EMBO/soda-nel huggingface dataset:
ECE2 regulates Neurogenesis and Neuronal Migration during Human Cortical Development (2020)
Figure 1

Figure 1: Neuronal mislocalisation and migration defects found in individuals with ECE2 variants are recapitulated in iPSC-derived in vitro models. A, Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles. B, ECE2 expression on RNA level. In situ hybridisation for ECE2 RNA in 50d old cerebral organoids (COs) shows higher signal in the cortical plate-like zone (CP') (Scalebar=100 µm). C, ECE2 expression on protein level. Immunohistochemistry (IHC) for ECE2 in 50d old COs shows accumulation in the CP' and at the apical surface (Scalebar=50µm). D, Scheme showing the electroporation of DNA into ventricles of COs and the organisation of different cell types within the germinal zone. DNA is injected into the ventricle-like lumen and taken up by aRG via their apical processes. 7 dpe, the transfected construct can additionally be found in IPs and neurons upon differentiation of transfected aRG (green) (VZ' = ventricular zone, SVZ' = subventricular zone, IZ' = intermediate zone, CP' = cortical plate, aRG' = apical radial glia, bRG' = basal radial glia, IP' = intermediate progenitor). E, COs transfected with micro-RNAs targeting ECE2 (KD) or scrambled negative control (CTRL) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD (Transfected cells are shown in green, NEUN+ neuronal nuclei in magenta. d=day. Scalebar=25 µm). F, Graph depicting the number of ectopically located NEUN+ cells in CTRL and KD electroporated COs using two different micro-RNAs targeting ECE2. Data shown as box plot (mean = red line, median = black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of ventricles analysed; ***P < 0.001 in Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pairwise Multiple Comparison). G, Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2-inhibitor PHOS [42]. H, Graphs showing different parameters of neuronal migration dynamics, tracked via live imaging. Upon ECE2 inhibition, significant decrease in velocity, increase in resting TP and increase in tortuosity are observed. Data shown as z-scores (CTRL = DMSO control; PHOS = Phosphoramidon; Resting TP = Resting timepoints; N=number of individual neurons analysed from 2 batches; Resting TP: *P=0.024; tortuosiy: *P=0.035, ***P < 0.001 in 2-tailed Chi-Sqare test). B, C, E, Ventricle-like lumen in COs is marked as V'.

See also Figure EV1.
ECE2 regulates Neurogenesis and Neuronal Migration during Human Cortical Development (2020)
Figure 2

Figure 2: Acute Ece2 KD in the developing mouse cortex causes ectopic rosettes and nodules. A, Quantification of distribution of GFP+ transfected cells 1dpe upon CTRL and Ece2 KD by IUE (n=3 CTRL and 4 Ece2 KD brains; *P=0.044 in One-way ANOVA and Tukey's Multiple Pairwise Comparison; data shown as mean±SEM). B-C, Ece2 KD leads to ectopic non-transfected Pax6+ progenitors 1 dpe (E13-E14). B, IHC staining of GFP (transfected cells) and Pax6 1 dpe showing ectopic Pax6+ progenitors (red circle) and a reduction in their apical localisation (yellow circle). C, High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected. D-J, E13-16 Ece2 KD. D, GFP-Pax6+ progenitors delaminate upon Ece2 KD (red arrows), leaving behind a region free of Pax6+ cells in the VZ (yellow circle) and forming ectopic rosettes (yellow box). E, G, Reduction in apical Arl13b and β-catenin in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD. F, Inside the nodule, which is surrounded by thickened GFP+ processes (pink arrows), Arl13b staining shows the formation of an ectopic apical surface (blue arrow; enlargement of boxed area from D and E). H, Deep layer and I, upper layer neurons localise next to ectopic rosettes, forming nodules of ectopic neurons (blue arrowheads for single ectopic neurons and blue circle for large nodule). J, Quantification at 3 dpe, counting the proportion of brains with reduced density of Pax6 in the VZ, ectopic progenitors or neurons, and changes in apico-basal polarity (including ectopic apical surface formation and reduction in apically localised apical markers; n=6 brains per condition; Exact binomial test: ****P < 0.0001). Scalebars in A, B, D, E, H, I=100 µm; in C, F, G Scalebar = 50 µm. V marks the position of the ventricle.

See also Figure EV2.
ECE2 regulates Neurogenesis and Neuronal Migration during Human Cortical Development (2020)
Figure 3

Figure 3: ECE2 KO and enzymatic inhibition cause a change in cell fate and neuronal positioning in COs. A, Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures. B, ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs (pink dotted line indicates position of the CP'). C, FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons (n=7-8 samples of 3 pooled organoids each from 3 different batches; **P < 0.01; ***P < 0.001 in One Way ANOVA and Tukey Pairwise Multiple Comparison). D, Example images of IHC for PH3+ cells in 60d old CTRL and ECE2 KO COs. E, Quantification of PH3+ cycling cells in 60d old COs, normalised to length of the apical surface of ventricle-like structures, shows an increase upon ECE2 KO (Data shown as box plots with median as black line, mean as red line; n=number of analysed ventricles from 2 batches of COs; One Way ANOVA: **P = 0.004). F, G, In ECE2 KO COs, more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2+ cell bodies in the progenitor zone (n=number of ventricles from 2 independent batches; two-tailed Mann Whitney U (U = 458) and chi-square test: χ2(2)=25.42, ****P < 0.0001).

B, D, F, Scalebars = 50µm; Ventricle-like structures in COs are marked with V'.

See also Figure EV3.
ECE2 regulates Neurogenesis and Neuronal Migration during Human Cortical Development (2020)
Figure 4

Figure 4: ECE2 KO affects neural progenitor polarity and actin and microtubule cytoskeleton in COs. A, Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). B, Fractionation of G- and F-actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G- at the expense F-actin upon ECE2 KO, suggesting a reduction in F-actin belt integrity (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). C, D, The thickness of the apical F-actin belt is increased in ECE2 KO COs. C, Example images of apical F-actin belt in 60d old CTRL and ECE2 KO COs with F-actin labeled by AlexaFluor594-conjugated Phalloidin (Scalebar=100 µm). D, Quantification of the thickness of apical F-actin belt in Fiji [86] by measuring the area of F-actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs (Box plots: mean=red line, median=black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of analysed ventricles in 2 batches; Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pariwise Multiple Comparison: ***P < 0.001). E, IHC for ARL13B and F-ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb-like structure of the apical adherens belt and of apico-basal polarity in terms of apically localised primary cilium in ECE2 KO COs (Scalebar = 10µm). F, G, The microtubule cytoskeleton is changed in COs upon ECE2 KO. F, Example images of ac-tub IHC in CTRL vs. ECE2 KO COs (Scalebar = 100 µm). G, Quantification of ac-tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO (n=number of analysed ventricles; *P = 0.019 in One Way ANOVA and Tukey Pairwise Multiple Comparison). H, I, Apico-basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs. KO COs (H; Scalebar = 100 µm). I, Quantification of germinal zones for normally high ("1") vs. reduced ("0") apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 (n=number of analysed ventricles from 2 batches; ****P < 0.0001 in exact binomial test). J, Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43-GFP reveals RG that delaminated (white arrowheads) and/or lost their bipolar morphology (red arrowheads) in the KO COs (Scalebar = 25 µm).

Ventricle-like lumen is marked by V'. See also Figure EV4.
ECE2 regulates Neurogenesis and Neuronal Migration during Human Cortical Development (2020)
Figure 5

Figure 5: Forced expression of ECE2 in the developing mouse brain and in COs causes mislocalisation of neural progenitor cells and neurons and rescues neuronal positioning in ECE2 KO COs. A-B, Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6+GFP+ neural progenitors with significantly reduced localisation to bin2 (A) and increased localisation of Ctip2+ deep layer neurons to bin2 with reduction in bin5 (B) (for binning strategy see Figure EV2B; n=3 CTRL and 5 ECE2 OX brains E13-14; data shown as bars with mean±SEM; *P < 0.05 and **P < 0.01 in Student's t-test). C, Example images E13-14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions (Scalebar = 100 µm; electroporated cells shown in green). D-F, E13-16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2+ (D) progenitors and Ctip2+ deep- (D) and Satb2+ upper layer neurons (E) (Scalebars = 100 µm; electroporated cells shown in green). F, Quantification at 3dpe, counting the proportion of brains with normal vs. abnormal positioning of Sox2+ neural progenitors and Ctip2+ and Satb2+ neurons and that of brains with intact vs. disrupted basal membrane (n=5 CTRL and 8 ECE2 OX brains; exact binomial test: ****P < 0.0001). G-L, ECE2 OX in COs (electroporated cells shown in green; Scalebars = 50 µm; n = number of CO ventricles analysed from 2 batches). G, Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3+ mitotic cells and NEUN+ neuronal nuclei. H, Quantification of PH3+ cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background (*P = 0.046 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). I, Quantification of GFP+ neurons as proportion of all GFP+ cells reveals an increase upon ECE2 OX in COs (*P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). J, Quantification of ectopic neurons localising to the VZ' relative to electroporated radial units (GFP+ cells in the VZ') reveals a significant increase upon ECE2 OX (***P < 0.001 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). K, L, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning. K, Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe. L, Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs, thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX (**P = 0.002 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). H-J; L: Outliers exceeding mean±2 standard deviations were removed. C-E, Ventricle marked by V. G, K, Ventricle-like lumen marked by V'. See also Figure EV5.
ECE2 regulates Neurogenesis and Neuronal Migration during Human Cortical Development (2020)
Figure 6

Figure 6: Proteomics reveal a role for ECE2 in ECM production and integrity. A, Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO (n=3 batches of 60d old CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off; names of example proteins are shown). B, Pie chart with proteins downregulated in ECE2 KO COs sorted by relevant categories (Number in the elements = number of proteins falling into each category, with numerous proteins falling into several ones) [50-52]. C, Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins (red) in the secretome (n=3 batches of CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off). Non-significantly changed proteins of interest are highlighted in green below the cut-off curves. D, GO cellular component analysis of proteins with dysregulated phosphorylation sites (either found excusively in CTRL or KO or up- or downregulated in ECE2 KO COs relative to CTRL) identified in whole CO proteomic analysis. E-G, Schemes summarizing identified phenotypes in in vivo and in vitro model systems upon acute ECE2 KD (E) or chronic inhibition or KO (F) or upon acute ECE2 OX (G) and thus the molecular action of ECE2 (Abbreviations: d = day, pe = post electroporation, VZ = ventricular zone, SVZ = subventricular zone, IZ = intermediate zone, CP = cortical plate, aRG = apical radial glia, bRG = basal radial glia, IP = intermediate progenitor, ECM = extracellular matrix).

See also Figure EV5 and Tables EV1-9.

Expanded view figure legends
Dynamic folding modulation generates FGF21 variant against diabetes (2020)
Figure 6

Figure 6. Anti-diabetic activity characterization of FGF21SS.

A. FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3-L1 adipocytes. Cells were cultured in RAW-CM to induce inflammation and insulin resistance, while vehicle, FGF21 or FGF21SS were added simultaneously. The insulin sensitivity was indicated by insulin-induced AKT phosphorylation using western blotting analysis. Error bars show the SEM of four independent experiments. *p < 0.05, **p < 0.01 (Student's t-test) vs RAW-CM treated only group.

B-D. Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. Body weight (B) was measured and plasma glucose (D) was measured every day. Plasma insulin (C) was analyzed at day 7 using insulin ELISA kit. Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.
Mrc1 and Rad9 regulate initiation and elongation 
of DNA replication in response to DNA damage (2018)
Figure 1

Figure 1 Genome-wide analysis of BrdU incorporation in cells exposed to MMS.

(A) Wild type, rad9∆ and mrc1∆ cells were arrested in G1 with α-factor and were released synchronously into S-phase with the addition of pronase, in medium containing BrdU and 0.1% or 0.033% MMS. Cells were collected at the indicated time points.

(B) Analysis of DNA content by flow cytometry.

(C) Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells.

(D) Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.
Mrc1 and Rad9 regulate initiation and elongation 
of DNA replication in response to DNA damage (2018)
Figure 2

Figure 2 Differential regulation of DNA replication and Rad53 activation by Rad9 and Mrc1 in MMS-treated cells.

(A) Wild type, mrc1∆ and rad9∆ cells were released into S phase in the presence of BrdU and 0.033% MMS, as described in Fig. 1A. DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase. Representative DNA fibers are shown. Red: DNA, green: BrdU. Bar is 20 kb.

(B) Distribution of center-to-center distances in cells released for 45 minutes into S phase.

(C, D) Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase, respectively.

(E, F) Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot (E) or with an in situ phosphorylation assay - ISA (F). The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.

Data information: In panels (B-D) median values are indicated in red. For each sample, at least 100 tracks were measured in two biological replicates. Asterisks indicate difference with wild type cells. ****: p<0.0001, ***: p<0.001, **: p<0.01, ns: non-significant, Mann-Whitney rank sum test.
Mrc1 and Rad9 regulate initiation and elongation 
of DNA replication in response to DNA damage (2018)
Figure 3

Figure 3 Pre-activation of the DDC allows Rad9-dependent repression of late origins.

(A) Wild type and rad9∆ cells were synchronized in G1 and released into S phase with the addition of pronase in medium containing IdU and 200 mM HU to activate the DRC. Then, DSBs were induced by the addition of 100 μg/ml Zeocin to activate the DDC. HU was washed away 45 minutes later to inactivate the DRC and cells were resuspended in fresh medium containing CldU and 100 μg/ml Zeocin, in order to keep the DDC active. Samples were collected at the indicated times.

(B) Analysis of Rad53 phosphorylation by Western blot. (C) Analysis of DNA content by flow cytometry.

(D) Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb.

(E) Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb.

(F) 2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing.

(G) Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates. ****: p<0.0001, Mann-Whitney rank sum test.
Mrc1 and Rad9 regulate initiation and elongation 
of DNA replication in response to DNA damage (2018)
Figure 4

Figure 4 Rad9 slows down fork progression in response to DSBs.

(A) Cells synchronized in G1 were released into S phase by pronase addition in the presence of 200 mM HU, to activate the DRC. 45 minutes later, 100 μg/ml Zeocin was added to activate the DDC. Still 45 minutes later, HU was washed away, zeocin kept, and different time points taken for analyses.

(B) 2D gel analysis of initiation and elongation in wild type and rad9∆ cells at the early origin ARS719 (probe 1) and at a locus 7 kb away of the origin (probe 2). Time in Zeocin after the HU wash is indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.

(C) 2D gel analysis of fork progression in wild type and rad9∆ cells through a 70-kb region of chromosome VI for which all active origins downstream of ARS607 have been deleted. The position of probes and the time after release from HU are indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.
Mrc1 and Rad9 regulate initiation and elongation 
of DNA replication in response to DNA damage (2018)
Figure 5

Figure 5 Rad9 regulates S-phase progression independently of Mrc1.

(A) Wild type, mrc1∆, mrc1AQ and rad9∆ cells synchronized in G1 with α-factor were incubated in the presence of BrdU and 100 μg/ml Zeocin for 45 minutes prior to release into S phase. Cells were collected at indicated time points.

(B) Analysis of DNA content by flow cytometry.

(C) Analysis of Rad53 phosphorylation in G1 prior to Zeocin addition (G1) and at indicated times. The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.

(D) DNA combing analysis of replication profiles 60 minutes after release into S phase in the presence of Zeocin. Representative DNA fibers are shown. Red: DNA, green: BrdU. The green channel is also shown separately. Bar: 20 kb.

(E) Distribution of BrdU tracks length in wild type, mrc1∆ and mrc1AQ cells. DNA fibers are fully labeled in the case of rad9∆ cells. Median values are indicated in red and correspond to the analysis of at least 150 tracks from two biological replicates. ns: non-significant Mann-Whitney rank sum test.
Mrc1 and Rad9 regulate initiation and elongation 
of DNA replication in response to DNA damage (2018)
Figure 6

Figure 6 Rad9 controls fork progression via Rad53.

(A) Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig. 5A and DNA content was analyzed by flow cytometry.

(B) Analysis of chromosome mobility by Pulse Field Gel Electrophoresis (PFGE) after staining of DNA with ethidium bromide. In G1, the chromosomes are linear and are sorted according to their length. Upon entry into S phase (30 min), they cannot enter the gel due to the presence of replication intermediates. Quantitation of chromosomes re-entering the gel shows completion of DNA replication in 40% of rad9∆, rad53-11 and rad9∆ rad53-11 mutants, but not in wild type cells and mrc1 mutants as determined for a representative experiment. Error bars indicate SD between the signal intensity determined for the three larger chromosomes (arrowheads).

(C) DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin. Representative images are shown. Red: DNA, green: BrdU. Bar is 20 kb.

(D) Length distribution of BrdU tracks measured at that time for wild type, rad9∆, rad53-11 and rad53-11 rad9∆ cells. Median lengths are indicated in red. For each sample, at least 200 tracks were measured from two biological replicates. ****: p < 0.0001. Mann-Whitney rank-sum test.