EMBO/soda-nel huggingface dataset:
Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy (2016)
Figure 3

Figure 3. Shh inhibits glutamate transporter activities and increases the extracellular glutamate levels.

(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6. (B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals. Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10) or transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1. Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18). (G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9. (H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student’s t test.
Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy (2016)
Figure 4

Figure 4. Inhibiting Shh pathway suppresses epileptogenesis in mouse epilepsy models.

Effects of Cyclo (A-C, n=14), 5E1 (D-F, n=16-20), or ablation of Smo in CaMKIIα-positive neurons (G-I), or ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice. In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20. In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20. (M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23. (N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student’s t test. (O) Schematic diagram depicting a working model for Shh-regulation of epileptogenesis. Epileptic activity triggers sequential responses, including Shh release, inhibition of glutamate transporter activity, and increase in extracellular glutamate, leading to epilepsy development.
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance (2021)
Figure 2

Figure 2. Genome-wide CRISPR Activation Screen Identifies BRAFi-Resistance Genes in Cutaneous Melanoma

(A) CRISPR-SAM workflow. Cell library was exposed to DMSO (solvent of BRAFi) or BRAFi 2 µM (vemurafenib (Vem) or PLX8394 (PB, Paradox Breaker)) during 14 days. 40x106 cells per arm. Experiments were done in duplicate.

(B) Plot showing sgRNAs detected in BRAFi-resistant cells versus in control cells (cell library exposed to DMSO) with a fold change >1.5. Raw data are available in Table EV5. (BRAFi for PBV). Appendix Figure S1 depicted the comparison Vem vs PB.

(C) sgRNAs counts in BRAFi-resistant cells versus in DMSO-exposed cells for EGFR and BIRC3. (BRAFi for PBV, Appendix Figure S1)

(D) Determination of differentiation states of 12 human BRAF(V600) melanoma cell lines from CCLE according to (Tsoi et al, 2018).

(E) BRAF(V600) melanoma cell lines from CCLE were distributed in two groups (Sensitive and "intrinsically" Resistant, n=6 cell lines per group) according to their BRAFi IC50 (µM, half maximal inhibitory concentrations) (Barretina et al, 2012). Error bars reflect mean + s.d..

(F) Volcano plot showing the expression levels of BRAFi-resistant genes (identified in 501Mel by CRISPR-SAM screen) in 12 human melanoma cell lines. The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines. In red: selected genes. EGFR is considered as positive control and SMAD3, BIRC3 and SLC9A5; selected as favorite genes for the next steps. SMAD3, BIRC3, SLC9A5 and AFAP1 are BRAFi-resistance genes and potent tumor-promoting genes (Fig. 1). Raw data are available in Table EV5. (BRAFi for PBV, Appendix Figure S1)

(G) Heatmap recapitulating the expression levels of the selected hits (red dots in Fig. 2F) in BRAFi-resistant and -sensitive cell lines. Markers of differentiation (MITF, MLANA, OCA2, TYRP1, TYR). In red: resistance genes already published (NRP1, AXL, ZEB1, LPAR1). Scale corresponds to Z scores.
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance (2021)
Figure 4

Figure 4. BRAFi-Resistance Genes Promote Tumor Growth

(A) Workflow to identify genes involved in tumor growth from BRAFi-persister cells. Cell populations were subcutaneously grafted on nude mice flanks (3x106 cells per mouse); Vem-persister cells (n=11 mice) and PLX8394 (PB)-persister cells (n=12 mice) (Tables EV2). (BRAFi for PBV, Appendix Figure S1)

(B) Tumor growth curves from BRAFi-persister cells (monitored during 5 months) (Table EV2). V for Vem-resistant cells and PB for PLX8394-resistant cells.

(C) Distribution of sgRNAs in BRAFi-resistant cells (before xenograft) and in the 7 tumors emerging from the BRAFi-resistant cells (log10(sgRNAs counts)). Blue and red lines indicated the enrichment ≥10 fold or ≥100 fold (tumors versus BRAFi-resistant cells (in vitro)). Raw data are available in Table EV7.

(D) From the seven tumors arising from BRAFi-resistant cells, the common genes (enriched) have been extracted. Ninety-seven genes including SMAD3, BIRC3 and SLC9A5 are detected in all these 7 tumors. Raw data are available in Table EV7.

(E) sgRNAs counts in tumors versus sgRNA detected in BRAFi-resistant cell library (in vitro) (respectively, black and orange points) for selected candidates. Each black point corresponds to one tumor. EGFR was the most potent BRAFi-resistant gene (Fig.2). Two replicates have been shown for CRISPR-SAM cell library (orange points). SMAD3, BIRC3 and SLC9A5 were identified as hits in the three screens (Fig.1, 2 and 4).
CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance (2021)
Figure 6

Figure 6. The Transcription Factor AhR Drives SMAD3 Expression

(A) AhR binding sites (xenobiotic responsive element (XRE); GCGTG) in human SMAD3 proximal promoter (-500_+1 bp).

(B) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.; Multiple comparisons have been done using ordinary one-way ANOVA, ***: p<0.001.

(C) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction.

501Mel cells have been treated as described in B. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.; Multiple comparisons have been done using ordinary one-way ANOVA, **: p<0.01, ***: p<0.001.

(D) AhR antagonist (CH-223191) reduces SMAD3 and TIPARP expression levels. SKMel28 cells (AhR wild-type) have been exposed to CH-223191 (5µM) or the solvent (DMSO) during 7 days. n=6 biologically independent experiments. Each histogram represents the mean + s.d.; Bilateral Student test (with non-equivalent variances): ***: p<0.001.

(E) Loss of AhR reduces SMAD3 expression levels. SMAD3 expression has been investigated in SKMel28 cells AhR wild-type (WT) or knock-out (KO). SKMel28R, have been obtained from SKMel28S by chronic exposure to non-lethal doses of BRAFi (Hugo et al, 2015b). R for BRAFi-resistant SKMel28 cells and S for sensitive. n=2 biologically independent experiments. Each histogram represents the mean + s.d..
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment (2016)
Figure 1

Figure 1. Loss of Eed affects H3K27 methylation and reduces proliferating cells in the intestinal epithelium.

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.

A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.

B) Western blot analysis with protein extracts obtained from ex vivo–purified intestinal crypts and probed with the indicated antibodies.

C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.

D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.

E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.

All scale bars represent 100μm.
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment (2016)
Figure 2

Figure 2. Loss of Eed perturbs cell differentiation in the intestinal epithelium.

A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.

C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.

D) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell–specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).

E) GFP expression from an Lgr5-eGFP transgene identifying LGR5+ ISCs in near-native agarose-embedded sections from Eed+/+ and Eed-/- mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.

All scale bars represent 100μm.
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment (2016)
Figure 3

Figure 3. Loss of Eed affects cell plasticity at the bottom of the crypt.

A) Radiation-induced intestinal regeneration: AhCre Eed+/+ and AhCre Eedfl/fl mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).

B) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post-irradiation.

C) Quantification (mean ± SD) of KI67 positive cells per crypt (B). More than 100 crypts were scored for each condition.

D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).

All scale bars represent 100μm.
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment (2016)
Figure 4

Figure 4. Loss of Eed induces transcriptional activation of genes involved in cell cycle arrest and secretory lineage specification.

A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.

B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in Eed-/- crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.

C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in Eed-/- crypts.

D) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.

E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.

F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.

G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment (2016)
Figure 5

Figure 5. Loss of Cdkn2a restores cell proliferation in Eed-/- intestinal crypts.

A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).

B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.

C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.

D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemical analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.

All scale bars represent 100μm.
PRC2 Preserves Intestinal Progenitors and Restricts Secretory Lineage Commitment (2016)
Figure 6

Figure 6. PRC2 directly maintains Atoh1 and Gfi1 repression

A) Expression analysis by qRT-PCR (mean ± SD) of the indicated intestinal epithelial cell markers regulated by the Wnt and Notch signaling pathways that are involved in stem cell homeostasis, regionalization, and Goblet cell differentiation (n=3).

B) Genomic snapshots of the indicated genomic loci for SUZ12 occupancy and H3K27me3 deposition in intestinal crypts of wild type mice (n=3).

C) Expression analysis by qRT-PCR (mean ± SD) of the Goblet cell markers (Atoh1, Gfi1, Spdef), early secretory precursor cells (Dll1), NOTCH targets (Hes1, Olfm4) and intestinal stem cells (Lgr5, Rnf43) in wild type intestinal crypts purified 38hrs after treatment with the NOTCH inhibitor DBZ. DMSO served as vehicle negative control (n=4).

D) ChIP analysis of Atoh1 and Gfi1 loci using H3K27me3 specific antibody. The same crypts described in C were used. H3K27me3 levels were normalized on Histone H3 density (percentage of H3 signal). Graphs show mean ± SD.

E) Graphic model highlighting the role of PRC2 in restricting secretory lineage differentiation and cell cycle checkpoint activation in TA cells.
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA (2018)
Figure 1

Figure 1. Formation of condensates of FtsZ·SlmA·SBS in crowding conditions.

Scheme of the E. coli division elements involved in the formation of condensates. The concentrations of FtsZ, SlmA and SBS used in this study were 12, 5 and 1 μM, unless indicated otherwise.

Representative confocal images of the FtsZ·SlmA·SBS condensates formed in 150 g/L dextran 500 or Ficoll 70, and absence of condensates in dilute solution. Scale bars: 20 μm, except for images at higher magnification in dextran (5 μm, second row).

Turbidity of FtsZ·SlmA·SBS in buffer (n = 3), in 150 g/L dextran 500 (n = 5) or Ficoll 70 (n = 3) and in 50 g/L PEG 8 (n = 3). Data correspond to the average ± S.D.
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA (2018)
Figure 2

Figure 2. Dependence of the formation of FtsZ·SlmA·SBS condensates on protein and salt concentrations.

Formation of condensates as a function of FtsZ and SlmA·SBS concentration, as measured by turbidity, in working buffer (300 mM KCl). SlmA concentration was fivefold that of SBS (except at 0.5 μM SBS, where SlmA concentration was 3 μM). Data are the average of 2 independent measurements. Errors (S.D.), symmetrical, are depicted as white discs.

Representative confocal images of the FtsZ·SlmA·SBS condensates at the specified salt concentrations. The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 µM, respectively. Scale bars: 5 μm.

Data information: All measurements in 150 g/L dextran 500.
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA (2018)
Figure 3

Figure 3. Dynamism of FtsZ·SlmA·SBS condensates.

Representative confocal images showing final state after addition of FtsZ-Alexa 488 to FtsZ·SlmA·SBS complexes (FtsZ labeled with Alexa 647) in 150 g/L dextran. Below, images showing the stepwise diffusion of FtsZ-Alexa 488 into the condensates containing FtsZ-Alexa 647 at the indicated times in seconds (time zero, beginning of visualization for that particular condensate) and corresponding intensity profiles at selected times in the green channel. The profile in the red channel is shown as a reference and varies slightly among images. Scale bars: 5 μm (top row) and 4 μm (bottom row).

Stepwise diffusion of FtsZ-Alexa 488 into FtsZ·SlmA·SBS condensates (FtsZ labeled with Alexa 647) at the indicated times in seconds (time zero, beginning of visualization for those particular condensates) in 100 g/L PEG. Scale bars: 4 μm.

Assembly of FtsZ fibers upon GTP addition (0.5 mM) to FtsZ·SlmA·SBS condensates and condensates formed after FtsZ fibers disassembly at the indicated times in minutes (time zero, GTP addition) in 150 g/L dextran. Scale bars: 5 μm. Scheme of the dynamic process on the right. The number of condensates decreases upon fibers formation, and they rearrange upon GTP depletion and fibers disassembly.
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA (2018)
Figure 4

Figure 4. Microfluidic encapsulation of FtsZ·SlmA·SBS in the PEG/dextran LLPS system inside microdroplets stabilized by the E. coli lipid mixture.

Scheme of the encapsulation setup and illustration, on the right, of the distribution of species within the encapsulated LLPS system.

B-D. Representative confocal images of the microdroplets without (B) and with GTP (C and D). The concentrations of FtsZ, SlmA and SBS were 12, 5 and 1 μM, respectively (B and C) or 6, 3 and 0.5 μM respectively (D). 1 mM (C) or 2 mM GTP (D). Scale bars: 40 μm except in images on the far right (20 μm), which are either a magnification of the indicated region in the merged image (B) or independent images at higher magnification (C, D).
Bacterial FtsZ protein forms phase-separated condensates with its nucleoid-associated inhibitor SlmA (2018)
Figure 5

Figure 5. Microfluidic encapsulation of FtsZ·SlmA·SBS in the PEG/DNA LLPS system inside microdroplets and GTP induced FtsZ fiber formation.

Scheme of the encapsulation procedure followed for the PEG/DNA LLPS system and illustration, on the right, of the distribution of species within the encapsulated LLPS system.

B,C. Representative confocal images of the microdroplets stabilized by the E. coli lipid mixture containing the biphasic PEG/DNA mixture and the FtsZ·SlmA·SBS complex without and with 2 mM GTP, respectively. Last image in (B) focuses in the lipid interface to show the high density of condensates. Scale bars: 20 μm except in images on the far right (10 μm), which are an independent image at higher magnification (B) or a magnification of the indicated region in the merged image (C).
Extended synaptotagmin regulates contact site structure and lipid transfer function in vivo (2020)
Figure 1

Figure 1: Single gene encoding Esyt expressed in Drosophila melanogaster.

(A) Schematic showing the phylogeny of extended synaptotagmin in different organisms: Drosophila melanogaster (Dm), Saccharomyces cerevesiae (Tricalbin), Homo sapiens (Hs), Mus Musculus (Mm), Rattus norvegicus (Rn), Caenorhabditis elegans (Ce), Danio rerio (Dr) and Xenopus tropicalis (Xt). Bold lines - Sub nodes. Numbers on the nodes - Bootstrap value. Colours - Esyt1, Esyt2 and Esyt3 clusters.

(B) Schematic of dEsyt-PA protein domain structure aligned with mammalian Esyts and yeast tricalbins (Image generated using IBS, Illustrator for Biological sciences Version 1.0 software http://ibs.biocuckoo.org/)(Liu et al., 2015). Amino acid numbers in each protein are indicated. SMP-SMP domain. C2A, B, C, D, E are the various C2 domains.

(C) Quantitative real time PCR analysis showing head enrichment of dEsyt; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).

(D) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD. Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).

Data information: Bar graphs with mean ± SD are shown. Statistical tests: (C and D) Students unpaired t test (two tailed). ns - Not significant. ****p value <0.0001.