EMBO/soda-nel huggingface dataset:
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 8

Figure 8. RSK-EphA2-pS897 inhibition specifically sensitizes GPRC5Ahigh HGSC cells to platinum chemotherapy.

A Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm.

B Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient-derived HGSC cells and CAFs.

C GPRC5A in earlier (top) and later passage (bottom) HGSC cells. See normalized GPRC5A relative to OCKI_p01 below the immunoblot images.

D Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots.

E Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3.

F, G Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFs (F; N = 3) and 10 µM BI-D1870 in mesothelial cells (G; N = 3).

H Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot.

I Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.

Data information: In (E-G) data are presented as mean (SD). P-values provided in Appendix Table S10, Student's t-test.
Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer (2020)
Figure 9

Figure 9. High GPRC5A is associated to poor survival and chemotherapy resistance in HGSC patients.

A Flow diagram of patients for the HGSC TMA.

B Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors. For maximum intensity scores an optical 4-point scale (0: negative, 1: mild, 2: moderate, 3: high) was used. For survival analyses a 2-point scale (low: scores 0-2, high: score 3) was used. Scale bars: 200 μm.

C, D Kaplan Meier survival curves illustrate the overall (OS) and progression-free (PFS) survival of patients with high or low GPRC5A. In primary tumors, mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow (C). In patients with metastatic tumors, mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow. Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow (D). No significant difference in mean PFS in primary tumor.

E Correlation of maximum GPRC5A intensity with objective response rate (ORR) and treatment sensitivity (platinum alone or in combination with taxane).

F-I Kaplan Meier survival estimates for the PFS of patients with high or low (top 25% vs bottom 25%) GPRC5A (F), EPHA2 (G), GPRC5A+EPHA2 (H) and GPRC5A+EPHA2+RSK1(RPS6KA1)+RSK2(RPS6KA3) combined (I) mRNA expression. Mean PFS for GPRC5A+EPHA2high was 15 months vs 26 months for GPRC5A+EPHA2low (H). Mean PFS for GPRC5A+EPHA2+RPS6KA1/3 high was 14 months vs 31 months for GPRC5A+EPHA2+RPS6KA1/3low (I).

Data information: In (C-D and F-I) Log Rank test was used. In (E) Pearson Chi-square test was used.

Table 1. Patient information

HGSC originA: O, ovary; FP, fallopian tube. Abbreviations: CCC, clear cell carcinoma; WT, wild type; Mut, mutant; CR, complete response; PR, partial response; PD, progressive disease; NED, no evidence of disease; NA, no available data.

Extended View Figure Legends
BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy (2021)
Figure 1

Figure 1. Clinical Characteristics of P1.

(A) Left quadriceps muscle biopsy performed at age 10 months (i-iii) demonstrating a dystrophic process with variation in fiber size (black arrow), increased connective tissue (blue arrow) and rare internalized nuclei (white arrow) on hematoxylin and eosin (H&E) staining (i) and Gömöritrichrome staining (ii) and type 1 fiber atrophy on nicotinamide adenine dinucleotide (NADH) staining (iii). Muscle biopsy in an unaffected (control) individual (iv-vi). Scale bar: 100μm.

(B) Western blot data (n=1) with quantification demonstrated reduced α-Dystroglycan (α-DG) in P1's muscle compared to control.

(C) Western blot data with quantification demonstrated reduced α-Dystroglycan in fibroblasts from P1 and P2 compared to control (c), although not to the extent of the reduction observed in fibroblasts from a patient with a confirmed α-dystroglycanopathy (α-DG pt) due to biallelic pathogenic variants in LARGE (n=1).

(D) Brain MRI findings in P1 at 28 months of age (top) revealing normal appearing cortex, cerebellum and pons on T1-weighted images and T2-weighted image. There is evidence of mild thinning of the corpus callosum (arrow). Brain MRI in an unaffected (control) individual at 30 months of age (bottom) demonstrating normal sized corpus callosum.
BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy (2021)
Figure 2

Figure 2. RNA sequencing analysis of the BET1 c.202G>C; p.(Asp68His) missense variant.

(A) Schematic depiction of BET1 indicating the position and conservation of the variants identified in this study. TMD = transmembrane domain.

(B) Sashimi plots comparing muscle RNA sequencing reads in P1 (red) and two control muscle samples (blue and green) at the exons 3-5 of BET1.

(C) Schematics of normal BET1 exon 3-4 splicing (top) and the various aberrant splice products associated with the paternally inherited p.(Asp68His) variant in P1. Splice variant one utilizes normal splicing generating a full-length BET1 protein with the Asp68His substitution. Splice variant two skips exon 4 to non-coding transcript (NR_133908) exon 5, resulting in a shift of the reading frame and a premature termination codon after 18 amino acids. Splice variant three utilizes a cryptic splice acceptor within exon 4, deleting the first 34 nucleotides of exon 4, and producing again a frameshift and a premature termination. The maternally inherited frameshift variant c.134delC introduces a premature stop codon in exon 2 and is predicted to induce nonsense mediated decay (NMD).
BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy (2021)
Figure 3

Figure 3. Analysis of BET1 expression and localization in patient fibroblasts and ER-to-Golgi transport in BET1 knockdown HeLa-cells.

(A) Immunoblot of patient fibroblast cell lines (P1 and P2) and three independent controls (C1-3). Immunoblots were probed with the indicated primary antibodies. Quantification of BET1 protein levels normalized to GAPDH. Data represented are mean ± SD of three independent biological experiments. Significant differences of P1 BET1-levels were checked compared to each control (***=p<0.001, one-way-ANOVA followed by Tukey`s post-hoc test).

(B) Subcellular localization of BET1 in control and patient fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of BET1 (red) and cis-Golgi marker GM130 (green). Scale bar: 20µm.Colocalization of BET1 with GM130 was determined using Pearson's correlation coefficient (PCC) measured per cell (right panel; C1: n=63, C2: n=50, C3: n=49 and P2: n=33) (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM.

(C) Representative immunofluorescence images of BET1 (red) and ER marker PDI (green). Scale bar: 20 µm. Right panel shows PCCs for BET1 versus PDI (C1: n=61, C2: n=42, C3: n=59 and P2: n=32 of two biological replicates). (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM.

(D) HeLa-cells stably expressing the pC4-reporter were transfected with control siRNA (ctrl) or two independent siRNAs (#1 and #2) directed against BET1 (siRNA #1 is shown in figure EV 2B). 2d post-transfection the pC4-secretion assay was performed for 0 and 10 min post induction of secretion. Representative images are shown (upper panel ctrl and lower panel #2 siRNA, respectively. Scale bar: 20 µm. Right panels represent magnifications of dashed areas.

(E) Quantification of colocalization using the Pearson`s correlation coefficient (PCC) of the pC4-construct (GFP) and the cis-Golgi marker GM130 (0 min: ctrl n=63, #1 n=56, #2 n=64; 10 min: ctrl n=104, #1 n=126, #2 n=110). Shown are means ± SEM. Colocalization is significantly reduced by BET1 knockdown (siRNA #1 and #2) compared to control transfected cells. Significant differences of PCC (pC4:GM130) after 10 min induction with solubilizer were analyzed with a one-way-ANOVA followed by Dunnett`s post-hoc test. ***=p≤0.001. 0 min: ctrl 0.265±0.016, #1 0.268±0.017, #2 0.264±0.018; 10 min: ctrl 0.798±0.009, #1 0.726±0.011, #2 0.715±0.011.

(F) Quantification of knockdown efficiency of BET1 in HeLa-pC4 cells by immunoblots. Data represented are mean ± SD of three biological replicates. ***=p<0.001 compared to ctrl cells, one-way-ANOVA followed by Dunnett`s post-hoc test).
BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy (2021)
Figure 4

Figure 4. In silico characterization of the SNARE complexes bearing the Asp68His and the Ile51Ser variant.

(A) Representation of the SNARE complex with Asp68 highlighted (top). BET1Asp68 is involved in a hydrogen bond with SEC22bLys169, with the most probable conformation of His68 in the mutant variant (bottom).

(B) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas. Parameter occupancy could be more than 100% if several stable hydrogen bonds could be established by residue. Bars represent average

fraction of the simulated time the residue is involved in a hydrogen bond(s). Data represented are mean ± SD.

(C) Sequence conservation of the corresponding regions of BET1 and SEC22b animal proteins. The relevant residues involved in hydrogen bond network are highlighted by color, hydrogen bonds are shown.

(D) Representation of the SNARE complex with Ile51 (upper) and Ser51 (lower) residues highlighted.

(E) Sequence conservation of the corresponding regions of BET1 and SEC22b and the relevant residues involved in the hydrophobic patch formed by Ile51 BET and SEC22bI148.
Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord (2021)
Figure 1

Figure 1. Integrated snRNA-seq data reveals transcriptomic features and changes in the proportions of neural cells in early and mid-gestation human spinal cord. (A) A schematic diagram of the single-cell sequencing process. (B) Visualization of major classes of spinal cord cells by integrated UMAP plots of cell clusters. Cell types are labelled and circled with dotted lines (top). Violin plots show the most distinct and commonly used marker genes for each cell cluster (bottom). Cell-type annotations are listed on the right and shown in varying colours. (C) Three-dimensional visualization of neural subtype developmental trajectories for individual samples from GW7, GW10, GW14, GW18, and GW23. Stage-dependent differentiation trends changed with development. The red arrow indicates the proliferative neural progenitor cell cluster (MKI67, PAX6, NES, and SOX2). Neural cell types are named and circled with dotted lines. Gray represents non-neural cell types (e.g., vascular and meningeal). (D) Immunofluorescence staining of the spinal cord at GW7, GW8, GW10, GW11, and GW23 for RBFOX3 (red), OLIG2 (green), and GFAP (white). The appearance of astrocytes and oligodendrocytes was observed from GW8 onward. Scale bars: 100 μm.
Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord (2021)
Figure 2

Figure 2. Integrated trajectory and expression feature analysis of neuronal cell developmental trajectory in GW7, GW8, GW10, and GW11 samples. (A) UMAP plot of cells integrated from datasets of GW7, GW8, GW10, and GW11 samples (GW7S-NC, GW8S-02NC, GW10C-01NC, GW10T-01NC, GW10L-01NC, GW10C-02NC, GW10T-02NC, GW10L-02NC, GW11C-NC, GW11T-NC, GW11L-NC) showing neuronal differentiation tendency. Cell types are labelled (left). Heatmap showing differentially expressed genes (DEGs) in neuronal restricted progenitors (NRP) and immature dorsal neurons (IDN) (right). (B) Visualization of selected top gene ontology (GO) enrichment terms for NRP and IDN clusters based on DEG analysis. (C) Immunofluorescence staining of the spinal cord from GW7, GW8, and GW10 for a dorsal marker (PAX7, green), neuronal precursor marker (ASCL1, red), and nuclei marker (DAPI, blue). Images show the neuronal differentiation process on the dorsal side of the spinal cord. Scale bar: 100 μm. (D) Stacked proportional changes of neural progenitor cells (NPC) and neuronal cell classes from GW7-GW11. Cell types were identified as: proliferative neural progenitors (Pro-NPC), glial restricted progenitors (GRP), IDN, DI4 interneurons (DI4), DI5 interneurons (DI5), motor neuron (MN), DI1 and DI2 interneurons (DI1/2), DI3 interneurons (DI3), DI6 interneurons (DI6), and V0 and V1 interneurons (V0/1). (E) Three-dimensional developmental trajectories for snRNA-seq samples from the GW10 cervical sample (top view in the upper right corner). Cells are classified by differentiation stage according to expression of specific markers (neuronal progenitor: PAX3 and PAX7; DI5 interneuron: POU4F1 and TLX3; V3 interneuron: SIM1; and motor neuron: MNX1). Cells in the neuronal development trajectory are coloured by gene expression. Distances from progenitor cells to differentiated neurons represents the differing maturation status between dorsal neurons (DI4, DI5) and ventral neurons (MN, V3). Cells with no detectable expression for a given gene were omitted from the plot.
Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord (2021)
Figure 3

Figure 3. Neural cell type integration analysis and characterization. (A) UMAP plot of snRNA-seq data from 64,381 combined neural cells from GW7 to GW23. Cell cluster phenotypes are labelled and the red arrow indicates the EGFR-expressing glial cluster (left). Selected UMAP plots from different development stages (GW7, GW11, GW18, and GW23) represent the neural development trend (right). Gray represents non-specific cell types. (B) The stacked proportions of neural cell classes showing changes in stage-dependent differentiation trends with developmental stage. (C) Violin plot showing the expression of selected genes. EGFR+ Cluster 14 is circled with a red dotted line. (D) Visualization of selected top gene ontology (GO) enrichment terms of astrocyte clusters based on DEG analysis.
Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord (2021)
Figure 4

Figure 4. Characterization of GW7 progenitor cell populations and EGFR-expressing transitional glial cells. (A) UMAP plot of Cluster 0 and Cluster 14 populations from the GW7 sample. (B) Heatmap of differentially expressed genes (DEGs) in Clusters 0 and 14 at GW7. On the right of the heatmap are the top-twelve DEGs. Selected top gene ontology (GO) terms related to the corresponding DEGs (right). (C) Single-cell resolution trajectory of EGFR+ transitional glial cells (Cluster 14) was constructed from sample (GW11T-NC, GW14T-NC, GW18T-NC, GW20T-NC, GW21T-NC) during the transitional period using Monocle. Linear arrangement of the transition trajectory with developmental stages represents the transition process of a regulatory cell type. The arrow indicates the developmental trend. (D) Violin plots produced by VlnPlot function of Seurat package representing changes in expression of EGFR, DLL1, MIAT, and FAM189A2 in cluster 14 with development based on snRNA counts data. (E) Overview of selected statistically significant interactions between neural cell types according to cell-cell communication analysis from statistical test of CellPhoneDB v.2.0. Size indicates P value and colour indicates the means of the average expression level of the receptor-ligand pairs. The EGFR+ glial cell type exhibited the most prominent interactions with Delta-Notch and EGFR signalling within the network.
Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord (2021)
Figure 5

Figure 5. Expression profile of EGFR-expressing glia during spinal cord development. (A) Schematic diagram showing the spatial and temporal dynamics of EGFR-expressing cells during spinal cord development. The solid green line represents EGFR-expressing radial glial cells, while the green dotted line represents EGFR-expressing transitional glial cells. The blue line represents the gray matter boundary. (B) Immunofluorescence staining of spinal cord sections at GW7, GW11, GW17, and GW23 for EGFR (green) and nuclei (DAPI, blue). EGFR expression on the ventral side was observed in GW7 and GW11 spinal cord. EGFR expression on the dorsal side was observed in GW11, GW17, and GW23 spinal cord. Dorsal EGFR expression was mainly concentrated within the dorsal horn area. Scale bar: 100 μm. (Top) Higher-magnification view of boxes at bottom. White dashed lines indicate dorsal horn borders. (C) UMAP plot of GW11 sample. Dot plot shows the expression of featured genes in the EGFR-expressing progenitor cluster mapped on the UMAP plot. Expression of PAX3 and OLIG2 display a complementary pattern, while PDGFRA was expressed at a low level within the EGFR+ glial cluster. (D) Representative immunofluorescence staining of a GW11 spinal cord section. Most dorsal EGFR-expressing cells at GW11 co-expressed GFAP and SOX9, but not OLIG2. Scale bars: 100 μm.
Single-cell analysis reveals dynamic changes of neural cells in developing human spinal cord (2021)
Figure 6

Figure 6. Comparison of EGFR-expressing cell features between GW11 and GW17 spinal cord. GW11 EGFR-expressing cells exhibited radial glia morphology and most co-expressed SOX9 (A) but not OLIG2 (B). However, EGFR co-expression with SOX9 or OLIG2 was found in GW17 EGFR-expressing cells (C, D). White dashed lines marked dorsal horn borders. Higher-magnification view of boxes on left. Scale bars: 100 μm.

Expanded View Figure Legends
STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons (2022)
Figure 1

Figure 1: DRG and SCG neurons express novel mechanisms to detect noxious cold.

(A,B) Representative images of changes in intracellular calcium, as measured in fluorescence ratio images (F340/380) (A) and traces (B). Examples show a TRPM8-expressing DRG neuron (left in A, top in B) responding to both the TRPM8 agonist menthol (300 μM) and to a cold stimulus from 32°C to 4°C (temperature trace bottom in B),and a neuron that responds to cold (right in A, middle in B) but does not express any of TRPM8, TRPA1 or TRPC5, as shown by lack of response to the TRPM8 agonist menthol (300 μM), the TRPA1 agonist AITC (50 μM) or the TRPC5 agonist rosiglitazone (100 μM). Calcium increase is due to influx from extracellular solution (see Fig. 2A, B). (C) Venn diagram showing the proportion of neurons that responded to at least one of the three TRP agonists menthol, AITC or rosiglitazone (grey, 44%) and those that responded to cold (blue, 14%). Neurons that responded to cold, but not to any TRP agonist (5%) were classified as "novel cold-sensitive neurons" (n=859 neurons imaged on 3 separate days). (D) Frequency histogram showing cold activation thresholds of novel cold-sensitive DRG neurons (left, mean cold threshold 15.1±1.0°C, n=40 neurons) and TRPM8+ neurons (right, mean cold threshold 23.3±2.1°C, n=15 neurons). (E,F) Similar experiments to (A) and (B) showing an SCG neuron responding to the TRPA1 agonist AITC (left in E and top in F) and a novel cold-sensitive SCG neuron that responds to cold but does not express any of TRPM8, TRPA1 or TRPC5 (right in E and middle in F). Many glial cells are also visibly activated by AITC but not by cold. (G) Similar Venn diagram to C for SCG neurons (n=155 neurons). Few responded to any of the TRP agonists (grey, 6%), while many neurons responded to cold (blue, 44%), of which most responded only to cold (39% "novel cold-sensitive neurons"). (H) Similar histogram to (D) showing cold activation thresholds of novel cold-sensitive SCG neurons (mean cold threshold 13.1±0.8°C, n=61 neurons). See EV1 for further data on distribution of cold-sensitivity in neurons of the DRG and SCG.
STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons (2022)
Figure 3

Figure 3: Dissection of mechanisms underlying the novel cold-induced Ca2+entry.

(A) Example of concurrent electrophysiological and Ca2+ imaging of a cold-sensitive SCG neuron in which the calcium channel blocker verapamil enhances the cold response under voltage clamp at -60mV. Larger increase in [Ca]i seen in current-clamp mode (4th cold ramp). Similar results obtained in 4 SCG neurons. (B) Example of cold-sensitive SCG neuron in which the calcium channel blocker verapamil inhibits cold response under current clamp. Smaller cold response seen under voltage-clamp (4th cold ramp). Similar results obtained in 5 SCG neurons. (C) Mean cold-sensitive increase in calcium-dependent fluorescence ratio F340/380 in SCG neurons is significantly smaller in neurons recorded in voltage-clamp at -60mV (0.56 ±0.08, mean ± SEM, n=21 neurons) than in neurons recorded in current-clamp mode (1.16 ±0.29, n=11 neurons, p=0.0165, unpaired t-test). (D) Frequency distribution showing the effect of verapamil (100μM, blue bars) on cold activation thresholds of SCG neurons. Mean cold threshold before (10.8±0.2°C) and after (9.4±0.1°C) addition of verapamil, n=301 neurons, p<0.0001. (E) Representative calcium imaging trace showing block of cold responses with ORAI channel antagonist YM58483 (3μM) in SCG neuron in the presence of CaV antagonist verapamil (100μM). (F) Summary of results of experiments on SCG neurons (mean ±SEM, n=115 neurons, 4 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test), with only partial recovery. Temperature trace below. (G) In neurons with a small response to cold (F340/380 increase <0.2; n=24 neurons), many responses are potentiated by verapamil (31% of cold-insensitive neurons) and are fully blocked by ORAI channel antagonist YM58483 (p<0.0001, RM one-way ANOVA + Dunnett's test). (H) Similar histogram to (D) showing the effect of verapamil (100μM, blue bars) on cold activation thresholds of novel cold-sensitive DRG neurons. Mean cold threshold without verapamil (15.1±1.0°C, black bars, n=40), and in the presence of verapamil (10.1±1.6°C, blue bars, n=26, p=0.0080). (I) Similar experiment to that shown in (E) but in DRG neurons. Block trace shows DRG neuron responding with a calcium increase to menthol and thus expressing TRPM8; cold-dependent calcium increase is partially blocked by verapamil (100μM) but not by ORAI blocker YM58483. Blue trace shows neuron not responding to either menthol or AITC but activated by cold (novel cold-sensitive neuron) in which response is blocked by ORAI channel antagonist YM58483. Temperature trace below. (J) Summary of results of experiments on novel cold-sensitive DRG neurons (mean ±SEM, n=28 neurons, 3 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test).