EMBO/soda-nel huggingface dataset:
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 6

Fig 6. Cyclin F regulation of DNA replication and DNA repair genes is dependent on E2F7/8

Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.

KEGG pathway analysis of genes downregulated by cyclin F knockdown, and rescued by additional E2F/8 depletion. Bars represent -log P values, such that larger values mean stronger statistical significance. The cut-off P value 0.05 is shown as a red dotted line.

qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.

Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 7

Fig 7. Degradation of atypical E2Fs maintains DNA damage repair

Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.

Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.

Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).

Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.
Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism (2019)
Figure 1

Figure 1

Opposite mRNA signatures of Grn-/- / Trem2+/+ and Trem2-/- / Grn+/+ microglia.

A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b- positive Grn-/- microglia in comparison to wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed, are significantly changed, and from these 58 genes are up- and 58 genes are down regulated. Eight genes with the highest fold change are highlighted.

B. Heatmap of significantly affected genes (p < 0.05) in FCRLS- and CD11b-positive Grn-/- microglia in comparison to Grn+/+ microglia isolated from 5.5-month-old mice (n = 5 per genotype). For the significantly affected genes of the Grn-/- microglia, mRNA expression data of the Trem2-/- microglia in comparison to the corresponding wt microglia were taken from previously published data (Mazaheri et al, 2017). The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation (n ≥ 5 per genotype). * labelled genes were not analyzed or below detection limit in Trem2-/- microglia mRNA expression data set (Mazaheri et al, 2017).

C. Changes in gene expression of 359 detected genes in Grn-/- microglia and Trem2-/- microglia. Note that from the 38 genes significantly altered in both genotypes, 32 genes are regulated in opposite direction.

D. Gene expression levels of FCRLS- and CD11b-positive Grn-/- microglia normalized to wt in comparison to the published normalized data set of Trem2-/- microglia (Mazaheri et al, 2017).

Data information: For statistical analysis the unpaired two-tailed Student's t-test was performed between Grn-/- microglia in comparison to Grn+/+ microglia or Trem2-/- microglia in comparison to Trem2+/+ microglia: not significant p > 0.05; significant p < 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Source data including exact p‐values and n numbers are available online for this figure.
Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism (2019)
Figure 2

Figure 2

Expression of microglial marker proteins characteristic for the homeostatic or disease associated state.

A. Immunoblot analysis of ApoE, CLEC7A, TREM2 and CD68 in lysates of acutely isolated microglia from Grn-/- and Grn+/+ mice (9 months of age, n=3 per genotype, female). IBA1 was used as loading control.

B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD.

C. ELISA mediated quantification of sTREM2 in brain homogenates of Grn-/- and Grn+/+ mice Data are shown as mean ± SEM (n=3-6).

D. ELISA mediated quantification of sTREM2 in serum of Grn-/- and Grn+/+ mice. Data are shown as mean ± SEM (n=4-13).

E. Microglial expression of P2RY12 in cortical sections of wt, Grn-/-and Trem2-/- mice. (9 months of age, female)

F. Quantification of P2RY12 positive microglia. Data are shown as mean ± SD (n=3 per genotype, female, except one male for Trem2-/-).

G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12 and TREM2 in lysates of BV2 wild type (Grnwt) and PGRN deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control.

H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD.

I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SD

J. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.

Data information: For statistical analysis the unpaired, two-tailed Student's t-test was performed between Grnmut or Grn-/- in comparison to Grnwt or Grn+/+: n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. For F the one-way ANOVA with Tukey post-hoc test was used.

Source data including exact p‐values, n numbers, and sex if applicable are available online for this figure.
Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism (2019)
Figure 3

Figure 3

Enhanced phagocytosis, migration and clustering around amyloid plaques upon PGRN deficiency.

A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 minutes (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± S.D (n=3).

B. Enhanced migration of CD68 positive, PGRN deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn+/+ and Grn-/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels).

C. Quantitative analysis reveals that Grn-/- microglia migrate larger distances than Grn+/+. Data are shown as mean ± SD (n=3 independent experiments).

D. Quantification displays an increased number of migrating Grn-/- cells compared to Grn+/+ cells. Data are shown as mean ± SD (n=3 independent experiments).

E. Enhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglia clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn+/+ mice. Right: Age-matched APPPS1/Grn-/- mice display microglial hyper-clustering around amyloid plaques compared to APPPS1/Grn+/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10µm and 3µm in inset.

F. Immunohistochemical quantification of IBA1 positive microglial cell number per plaque

(APPPS1/Grn+/+ n=4, APPPS1/Grn-/- n=4; 4-month-old; two males and two females per genotype) normalized to APPPS1/Grn+/+ mice. Approximately 20-50 amyloid plaques plaques were counted per mouse in 3 cortical sections to calculate the number of IBA1-positive microglia per plaque. Data are shown as mean ± SD.

Data information: For statistical analysis the unpaired two-tailed Student's t-test was performed (A, C, D) between PGRN deficient Grnmut BV2 cells or Grn-/- microglia and Grnwt BV2 cells or Grn+/+ microglia. For F the Mann-Whitney U test, two-tailed was used. Significance is indicated by * p < 0.05; ** p < 0.01; *** p < 0.001.

Source data including exact p‐values and n numbers are available online for this figure.
Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism (2019)
Figure 4

Figure 4

Enhanced microglial activity in Grn-/- mice and in a patient with Grn haploinsufficiency.

A. TSPO-µPET in rodents: Axial slices (A1, A2, A3) of averaged %-TSPO-PET differences between Grn-/-or Trem2-/- mice and wt indicate increased microglial activity in the cerebrum of Grn-/- mice (hot color scale) and decreased microglial activity in the cerebrum of Trem2-/- mice (cold color scale). Scatter plot depicts single TSPO-µPET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=8 at 11 months; wt n=10 at 8-11 months). Statistics derive from one-way ANOVA with Tukey post-hoc test.

B. PGRN serum levels of the patients subjected to TSPO-PET confirmed the GRN mutation and non-GRN mutation carrier. PGRN was measured by ELISA and normalized to serum levels of a healthy control.

C. TSPO-PET in human FTLD patients: Visual interpretation of axial TSPO-PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices (white arrows) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation. PET results are illustrated upon an MRI template; 60-80 min p.i. emission recording; global mean scaling. Both patients were low-affinity binder.

Data information: Statistics derive from one-way ANOVA with Tukey post-hoc test (A).
Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism (2019)
Figure 5

Figure 5

Cerebral hypometabolism upon loss of GRN and TREM2.

FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).

Data information: Statistics derive from one-way ANOVA with Tukey post-hoc test.
Structural Basis for the Activation and Suppression of Transposition during Evolution of the RAG Recombinase (2020)
Figure 4

Figure 4. Identification of the RAG TCC

(A) Diagram of the disintegration reaction and substrate used in (B).

(B) Disintegration reaction (1 hour) performed by RAG1 aa 216-1008 R848M/E649V, RAG2 aa 1-361 and full length human HMGB1 with Mg2+ or Mn2+ at the indicated temperatures. Denaturing gel displays the fluorophore-labeled DNA strand from the RAG STC before (16 nt band, lane 2) and after (37 nt) the disintegration reaction. Lane 1, fluorophore-labeled 37 nt DNA marker. Representative of 2 independent experiments.

(C,D) Cryo-EM maps of RAG TCC (C) at 3.8 Å and Paired STC (D) at 2.9 Å shown in both top and front views with protein colored white, target DNA colored red, and signal DNA colored blue. Insets highlight the different connectivity between signal and target DNA in the TCC and STC.
Structural Basis for the Activation and Suppression of Transposition during Evolution of the RAG Recombinase (2020)
Figure 7

Figure 7. A jawed vertebrate-specific extended loop on RAG2 contacts target DNA and inhibits transposition

(A) Comparison of RAG2 and BbeRAG2L structures. Red color indicates the amino acid 333-342 RAG2 loop and the equivalent loop of BbeRAG2. The four amino acid deletion, (∆L in (D)), is indicated with green brackets. Due to areas of low resolution in the BbeRAG2L structure, some regions that are likely to be beta strands cannot be modeled as such.

(B) Sequence alignment of RAG2 and RAG2-like proteins in the vicinity of mouse RAG2 loop 333-342. Green shading, four amino acids deleted at tip of loop. Red letters in blue box, relatively well-conserved residues; white letters on red background, highly conserved residues.

(C) Cryo-EM map (transparent) and cartoon model of RAG2 showing contact between the extended loop (red) and target site DNA (yellow; stick model).

(D) Results of in vivo plasmid-to-plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking (∆L) the four amino acids at the tip of the 333-342 loop, performed as described in (Zhang et al., 2019); (****, p<0.0001). Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented.

(E) Results of in vivo plasmid-to-genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated. As previously demonstrated (Zhang et al., 2019), R848 (present in WT RAG1) and RAG2 acidic hinge residues 362-383 each potently suppress transposition. Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1-361, which is further increased by ∆L; (***, p=0.001). Four biological replicates for each condition.

(F) In vivo recombination activity in HEK293T cells of mouse wild-type RAG1 or RAG1 R848M with RAG2 1-361 or 1-361 ∆L. (p=0.42 for wild-type RAG1 group, p=0.41 for RAG1 R848M group). Three biological replicates for each condition.

Data information: Data in D-F are depicted as mean +/- SEM. Unpaired, two-tailed t-test.
The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes (2021)
Figure 1

Figure 1: Lack of Mecp2 does not affect the commitment of NPCs but delays their differentiation

A: Schematic representation of the differentiation protocol and the cell types composing cultures differentiated from NPCs (adapted from Carlessi et al, 2013).

B-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm.

K: Representative immunostaining of Neun (green) and Tuj1 (red) positive neurons at DIV14. Scale bars: 15 µm.

L,M: Graphs show the percentage of Neun and Tuj1 positive cells and the average Neun intensity in wt and null samples (L: mean ± SEM; M: central band shows the median, boxes show the lower and upper quartiles, whiskers show max and min values). The percentage of cells positive for each marker was counted over the total of DAPI positive cells. Student's t-test highlighted a significant decrease of Neun intensity in null samples. **: p-value<0.01. Sample size: n≥10 wells obtained from two independent preparations.

N,n': The binned distribution of Neun intensity is plotted as both frequency distribution graphs, in panel N, and cumulative probability plot in panel n'. The null neurons distribution is significantly shifted towards low levels of Neun intensity, Kolmogorov-Smirnov test **: p-value<0.01. Each value in panel N is represented as mean ± SEM obtained by measuring 900 cells from n ≥10 wells derived from two different preparations.

O: Neun expression levels assessed at DIV0, DIV3 and DIV14 are represented as 2-∆ct. Box plots show the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 5 samples obtained from two independent preparations. Two-way ANOVA indicated both a significant genotype effect (F(2,28)=19,60; p<0.0001) and time effect (F(2,28)=93,70; p<0.0001). Bonferroni post-hoc test revealed significant differences between genotypes at DIV 14 but not at DIV0 and DIV 3. ****: p-value<0.0001.

P: Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14. For each gene Two-way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Values are represented as mean ± SEM. Sample size: ≥5 samples obtained from two independent preparations.
The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes (2021)
Figure 2

Figure 2: Neurons derived from Mecp2 null NPCs are morphologically more immature than wt controls and establish less active networks

A,B: Wt or Mecp2 null NPCs plated on the grid of 64 MEA electrodes. Scale bars: 50 µm.

C-G: Line graphs represent the changes through time of 5 parameters describing neuronal activity. Difference between genotypes and through time were analysed using Two-way ANOVA for repeated measures and Bonferroni post-hoc test; * refers to differences between genotypes (*: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001); # refers to differences between the two time points (#: p-value<0.05; ##: p-value<0.01). Sample size: n=8 wells deriving from three independent preparations. Values are represented as mean ± SEM.

H-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations.

L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm.

M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.
The enhancement of activity rescues the establishment of Mecp2 null neuronal phenotypes (2021)
Figure 3

Figure 3: Early exposure to CX546 rescues selected morphological and transcriptional defects of null neurons

A: Schematic representation of the CX546 treatment. The drug was added to the cell medium from DIV3 to DIV6 (early) and from DIV7 to DIV10 (late); a complete wash-out of the medium was performed at the end of each treatment (DIV7 and DIV11 respectively).

B,C: Five morphological parameters were measured in untreated and treated null neurons at DIV14. One-way ANOVA indicated a significant treatment effect for the maximal dendritic length (F=6,306;p=0,0039), total dendritic length (F=4,880;p=0,0126), number of intersections (F=10,70; p=0,0003) and soma area (F=7,721;p=0,0015). Tukey's multiple comparison test was used to assess differences between null neurons and early and late treated neurons. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001. The variation of each of the selected parameters is summarized in the radar plot (C). Sample size (panels B,C): n≥18 wells for ctrl group and n≥10 wells for the treatment groups deriving from three independent preparations; for each well we measured at least 15 cells. Each point is representative of a single well. Values are represented as mean ± SEM.

D-H: The three PCA analyses are based on the 43 genes selected in Appendix Figure S2 and highlight the transcriptional differences between wt (in blue), ko (in red) and ko CX546 treated (light and dark green for early and late treatment respectively) samples. Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.