EMBO/soda-nel huggingface dataset:
Differentiation-related epigenomic changes define clinically distinct keratinocyte cancer subclasses (2022)
Figure 1

Figure 1. Single-cell multi-omics characterization of human epidermal differentiation.

A Joint UMAP plot depicting both scATAC-seq and scRNA-seq data from 5,355 keratinocytes from sun-protected human epidermis (n=2). Color depicts the unsupervised clustering (left) as well as cell type annotation based on the reference scRNA-seq dataset (right).

B Representative examples of chromatin accessibility and gene expression of KRT5 and KRT10, two key epidermal differentiation-related genes, in the main keratinocyte populations.

C Co-accessibility at the human epidermal differentiation complex (EDC) in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Only connections (arcs) with a co-accessibility score above 0.25 are plotted. Grey boxes below tracks represent scATAC-seq peaks.

D Left: Heatmap displaying the differentially accessible peaks between undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Number of Tn5 insertion sites in each region were scaled by row. Right: DNA sequence motifs for the top six overrepresented transcription factor (TF) motifs in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocyte-specific accessible peaks.

E Heatmaps displaying the predicted top five transcription factors in each cell cluster using chromVAR motif activity (left) and gene expression (right).

F chromVAR deviations (in quantiles) and gene expression for representative enriched TF projected onto the joint UMAP plot of keratinocytes from the single-cell multi-omics dataset.

G Heatmap showing the top five active regulons in each cell cluster, based on the expression data of the multi-omics dataset. TF regulons also identified by other approaches are highlighted.
Differentiation-related epigenomic changes define clinically distinct keratinocyte cancer subclasses (2022)
Figure 4

Figure 4. AK/cSCC cell-of-origin-based subclasses present distinct mitotic ages and EMT-related methylation features.

A Boxplots representing the total number of cumulative stem cell divisions per sample (mitotic age) as calculated with epiTOC2 in each AK/cSCC cell-of-origin-related subclass.

B Boxplots representing the stem cell division rate per stem cell and year in each entity and cell-of-origin-based subclass.

C Upper: Heatmaps displaying the unsupervised clustering of 67 AK/cSCC and healthy controls based on the methylation patterns of the MIR200C/141 cluster and the MIR205 gene. Lower: Bar plots showing the quantification of the methylation values at CpGs located at promoter regions in each cell-of-origin-based subclass. For the MIR200C/141 cluster, probes located at the regulatory CpG island are depicted with green arrows. For the MIR205 gene, all probes shown in the heatmap are part of the promoter region and were used for quantification.

D Left: Representative images of EpSC-like (n=5) and Keratinocyte-like (n=5) cSCC tumors stained for the EMT-marker ZEB2 (green) and the keratinocyte marker TP63 (red). Nuclei were counterstained with DAPI. Images are shown at 40x original magnification. Scale bar, 50 µm.

Right: Quantification of ZEB2-positive tumor cells (TP63-positive) in three independent regions per sample. Each dot represents a region with at least 500 tumor cells (TP63-positive) counted.

Data information: In Boxplots in A-B, the central bar, boxes and whiskers represent the median, first and third quartiles, and 1.5-time interquartile range (IQR), respectively. Barplots in C represent the mean and error bars represent the standard error of the mean (SEM). Statistical analyses in A-C were performed using a Wilcoxon Rank Sum test, comparing each subclass (EpSC-like: n= 22; keratinocyte-like: n= 33) to healthy samples (n= 12), or between cell-of-origin-related subclasses (depicted by a line). Statistical analysis in D was performed using an unpaired two-sided t-test (EpSC-like: n= 5 samples, 15 technical replicates; keratinocyte-like: n= 5 samples, 15 technical replicates). Central bar represents the mean and error bars represent the standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001. AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma.
Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 1

Figure 1. Trafficking Phosphoproteomics Reveals FGFR2b Recycling-dependent Outputs

A. Overview of the Trafficking Phosphoproteomics Approaches (TPAs).

B. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.

C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test).

D. Confluence over time of BT20, T47D, and BT549 transfected with HA-FGFR2b stimulated with FGF7 (dark green) or FGF10 (burgundy). Data represent the mean ± SD of N=3 compared to FGF10. P value = <0.05 *, <0.01 **, <0.001*** (Student's t-test).

E. Hierarchical clustering of the phosphorylated sites differentially quantified T47D stimulated with FGF7 (green) or FGF10 (burgundy). Specific clusters are highlighted with black lines. The intensity of phosphorylated sites is presented on the logarithmic scale with intensity below and above the mean color-coded in blue and red, respectively.

F. Enriched terms in the selected clusters of E.

G. Network of phosphorylated proteins belonging to "protein localization" in FGF10 clusters, based on STRING, visualized in Cytoscape, and color-coded based on cell components. The squared shape represents phosphorylated proteins found in the database DISEASES. The recycling adaptors TTP and RCP are highlighted in grey.

H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.

I. The presence (total), internalization (internalized), and recycling (cell surface) of transfected HA-FGFR2b in BT549 upon stimulation were quantified as in (Francavilla et al., 2016) and in the section "Quantification of the Recycling assay". Values represent the median ± SD of N=3. Representative pictures upon FGF10 stimulation are shown in H.
Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 2

Figure 2. TPA2-3 Unveil FGF10-dependent EGFR_T693 Phosphorylation

A. Experimental design of TPA2.

B. Hierarchical clustering of the phosphorylated sites differentially quantified in BT549 transfected with HA-FGFR1c, 2b, or 4 and stimulated for 8 or 40 min. with Enkamin-E, FGF10, or FGF1, respectively. Early Signalling and Recycling Receptors clusters are highlighted in light and medium blue, respectively. The intensity of phosphorylated sites is presented on the logarithmic scale with intensity below and above the mean color-coded in blue and red, respectively.

C. STRING-based and Cytoscape-visualized network of the phosphorylated proteins belonging to "endocytosis" (clusters medium blue in b). The diamond shape represents phosphorylated proteins found in TPA1. Kinases are highlighted in burgundy. The border is color-coded based on the substrate motifs.

D. Experimental design of TPA3.

E. Hierarchical clustering of the phosphorylated sites differentially quantified in T47D stimulated with FGF10 and depleted or not of TTP or RCP. The clusters for TTP Adaptors, RCP Adaptors or Recycling Adaptors are highlighted in orange, brown, and light green, respectively. The intensity of phosphorylated sites is presented on the logarithmic scale with intensity below and above the mean color-coded in blue and red, respectively.

F. STRING-based and Cytoscape-visualized network of phosphorylated proteins belonging to the Recycling Adaptors cluster (light green in E) and found in TPA1. Kinases are highlighted in burgundy. The border is color-coded based on the substrate motifs.

G. Venn diagram showing the phosphorylated proteins belonging to "establishment of localization" identified in TPA1-3 (left). STRING-based and Cytoscape-visualized network of the 22 proteins identified by the 3 TPAs (centre). Phosphorylated sites quantified on EGFR (right). T693 is highlighted in yellow.

H-J. Lysates from (H) T47D, BT20, and HA-FGFR2b-transfected BT549 stimulated or not with FGF7, FGF10, EGF, and TGFα for 40 min.; (I) breast cancer organoid cultured from the PDX tumour BB6RC37 and grown for the last 24 hours as indicated; (J) control or 40 min. FGF10- or TGFα-stimulated T47D left untreated or depleted of TTP, followed or not by transfection with siRNA resistant Flag-TTP (Flag-TTP*) or depleted of RCP followed or not by transfection with siRNA resistant RCP-GFP (RCP-GFP*) were immunoblotted with the indicated antibodies. **, non-specific band (J).
Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 3

Figure 3. FGF10-dependent EGFR_T693 Phosphorylation Requires FGFR and ERK

A. List of compounds and their targets.

B-D Lysates from BT549 transfected with HA-FGFR1c, 2b, or 4 and stimulated for 8 or 40 min. with Enkamin-E, FGF10, or FGF1, respectively, followed by treatment with either DMSO or the FGFR inhibitor PD173074 (B); BT549 transfected with HA-FGFR2b or HA-FGFR2b_Y656F/Y657F and stimulated with FGF10 for 40 min. (C); T47D treated with DMSO, PD173074 or the EGFR inhibitor AG1478 and stimulated or not with either FGF10 or TGFα for 0, 8, and 40 min. (D) were immunoblotted with the indicated antibodies.

E. Protein sequences surrounding T693 (based on Uniprot P00533) of members of the ErbB family were aligned using CLUSTAL O (version 1.2.4). The red box indicates that the amino acid T is followed by a conserved proline. Asterisks below the sequences indicate identical amino acid residues; double dots indicate conserved amino acid residues; single dots indicate semi-conserved substitutions.

F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.
Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 4

Figure 4. FGF10 Primes EGFR Responses.

A-E Lysates from T47D stimulated or not with FGF10, EGF or TGFα for different time periods (A); pre-treated or not with FGF10 for 40 min. and either stimulated or not with FGF10, EGF or TGFα for different time periods (B) or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.

F. Heatmap of the mRNA relative expression of the indicated ERK targets compared to control and quantified by qPCR. N=3. The minimum and maximum fold-induction is color-coded in white and burgundy, respectively.

G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).
Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 6

Figure 6. EGFR_T693 Phosphorylation Controls CDK1 Phosphorylation

A. Experimental design of MS-based quantitative phosphoproteomics analysis of wt- and T693A-expressing T47D cells stimulated with FGF10 for 40 or 60 min.

B. Hierarchical clustering of the phosphorylated sites differentially quantified in wt- and T693A-expressing T47D stimulated or not with FGF10 for 40 or 60 min.. Four clusters for plasma membrane Response (PMR), Acquired Response (AR), Late Response (LR), and T693 Phosphorylation Dependent Response (PDR) are highlighted with black lines and color-coded on the right with blue, medium, or dark blue and yellow, respectively. The intensity of phosphorylated sites is presented on the logarithmic scale with intensity below and above the mean color-coded in blue and red, respectively.

C. KEGG pathways enriched in each cluster.

D. STRING-based and Cytoscape-visualized network of phosphorylated kinases color-coded based on clusters shown in Figure 6C. The border of known regulatory sites is coloured in burgundy. The shape depends on the known function of the regulatory site.

E, F Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.
Reciprocal Priming between Receptor Tyrosine Kinases at Recycling Endosomes Orchestrates Cellular Signalling Outputs (2021)
Figure 7

Figure 7. FGF10-induced Cell Cycle Progression Depends on EGFR_T693 Phosphorylation

A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).

E. Relative invasion of wt- or T693A-transfected BT20 cells into fibronectin-supplemented collagen I was quantified as described in Material and Methods. Graph depicts mean ± S.E.M. of N=8. *P value=<0.05 (Student's t-test).

F. Representative images of E. Black indicates cells. Confocal depth is indicated between panels. Scale bar, 250 μm.

G. Model of reciprocal priming between FGFR and EGFR, based on this study.
A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC (2021)
Figure 1

Fig. 1 XMU-MP-5 is a potent and selective ALK inhibitor.

A Chemical structure of XMU-MP-5.

B Antiproliferative activities of XMU-MP-5 and crizotinib against EML4-ALK Ba/F3 and parental Ba/F3 cells, respectively. Cells were treated with increasing concentration of each drug for 48 hours and then analyzed by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3) .

C XMU-MP-5 downregulates ALK signaling pathways in EML4-ALK Ba/F3 cells. Cells were treated with XMU-MP-5 or crizotinib for 4 h, and then analyzed by immunobloting.

D The KINOMEscan result of XMU-MP-5 against a panel of 468 kinases. Selectivity score S(10) and kinase hits of XMU-MP-5 with ctrl% ≤10% were shown. ALK and its mutants were highlighted in blue (see Appendix Table S1 for the full list and see Experimental Section for the detailed kinase group names).
A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC (2021)
Figure 2

Fig. 2 The antiproliferative activity of XMU-MP-5 against H3122 cells both in vitro and in vivo.

A H3122 cells were treated with XMU-MP-5 or crizotinib for 72 hours. Cell viability was measured by MTS assay. Each concentration was performed in triplicate. Data were presented as Mean ± SEM from three independent experiments (n = 3)

B XMU-MP-5 inhibited colony formation of H3122 cells. H3122 cells were treated with XMU-MP-5 or crizotinib for 18 days and then stained with crystal violet.

C XMU-MP-5 suppressed ALK signaling pathway in H3122 cells.

D XMU-MP-5 induced cell apoptosis in H3122 cells. Cells were treated as indicated for 72 hours and then assayed by Annexin-V-FLUOS/PI staining.

E Comparison of tumor volume of different treatment groups in 11 days. BALB/c nude mice bearing H3122 xenograft tumors were treated with vehicle, XMU-MP-5 and crizotinib by tail vein injection once daily. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test. N.S., P = 0.1063; **P = 0.0097.

F Comparison of tumor weight of each group at end point. Data were shown as Mean ± SEM (n = 6). Statistical comparasions were performed using a one-way ANOVA with Dunnett test. *P = 0.0177, **P = 0.002.

G Representative images of tumors analyzed by H&E and IHC staining. Scale bars, 100 μm.
A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC (2021)
Figure 4

Fig. 4 XMU-MP-5 overcomes secondary mutations on ALK.

A Cell viability was measured by MTS assay. Each concentration was performed in triplicate. The exact IC50 value was presented as Mean ± SEM from three independent experiments (n = 3) and shown in Appendix Table S6. IC50 fold changes of wild type and mutant EML4-ALK Ba/F3 cell lines to parental Ba/F3 cell line of XMU-MP-5 and crizotinib were determined.

B Heatmap of the anti-proliferative activity of ALK inhibitors across Ba/F3 cell lines with different ALK mutations.

C, D In vivo activity of XMU-MP-5 against L1196M xenograft mouse model. BALB/c nude mice bearing L1196M Ba/F3 xenograft tumors were treated with vehicle, 30 mg/kg or 60 mg/kg XMU-MP-5 by tail vein injection once daily. Tumor volume (C) and tumor weight (D) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (C), N.S., P = 0.1482; *P = 0.037. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (D), N.S., P = 0.2162; *P = 0.0122.

E, F Efficacy of XMU-MP-5 in G1202R Ba/F3 xenograft mouse model. BALB/c nude mice bearing G1202R Ba/F3 cells xenograft tumors were administrated with vehicle or XMU-MP-5 by tail vein injection twice daily. Lorlatinib was used as a positive control by oral administration. Tumor volume (E) and tumor weight (F) were shown as mean ± SEM (n = 6). Statistical comparasions were performed using a two-tailed, unpaired Student's t test for (E), Vehicle vs XMU-MP-5 30 mg/kg, *P = 0.0433; Vehicle vs XMU-MP-5 30 mg/kg BID, **P = 0.0093; Vehicle vs Lorlatinib, **P = 0.0029. Statistical comparasions were performed using a one-way ANOVA with Dunnett test for (F). Vehicle vs XMU-MP-5 30 mg/kg, ***P = 0.0008; Vehicle vs XMU-MP-5 30 mg/kg BID, ***P = 0.0003; Vehicle vs Lorlatinib, ***P = 0.0002.
A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC (2021)
Figure 5

Fig. 5 XMU-MP-5 treatment leads to tumor regression in GEM models.

A Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10A. Mice were treated with 30 mg/kg XMU-MP-5 twice daily for 3 weeks. H: heart; arrow indicated tumor. Scale bars, 200 μm.

B IHC analyses of lungs from EML4-ALK transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.

C Representative CT images (upper panel) and H&E staining (lower panel) of lungs from EML4-ALK(L1196M) tumor-bearing mice are shown. The complete set of CT images refers to Appendix Figure S10B. Mice were treated with 40 mg/kg XMU-MP-5 twice daily for 1 week. H: heart; arrow indicated tumor. Scale bars, 200 μm.

D IHC analyses of lungs from EML4-ALK(L1196M) transgenic mice. pALK and Cl-caspase 3 were probed. Scale bars, 100 μm.
Deletions in CWH43 Cause Idiopathic Normal Pressure Hydrocephalus (2020)
Figure 1

Figure 1: iNPH-associated deletions disrupt the ability of Cwh43 to regulate the localization of GPI-anchored receptors.

A. Sanger DNA sequencing data from two iNPH patients confirming the presence of heterozygous CWH43 deletions. Arrows identify location of the deletion.

B. Diagram of the domain structure of the Cwh43 protein illustrating the location of the damaging CWH43 deletions. Lower panel details the disruptive effect of the k696Asnfs mutation on the C-terminal endoplasmic reticulum export signal (YF), replacing it via a frameshift with a 23 amino acid sequence that contains an endoplasmic reticulum retention signal (KKKS).

C. Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP-Cwh43, GFP-Cwh43-k696fs or GFP-Cwh43-Leu533ter fusion proteins. To label the ER, HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP-calreticulin fusion protein containing an ER retention signal (RFP-calreticulin-KDEL). To label the Golgi apparatus, cells were immunostained for golgin-97 (red). When compared with wild type GFP-Cwh43, GFP-Cwh43-k696fs and GFP-Cwh43-Leu533 showed decreased association with the Golgi apparatus (but not the ER) and were diffusely distributed throughout the cytoplasm. Scale bar is approximately 5 µm.

D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.
Deletions in CWH43 Cause Idiopathic Normal Pressure Hydrocephalus (2020)
Figure 2

Figure 2: Expression of Cwh43 mRNA and protein in the mouse brain.

mRNA in situ hybridization images showing expression of Cwh43 mRNA in the mouse brain. Enclosed areas containing potions of the ventricle, hippocampus and dorsal thalamus are shown at higher magnification on the right. Arrowheads point to choroid plexus. Scale bar is approximately 400 µm.

Fluorescence immunohistochemistry of the ependymal surface of the lateral ventricle of the mouse brain. Cilia are visualized using an antibody for acetylated alpha tubulin (green). Cwh43 is visualized using a specific anti-Cwh43 antibody (red). Nuclei are counterstained using DAPI (blue). Arrowheads point to motile cilia and scale bar is approximately 5 µm.

Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.