F) Western blot of WT endothelial cells treated with unconditioned or microglia conditioned edia (MCM) produced from Csf-1r+/- or PLX3397- inhibited microglia.

G) Western blot of Csf-1r+/- endothelial cells treated with unconditioned, Csf-1r+/- MCM or PLX3397 MCM.

H-K) qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).

L-O) qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of changes relative to respective untreated Control. (** p < 0.005, * p < 0.05. Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM).
Attenuated CSF-1R signalling drives cerebrovascular pathology (2020)
Figure 6

Figure 6. Loss of CSF-1R reduces BBB integrity

A-C (A) IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.

D-H (D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), (G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.

I) T1-weighted DCE-MRI of an ALSP patient with the ΔA781_N783 CSF-1R variant. Colourmetric scale indicates the slope of the quantified Gd-BOPTA accumulation, as an indicator of BBB permeability.

J) Susceptibility weighted imaging (SWI) of an ALSP patient with the ΔA781_N783 CSF-1R variant showing iron deposition.

Data information: Note that Figures 6C and F and are shown again in EV1 for ease of comparison.

Supplementary Figures
Non-apoptotic caspase activation preserves Drosophila intestinal progenitor cells in quiescence (2020)
Figure 4

Figure 4. Dronc is preferentially accumulated and activated in progenitor cells.

A. Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes), Prospero (red nuclei of EEs) and Dronc-TurboID (green shows Streptavidin labelled proteins by Dronc-TurboID); note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells (white arrows indicate an intestinal progenitor cell upregulating Dronc). Genotype: Dronc-TurboID-V5 (Masayuki Miura)/Tm6B

B. Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes) and Actin-Dronc-GFP-Myc (green, anti-GFP); note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells (arrow). Genotype: w1118; Actin-Dronc-GFP-Myc (attP-VK37)/Cyo

C. High magnification of the picture shown in B. The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP+ EBs. Genotype: w1118; Actin-Dronc-GFP-Myc (attP-VK37)/Cyo. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc.

D. Transversal Z-section of the cells shown in C. Genotype: w1118; Actin-Dronc-GFP-Myc (attP-VK37)/Cyo. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc.

E. The enteroblast marker Su(H) (red, immunostaining against Beta-galactosidase) strongly co-localises with high levels of Dronc expression (green, immunostaining against GFP); white arrow indicates the enlarged area depicted in the insets. Genotype: w1118, Su(H)GBE-LacZ (Irene Miguel Aliaga); Actin-Dronc-GFP-Myc (attP-VK37)/Cyo

F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.

Data information: All of the experiments described in the figure apart from F were performed in Oxford medium following an experimental regime that protects the epithelial integrity at 29 Co. The experiment shown in F was conducted at 25 Co.Genotype: Females w1118Su(H)GBE-LacZ/y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF
Homologous recombination and Mus81 promote replication completion in response to replication fork blockage (2020)
Figure 1

Figure 1. HR-mediated replication restart occurs by a BIR-like mechanism.

(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.

(C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed.

(D) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed.

(E) Schematic representation of the BIR assay. Two fragments of the LYS2 gene sharing 2.1 kb of homology (lys and ys2) were integrated on chromosomes V and XI, respectively. Induction of HO endonuclease expression under control of the GAL1 promoter produces a DSB next to the lys fragment that can be repaired by BIR. BIR events can be scored by selecting survivor colonies, which harbor a functional LYS2 gene.

(F) BIR frequencies (Lys+ survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean ± SD from 3 to 6 independent experiments. ns, non-significant difference, * P-value = 0.0134, Mann-Whitney unpaired t test.
Homologous recombination and Mus81 promote replication completion in response to replication fork blockage (2020)
Figure 2

Figure 2. Mus81 is required in the restart pathway involving both Rad51 and Pol32.

(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.

(C) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed.

(D) Synthetic combinations of rad3-102 mus81∆ with rad51∆ and pol32∆. Tetrads dissected on YPAD medium are shown. Triangles indicate either a severe growth defect or lethality.
Homologous recombination and Mus81 promote replication completion in response to replication fork blockage (2020)
Figure 3

Figure 3. Mus81 is not required for S phase progression in CPT-treated and rad3-102 cells.

(A) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad52∆, rad51∆, mus81∆ and pol32∆ cells and further released into S phase. Cells were synchronized in G1 with α -factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Three biological replicates have been performed.

(B) (C) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad3-102, rad3-102 rad51∆, rad3-102 mus81 and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G1 with α-factor, untreated or irradiated with 20 J/m2 UV-C, let in G1 for 2h, and released into S phase. Three biological replicates have been performed.

Data information: Asterisks indicate the progression of cells in S phase.
Homologous recombination and Mus81 promote replication completion in response to replication fork blockage (2020)
Figure 4

Figure 4. Rad52 and Rad51 are required for HR-mediated replication fork protection.

(A) Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52∆, rad51∆, mus81∆ and pol32∆ cells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse-labeled with 50 µM EdU for 20min. DNA fibers were combed on silanized coverslips and EdU-labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann-Whitney unpaired t test, **: p=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA.

(B) Schematic representation of replication intermediates visualized by 2D gels.

(C) Analysis of replication intermediates by 2D gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81∆ and rad3-102 mus81∆ cells. Cells were synchronized in G1 with α-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed.

Data information: Accumulation of recombination molecules is indicated by white arrows.
Homologous recombination and Mus81 promote replication completion in response to replication fork blockage (2020)
Figure 5

Figure 5. Mus81 is involved in the processing of recombination intermediates in G2/M after replication restart.

(A) Analysis of nuclear Rad52-GFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-GFP; red, mCHERRY-Pus1 (nuclear compartment marker). Scale bar = 5 µm.

(B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed.

(D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed.

(E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed.

(F) UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed.
Homologous recombination and Mus81 promote replication completion in response to replication fork blockage (2020)
Figure 6

Figure 6. Recombination intermediates generated by replication restart accumulate in the absence of Mus81.

(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.

(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.
A non-canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation (2015)
Figure 1

Figure 1. Plk4 kinase activity is required for the pericentriolar distribution of centriolar satellite components

A. Evaluation of siRNA-mediated depletion. U2OS cells were treated with control, Plk4 or hSAS-6 siRNA and immunoblotting was performed with the indicated antibodies. Asterisk indicates non-specific bands. The positions of molecular weight markers (kDa) are shown on the right.

B. U2OS cells were transfected with the indicated siRNAs. Cell peripheries are marked with dotted lines. Enlarged single cell images (marked with arrowheads in the top row) are shown at the bottom. Scale bars, 5 μm (bottom),10 μm (top).

C, D, E. Quantification of the dispersion of satellite components upon depletion of Plk4 but not hSAS-6 knockdown. The results of PCM1 (C), hMsd1/SSX2IP (D) or BBS4 (E) are shown. Data represent the mean + SD (>200 cells, n=3). Statistical analysis was performed using two-tailed unpaired student’s t-tests. **p<0.01, n.s. (not significant).

F, G. U2OS cells were arrested in G1 and treated with control or Plk4 siRNA, followed by immunofluorescence microscopy by using indicated antibodies. Enlarged images (marked by arrowheads) are shown on the right. Quantification of PCM1 distribution is shown in G. >200 cells were counted and classified into two categories: normal (around and away from the centrosome) or dispersed. Data represent the mean + SD (n=3). Statistical analysis was performed using two-tailed unpaired student’s t-tests. **p<0.01. Scale bar, 1 μm (F, right), 10 μm (F).

H, I. siRNA-treated U2OS cells were transfected with empty vectors (EV) or plasmids containing Plk4 or siRNA-resistant Plk4-myc WT* or KD* and immunostained with the indicated antibodies. Cell peripheries are marked with solid lines, in which cells with yellow lines are successfully transfected (positive in myc signals), while those with white lines represent non-transfected cells. Quantification of PCM1 distribution is shown in I. Data represent the mean + SD (>200 cells, n=3). Statistical analysis was performed using two-tailed unpaired student’s t-tests. ****p<0.0001, **p<0.01, n.s. (not significant). Scale bar, 5 μm (H).
A non-canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation (2015)
Figure 2

Figure 2. PCM1 physically interacts with Plk4 and is phosphorylated at S372

A, B. Either empty vector plasmids (EV) or those producing EGFP-PCM1 (A) or myc-Plk4 (B) were transfected into U2OS cells. Immunoprecipitation (IP) was performed with anti-GFP (A) or anti-myc antibodies (B), followed by immunoblotting with the indicated antibodies. DIC, the dynein intermediate chain.

C. Bacterially expressed and purified His-PCM1 (a.a. 1-1128) was incubated with MBP alone or MBP-Plk4 (WT or KD) in vitro, and pulled down by magnetic beads coupled with an anti-MBP antibody, followed by staining with Coomassie Blue (left) or immunoblotting with an anti-His antibody (right). Asterisks show non-specific bands. The mobility difference between Plk4-WT and Plk4-KD was reported previously [43, 48]. We reproducibly observed the upward mobility shift of His-PCM1 upon binding to MBP-Plk4-WT, but not Plk4-KD.

D. Overall structure of PCM1 and the Plk4-mediated phosphorylation site. The conserved serine residue that is phosphorylated in a Plk4-dependent manner is indicated in red.

E. Total cell extracts were prepared from U2OS cells treated with PCM1 siRNA and transfected with RNAi-resistant EGFP-PCM1-WT*. Cells were cultured asynchronously (As) or arrested in G1, G2 or M phase, followed by immunoprecipitation with an anti-GFP antibodies. Pulled down samples were immunoblotted with the indicated antibodies.