EMBO/soda-nel huggingface dataset:
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 6

Figure 6 Tumor heterogeneity amongst the major breast cancer subtypes.

A-C t-SNE plots of combined scRNA-seq profiles of total cells from 8 TNBC tumors, 6 HER2+ tumors and 13 ER+ tumors respectively. Integration and cluster sizes for the same cells shown in Fig EV2B-D. Cells colored by cluster (top left panels), EPCAM expression (top right), cancer subtype marker (bottom left) or MKI67 expression (bottom right). Dotted lines delineate epithelial cells (top panels) and cycling epithelial cells (bottom panels). Normal epithelial subsets (normal) are also demarcated by dotted lines in the upper-right panels of B and C.

D InferCNV plots to map inferred copy number variation (CNV) for the combined tumor scRNA-seq expression data for the epithelial clusters indicated in panels A-C. scRNA-seq data from normal breast epithelial cells (N-1105-epi) served as a reference for normalization. Each row represents a gene and each column cells from a cluster in a single tumor. The tumor clusters are color-coded as in A-C. Amplifications (red) and deletions (blue) are inferred from the gene expression. Tumor cells were distinguished from normal (N) cells by abundance of CNV.

E t-SNE plot of EPCAM+ epithelial cells from ER+ tumors (C). Top panel shows reclustering (resolution 0.05), bottom panel shows expression of MKI67.

F Heatmap of cells from clusters in (E) using genes from the PAM50 cancer subtype classifier.

G Enrichment of KEGG pathways in EPCAM+ clusters 1 to 3 in (E) for ER+ tumors (Fisher's exact test).

H Combined t-SNE transcriptome profiles of two distinct ER+ tumors isolated from the same breast of a patient: ER-0029-7C (blue) and ER-0029-9C (yellow). The corresponding t-SNE cell clusters are shown in the middle panel and expression of EPCAM is shown in the right-hand panel.
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 7

Figure 7 Deconvolution of the microenvironment in different breast tumor subtypes.

A-C t-SNE maps of reclustered EPCAM-negative non-epithelial cells identified in Fig 6 (A-C). Cluster resolutions 0.136, 0.1 and 0.1 respectively. The major cell clusters within the microenvironment were identified based on expression of lineage-specific genes. Heat maps of pseudo-bulk samples show marker genes for each cluster. The top 30 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Cluster 9 in HER2+ tumors (B) expressed myeloid and luminal epithelial markers, suggesting phagocytosis of the latter by macrophages. Color bars at the top of the heat map indicate the cluster of each sample; top genes that mark each cluster are indicated.

D t-SNE plots showing the expression of T lymphoid and myeloid markers shown in (A-C). Right panels: t-SNE plots showing the expression the proliferation marker MKI67 for the same clusters. Dotted lines highlight T and myeloid cells that express MKI67.
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 8

Figure 8 Presence of distinct proliferating immune cell subsets in TNBC and ER+ tumors.

A, B Image quantification showing number of CD8+Ki67+ cells (A) or CD68+Ki67+ cells (B) per mm2 of tissue from TNBC, HER2+ and ER+ tumors, either within the tumor region (K8/18+) or the stroma (defined as >5 μm from tumor border). Error bars represent s.e.m. CD8/Ki67: n=11 for TNBC, n=12 for ER+, n=5 for HER2+. CD68/Ki67: n=6 for TNBC, n=8 for ER+, n=5 for HER2+.

C-F Representative confocal images of ER+ (ER-0032) and triple negative tumors ( TN-0066) immunolabeled for K8/18 (yellow), CD68 (green) and Ki67 (red) (C, E) or K8/18 (yellow), CD8 (green) and Ki67 (red) (D, F). DAPI is shown in blue. Arrows depict proliferative T cells (CD8+Ki67+) or macrophages (CD68+Ki67+). Enlargements on shown in the right-hand panels. Scale bars, 200 µm for large tilescans; 50 µm for enlargements and smaller tilescans.
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 9

Figure 9 Analysis of the single-cell transcriptomes of primary tumors and infiltrated lymph nodes identifies clonal propagation of tumor cells.

A Combined t-SNE plots of matching tumor and lymph node samples from seven patients (6 female and 1 male) with ER+ disease. Patient 1: ER-0040-T and ER-0040-LN; Patient 2: ER-0056-T and ER-0056-LN; Patient 3: ER-0043-T and ER-0043-LN; Patient 4: ER-0064-T and ER-0064-LN; Patient 5: ER-0167-T and ER-0167-LN; Patient 6: ER-0173-T and ER-0173-LN and Patient 7: mER-0068-T and mER-0068-LN. The top panels show the combined cells marked according to primary tumor (blue) or involved LN cells (yellow). The middle panels show expression of the epithelial marker EPCAM and proliferation marker MKI67. The bottom panels indicate the major cell clusters and their identity based on expression analyses for lineage-specific markers.

B InferCNV plots were generated from the combined transcriptomes (A) to map copy number variation (CNV) in each chromosome. Tumor cells can be distinguished from normal (N) cells by abundance of CNV.

C Immunostaining of tumor and LN from patient ER-0064 for expression of ER, PR and pan-cytokeratin. Insets show sections stained with control isotype antibodies. PR, progesterone receptor. Scale bar, 100 μm.

Expanded View Figure Legends
A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort (2018)
Figure 2

Figure 2 - FGMYB genes are phylogenetically closely related to each other and expressed in female gametophytes

A Phylogenetic tree of R2R3-MYB proteins of clades 11, 12, and 14-16, as described by Bowman et al. (Bowman et al, 2017), from representative land plant species constructed using the maximum likelihood method based on conserved MYB domain sequences. See Source Data for the sequences used and accession numbers. Numbers at nodes indicate bootstrap values calculated from 1,000 replicates. The tree is drawn to scale, with branch lengths reflecting the number of substitutions per site. Scale bar, 0.5 substitutions per site. Arrows indicate FGMYB orthologues involved in embryo sac development in A. thaliana, MpFGMYB of M. polymorpha (this study), and the most similar P. patens genes shown in (B). The FGMYB clade is shaded in red, and a distantly related FOUR LIPS (FLP) clade is in blue.

B Schematic representations of the FGMYB polypeptide structures. R2R3 MYB domains are shown in red and a conserved amino-terminal motif of PpFGMYBs and MpFGMYB in orange.

C Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Constitutively expressed MpEF1α was used as a control.

D Real-time RT-PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by p <0.05 in a Tukey-Kramer test.

E A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.

F MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC) (Shimamura, 2016). Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.

G A transcriptional AtMYB64 reporter (AtMYB64-NYG) is specifically expressed in all four cell types of the A. thaliana embryo sac (Rabiger & Drews, 2013; Waki et al, 2013). Scale bar: 25 µm. Green, YFP fluorescence; white, cell walls.

H Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac (Rabiger & Drews, 2013). Scale bar: 25 µm. Green, Citrine fluorescence; white, cell walls.
Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs (2018)
Figure 1

Figure 1. Single-cell RNA-seq identified distinct cell populations in the VPR and DPR.

The VPR and DPR (right) were dissected from E10.5 Pdx1-GFP embryos (left). Scale bar: 500 µm. DPR, dorsal Pdx1-expressing region; VPR, ventral Pdx1-expressing region; Duo, duodenum bud.

FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR.

The ratios of GFPhigh cells to GFP+ cells in the VPR and DPR. The data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.

PCA plot of single-cell RNA-transcriptomes of GFP+ (circle), GFPlow (triangle) and GFPhigh (square) cells from the VPR (blue) and DPR (orange). Each point represents a single-cell. Three groups of cells are included in three colored circles.

Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters (a-d) of genes (p < 1 ×10-9, cell cycle-related genes are excluded). Each column is a single-cell, and each row represents one gene. Cluster-specific TFs are listed on the right. The genes in bold are known to have roles in pancreatic development. L, GFPlow; H, GFPhigh.

Selected GO categories enriched among the four clusters of genes in (E).

Figure 2. Three groups of cells represent distinct cell fates.

The VPR and DPR (right) were dissected from E10.5 Pdx1-GFP embryos (left). Scale bar: 500 µm. DPR, dorsal Pdx1-expressing region; VPR, ventral Pdx1-expressing region; Duo, duodenum bud.

FACS gating for sorting GFPlow and GFPhigh cells from the VPR and DPR.

The ratios of GFPhigh cells to GFP+ cells in the VPR and DPR. The data show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.

PCA plot of single-cell RNA-transcriptomes of GFP+ (circle), GFPlow (triangle) and GFPhigh (square) cells from the VPR (blue) and DPR (orange). Each point represents a single-cell. Three groups of cells are included in three colored circles.

Hierarchical clustering of 815 genes correlating with the first two PCs identified three groups of cell types and four major distinct clusters (a-d) of genes (p < 1 ×10-9, cell cycle-related genes are excluded). Each column is a single-cell, and each row represents one gene. Cluster-specific TFs are listed on the right. The genes in bold are known to have roles in pancreatic development. L, GFPlow; H, GFPhigh.

Selected GO categories enriched among the four clusters of genes in (E).
Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs (2018)
Figure 3

Figure 3. More endocrine and β-cells are generated in the dorsal pancreas.

A-I Flow cytometric analysis of endocrine progenitors and β-cells in the ventral pancreases (V. P.) and dorsal pancreases (D. P.). (A, D and G) Dissected ventral and dorsal pancreatic tissues. Scale bar: 500 µm. (B, E and H) FACS gating for sorting Ngn3-GFP+ or Ins1-RFP+ cells from the V. P. and D. P. (C and F) statistical analysis of percentages of Ngn3-GFPhigh cells among Ngn3-GFP+ cells in the V. P. and D. P. (I) Statistical analysis of percentages of Ins1-RFP+ cells among total pancreatic cells in the V. P. and D. P.

Data information: Data in C, F and I show the mean ± SEM; n = number of independent biological replicates, unpaired t-test. ***p-value < 0.001.

Differentially expressed genes among cells from VPR, DPR, the V. P. and D. P. at various developmental stages. The numbers indicate significantly highly expressed genes in the ventral (blue) or dorsal (orange) regions. The dotted lines represent the two-fold change threshold.

Figure 4. Three cell lineages among GFPlow VPR cells.
Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs (2018)
Figure 4

Figure 4. Three cell lineages among GFPlow VPR cells.

Flow cytometric analyses of Liv2+ cells in the VPR, DPR and the liver buds (LB). Liv2+ cells from the VPR-L (brown arrow), Liv2- cells from the VPR-L (yellow arrow) and Liv2+ cells from the LB (black arrow) were sorted for single-cell RNA-seq.

Immunofluorescence of PDX1 and Liv2 on sagittal section of E10.5 mouse embryo. The white and yellow arrows denote PDX1low/Liv2+ cells in in the liver and EHBD regions, respectively. Scale bar: 50 µm.

PCA plot of single-cell transcriptomes from the indicated cells (left). Each dot represents a single-cell. All cells are classified into three groups (right). The two panels represent the same PCA coordinates.

Hierarchical clustering of 694 genes correlated with the first two PCs identified three major distinct cell lineages (p < 1 × 10-6). Each column represents a single-cell and each row represents one gene. Cells were re-ordered according to the value of PC1 or PC2. Genes in clusters 'a, b' and 'c, d' were re-ordered by correlations with PC2 and PC1, respectively. The color of the cell type and cell source are indicated in (C). Lineage-specific TFs are listed on the right.

E, F The gene expression values of lineage marker genes were projected onto the PCA (E) and box (F) plots. Each point represents a cell. The y-axes of the box plots represent the expression levels (RPKM). Central line in the box represents median; box limits represent the first and third quartile; whiskers represent 1.5 × interquartile range. Unpaired Wilcoxon rank-sum test. **p-value <1 x 10-5; ***p-value <1 x 10-10.

PCA plot of single-cell transcriptomes from the indicated cells. Each dot represents a single-cell. The PCA was performed using the same PC1 and PC2 in (C).

Pdx1 and Sox17 expression levels in E9.5 VPR and DPR cells.

Inferred ventral and dorsal pancreatic lineage relationships analyzed by minimum spanning tree.

Figure 5. Hepatic potential of ventral Pdx1-expressing cells.

Sagittal section of E12.5 Pdx1-Cre; Rosa26-LacZ mouse embryo counterstained for ALB (brown). X-gal staining (blue) showing cells derived from Pdx1-expressing cells. The arrowheads indicate cells co-stained with X-gal and ALB (See Fig EV5D for more images of different sections). Scale bar: 50 µm (left) or 20 µm (right).

Figure 6. Working model of distinct ventral and dorsal pancreatic programs.
Single-cell transcriptomic analyses reveal distinct dorsal/ventral pancreatic programs (2018)
Figure 5

Figure 5. Hepatic potential of ventral Pdx1-expressing cells.

Sagittal section of E12.5 Pdx1-Cre; Rosa26-LacZ mouse embryo counterstained for ALB (brown). X-gal staining (blue) showing cells derived from Pdx1-expressing cells. The arrowheads indicate cells co-stained with X-gal and ALB (See Fig EV5D for more images of different sections). Scale bar: 50 µm (left) or 20 µm (right).
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf1

(a) Confocal micrographs of HeLa cells expressing yellow fluorescent protein (YFP) fused to Nap1, Sintbad or Tank and infected with S. Typhimurium, stained for ubiquitin (Ub) 2 h after infection. At least 100 cells were analyzed. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm; arrowheads, bacteria shown in insets. (b–e) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with lysates of 293ET cells expressing Nap1, Sintbad or Tank, each fused to luciferase (b), lysates of 293ET cells expressing the indicated Nap1 mutants fused to luciferase (c), lysates of E. coli expressing Nap1(1–85) fused to luciferase (d), and lysates of 293ET cells expressing Nap1, NEMO or Optineurin fused to luciferase (e). The reaction in d was complemented with lysates of 293ET and Sf9 cells obtained from the indicated number of cells. Fusion proteins are further characterized in Supplementary Figure 1. All data are representative of at least two independent experiments.
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf2

(a) Flowthrough from nickel-agarose column, separated by SDS-PAGE and stained with Coomassie blue. (b) Amino acid sequence of NDP52. Red and underlining indicates peptides identified by mass spectrometry. (c,d) Normalized ratio between luciferase activity bound to beads and present in lysates. The indicated purified GST fusion proteins coupled to beads were incubated with bacterially expressed Nap1(1–85) fused to luciferase. Reactions were complemented with lysates from 293ET cells that had been depleted with the indicated (α-) antibodies (c, top). Immunoblot of lysates after depletion (c, bottom). Reactions were complemented with the indicated amounts of lysates from E. coli cultures expressing myelin basic protein (MBP) fused to NDP52 (d). (e) Lysates of 293ET cells were immunoprecipitated (IP) using the indicated antibodies or an isotype control. Lysates and precipitates were blotted (IB) for the presence of TBK1, IKKε and IKKβ. (f) Lysates of 293ET cells were immunoprecipitated using the indicated antibodies or an isotype control. Precipitates were blotted for the presence of TBK1 and NDP52. Fusion proteins used in Figure 2 are further characterized in Supplementary Figure 1. Data in panels c–f are representative of at least two independent experiments. Ub, ubiquitin; PCNA, proliferating cell nuclear antigen.
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf3

(a) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to Cocoa, NDP52 and ubiquitin (Ub). (b) Colocalization, at the indicated time points after infection, of ubiquitin-positive S. Typhimurium with NDP52 or Cocoa. (c) Confocal micrographs of HeLa cells 2 h after infection with S. Typhimurium, stained with antibodies to NDP52 and LAMP1. (d) Colocalization, at the indicated time points after infection, of NDP52-positive S. Typhimurium with LAMP1. DAPI, 4,6-diamidino-2-phenylindole. All data are representative of at least two independent experiments. Error bars, s.d.; three coverslips per experiment, 50 marker-positive cells evaluated per coverslip. Scale bars, 10 μm; arrowheads, bacteria shown in insets.
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf4

Percentage of NDP52-positive S. Typhimurium among ubiquitin-coated bacteria, determined by microscopy. HeLa cells transduced with the indicated NDP52 domain-deletion mutants fused to green fluorescent protein were infected with S. Typhimurium and stained for ubiquitin 2 h after infection. ZnF, zinc finger domain. *P 0.01, Student's t-test. Mean and s.d. of triplicate coverslips from two independent experiments; more than 50 ubiquitin-positive bacteria were evaluated per coverslip.
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf5

(a,b) Confocal micrographs of HeLa cells expressing YFP-Nap1, YFP-Sintbad or YFP-Tank (a) or YFP-TBK1(K38M), a catalytically inactive mutant (b), stained with NDP52 antiserum 2 h after infection with S. Typhimurium. All data represent at least two independent experiments. DAPI, 4,6-diamidino-2-phenylindole. Scale bars, 10 μm; arrowheads, bacteria shown in insets.
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf6

(a) Lysates of cells transfected with the indicated siRNAs blotted for NDP52, TBK1, IKKε and proliferating cell nuclear antigen (PCNA). Control, a nonspecific band detected by the IKKε antibody. (b,c) Numbers of bacteria recovered from HeLa cells transfected with the indicated siRNAs and infected with S. Typhimurium. At the indicated time points after infection, cells were lysed and bacteria counted on the basis of their ability to form colonies on agar plates. Mean and s.d. of triplicate HeLa cultures and duplicate colony counts. (d) Numbers of bacteria recovered from HCT116 cells expressing the indicated shRNAs and infected with S. Typhimurium. At the indicated time points cells were lysed and bacteria counted by flow cytometry. Mean and s.d. of triplicate cultures. (e) HeLa cells transfected with the indicated siRNAs were infected with S. Typhimurium expressing green fluorescent protein (GFP). At the indicated time points after infection, cells were scored according to the number of bacteria they contained. (f) HeLa cells transfected with the indicated siRNAs were infected with S. Typhimurium expressing GFP. At the indicated time points after infection, cells were stained for ubiquitin and infected cells were scored if they contained ubiquitylated bacteria. All data represent at least two independent experiments. *P 0.05, Student's t-test.
The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria (2009)
figf7

(a–d) Analysis of HeLa cells stably expressing green fluorescent protein–LC3, infected with S. Typhimurium. (a) Confocal micrographs of cells stained 1 h after infection with NDP52 antiserum. Scale bar, 10 μm; arrowheads, bacteria shown in insets. (b) Colocalization of NDP52 with LC3-positive bacteria. (c,d) Counts of LC3-positive S. Typhimurium 1 h after infection in cells transfected with the indicated siRNAs. In c, cells were stained for ubiquitin (Ub); in d, red fluorescence protein (RFP)-expressing S. Typhimurium were used. Means of duplicate counts from four independent experiments; *P 0.05, Student's t-test. (e) Binding of NDP52 to GST-LC3 and GST-ubiquitin. Beads carrying the indicated purified GST fusion proteins were incubated with lysates of E. coli expressing NDP52. Bound proteins were eluted with glutathione and stained with Coomassie blue after SDS-PAGE. (f) Normalized ratio of luciferase-LC3 binding to beads. The indicated purified GST proteins coupled to beads were incubated with bacterially expressed LC3 fused to luciferase and the indicated volumes of lysed E. coli expressing NDP52. Data in a,b,e and f are representative of at least two independent experiments.