EMBO/soda-nel huggingface dataset:
A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction (2020)
Figure 6

Figure 6 - Microtubules (MTs) promote LUZP1 inhibition of myosin phosphatase.

Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range.

Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.

Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).

In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.

A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ-associated CRs to promote apical constriction.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 1

Figure 1 Overexpression of hTau activates STAT1 with an increased nuclear translocation in vitro.

(A, B) Overexpression of wildtype full-length human tau (hTau, also termed tau441 or tau40 or tau2N4R) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II, in which 96 transcription factors (Appendix Table S1 and S2) were monitored. The empty vector was transfected as a control (Ctrl).

(C-F) Expression of hTau (probed by HT7) increased total and the phosphorylated STAT1 at Tyr701 (pY-STAT1) in cell whole extracts (C, D) and the nuclear fraction (E, F) measured by Western blotting (n=4).

(G) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control (eGFP) (n=5). Scale bar, 10 μm.

(H) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction (Nu) measured by Western blotting.

(I) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay (n=4).

(J) Overexpression of hTau increased STAT1-DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). *, indicates STAT1/DNA complex.

Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 2

Figure 2 Overexpression of hTau upregulates phosphorylated STAT1 in vivo.

(A, B) AAV-hTau-eGFP (AAV-hTau) or the empty vector AAV-eGFP (1.13×1013 v.g./ml) was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, the increased levels of STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting (n=6, Mann-Whitney test).

(C, D) The increased STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction of 12-month-old hTau transgenic mice measured by Western blotting (n=4, Mann-Whitney test).

(E, F) The representative images of STAT1 and pY-STAT1 in the brain of AD patients probed by co-immunohistochemical staining and quantitative analysis (hematoxylin stains nuclei, purple; DAB stains the target proteins, brown; n=5-6 slices). Arrowheads indicated typical nuclear staining of STAT1/pY-STAT1.

(G) The increased AT8 (pS202/pT205), STAT1 and pY-STAT1 in cortex total extracts of AD patients measured by Western blotting (n=3, Student's t test).

(H) STAT1 mRNA was analyzed by qPCR in AAV-hTau or AAV-eGFP infected hippocampal tissues (n=6, Mann-Whitney test).

Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 3

Figure 3 Downregulating STAT1 ameliorates hTau-induced cognitive and synaptic impairments.

(A, B) AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by Western blotting and immunohistochemical staining.

(C) Downregulation of STAT1 ameliorated hTau-induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze (MWM) test (n=9-11 for each group).

(D-G) Downregulation of STAT1 ameliorated hTau-induced spatial memory deficit shown by the decreased latency to reach the platform quadrant (D), the increased crossing time in the platform site (E) and time spent in the target quadrant (F) measured at day 6 by removed the platform in MWM test; no motor dysfunction was seen (G) (n=9-11 for each group).

(H) Downregulation of STAT1 ameliorated hTau-induced contextual memory deficits measured at 24 h during contextual fear conditioning test (n=8 each group).

(I-L) One month after the virus infection, paired-pulse ratio (PPR) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice (I). The IO curve of fEPSP recorded on acute hippocampal slices (J). The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice (K). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (L). (n=5 slices from 4 mice for each group).

(M-P) One month after the virus infection, whole cell patch clamp was used to measure the function of NMDA (at +40 mV) and AMPA (at -70 mV) receptors on acute brain slices (400 μm). The insets show representative sample traces of EPSCs in virus infected neurons (M). The reduced NMDA and unchanged AMPA currents with a reduced NMDA/AMPA ratio were seen in hTau infected neurons, while knockdown of STAT1 restored the hTau-induced NMDA currents (N-P). (n=12 neurons from 4 animals for eGFP group; n=11 neurons from 4 animals for hTau group; n=13 neurons from 4 animals for hTau+CRE group).

Data information: Data were presented as mean ± s.e.m. for (C-H) and mean ± SD for others (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's post hoc test for C, two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for I-K, one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 4

Figure 4 Overexpressing hTau suppresses expression of NMDARs by upregulating STAT1 in mice.

(A, B) Overexpression of AAV-hTau decreased the protein levels of GluN1, GluN2A and GluN2B detected by Western blotting and qRT-PCR in the hippocampal CA3 of C57 mice, compared with the AAV-eGFP vector control.

(C, D) Simultaneous downregulation of STAT1 by infusing AAV-Cre in hippocampal CA3 of STAT1flox/flox mice abolished the hTau-induced inhibition in expression of NMDARs protein.

(E, F) Overexpression of AAV-hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1, GluN2A and GluN2B detected by qRT-PCR in the hippocampal CA3.

Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 5

Figure 5 Overexpressing hTau increases binding of STAT1 to NMDAR promoters and inhibits the expression of NMDARs.

(A) Overexpression of AAV-hTau increased binding of STAT1 to the promoter regions of GluN1, GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay (ChIP) (n=3 from three independent experiments).

(B) Overexpression of hTau or wildtype STAT1 (WT-STAT1) inhibits the transcription activity of NMDARs compared with the empty vector control (Ctrl) measured by luciferase activity assay in HEK293 cells (n=3 from three independent experiments).

(C-J) Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN1 (C), GluN2B (E) and GluN2A (G, I). The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group.

Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for A-J). *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 6

Figure 6 JAK2 activation mediates hTau-induced STAT1 activation.

(A, B) Overexpression of hTau in HEK293 cells for 48 h increased the activity-dependent phosphorylation of JAK2, JNK1 and ERK1 compared with the empty vector control (Ctrl) measured by Western blotting (n=3, student's t test).

(C-F) Pharmacological inhibition of ERK1 (C, D) or JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C, E) and the nuclear fraction (D, F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors.

(G-J) Pharmacological inhibition of JAK2 (G, H) or knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G, I) and the nuclear fraction (H, J) (n=3).

(K, L) The phosphorylated JAK2 level increased in the hippocampus of 12-month-old hTau transgenic mice (K), and the hippocampus of C57 mice infected with AAV-hTau (1.13×1013 v.g./ml) (L) (n=4, Mann-Whitney test).

Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.
Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression (2019)
Figure 7

Figure 7 Blocking STAT1 activation rescues hTau-induced synapse and memory impairments.

(A-F) AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (A) The representative fluorescence image confirms expression of AAV-hTau and AAV-Y701F-STAT1. Scale bar, 200 μm, or 100 μm for the enlarged. (B) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial learning deficits shown by the decreased escape latency during water maze training (n=7-10 each group). (C-E) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial memory impairment shown by the decreased latency to reach the platform (C), the increased crossing time in the platform site (D) and time spent in the target quadrant (E) measured at day 6 by removed the platform (n=7-10 each group). (F) Expression of Y701F-STAT1 did not change the swimming speed of the mice in water maze task (n=7-10 each group).

(G-I) Simultaneous expression of Y701F-STAT1 did not induced any further change on basal synaptic transmission (I/O curve) compared with expression of hTau alone, recorded in hippocampal CA3 (G). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (I). (n=5 slices from 4 mice for each group).

(J-L) Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein (I, J) and mRNA (K) expression measured by Western blotting and qRT-PCR in hippocampal CA3 of C57 mice (n=4).

Data information: Data were presented as mean ± s.e.m for (B-F) and mean ± SD for others (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for B, two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.

Expanded View Figure Legends
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 1

Figure 1 Workflow for the breast atlas and scRNA-seq profiling of normal breast epithelium.

A Schematic diagram showing workflow for scRNAseq of human specimens: normal and preneoplastic breast tissue, breast tumors (TNBC, ER+, HER2+, male breast tumors) and matching pairs of tumor and lymph node (LN) samples.

B Flow cytometry based on CD49f and EpCAM staining separates lineage-negative breast tissue cells into stromal (CD49f-EpCAM-) and epithelial cells, which includes basal (CD49f+EpCAMlo/-), luminal progenitor (LP) (CD49f+EpCAM+) and mature luminal (ML) (CD49f-EpCAM+) cells.

C t-SNE plot of the integrated scRNA-seq profiles of epithelial cells from 11 reduction mammoplasties. Cell colors correspond to tissue specimens.

D Same t-SNE plot as (C) but separated by hormonal status of the donor (8 premenopausal and 3 postmenopausal).

E Same t-SNE plot as (C) but colored by cell clusters (with Seurat cluster resolution set to 0.015).

F Ternary plot positioning each cell according the proportion of basal, LP or ML signature genes expressed by that cell. The three vertices of the plot correspond to cells expressing basal genes only, LP genes only or ML genes only. Cells expressing equal numbers of basal, LP and ML genes are in the center of the plot. The plot shows the same cells and cell colors as for (E), thus identifying green as basal, blue as LP and orange as ML populations, respectively.

G Diffusion map of the epithelial cells.

H t-SNE plot as in (E) colored by the expression level of a selection of basal, LP and ML marker genes. Red=high expression, grey=not-detected.

I The basal cell cluster shows a `tail` of atypical cells (highlighted in pink). These tail cells are in the centre of the diffusion plot, downstream of the other basal cells in pseudo-time and intermediate between the basal and two luminal lineages.
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 2

Figure 2 Transcriptional changes in the microenvironment of postmenopausal breast tissue.

A t-SNE map of combined scRNA-seq transcriptomes of total tissue cells from 13 reduction mammoplasties. Cell colors correspond to tissue specimens.

B Same t-SNE map as (A) but colored by cell cluster (with cluster resolution 0.05).

C Ternary plot positioning each cell according the proportion of basal, LP or ML signature genes expressed by that cell. The plot shows the same cells and cell colors as for (B).

D Re-clustered EPCAM-negative non-epithelial cells revealed seven clusters (resolution 0.05) representing immune and stromal cell lineages.

E Multidimensional scaling plot showing expression profile distances between the pseudo-bulk samples. Each dot corresponds to aggregated expression for a cell cluster for one patient. Cluster colors are overlaid from (D, right panel). Distances on the plot correspond to leading log2-fold-change, defined as the average log2-fold-change for the 500 most differential genes between each pair of profiles.

F Heat map of pseudo-bulk samples showing marker genes for each non-epithelial cluster. The top 20 marker genes were identified for each cluster by differential expression analysis of the pseudo-bulk read counts. Color bars at the top of the plot indicate the cluster and menopausal status (blue/yellow for pre/post menopause) of each sample.

G Same t-SNE map of non-epithelial cells as in (D) but colored by menopausal status. Barplot shows relative cluster sizes (percentage of total cells) for each status condition. Clusters 1 and 2 have significantly different sizes in post-menopause samples after allowing for inter-patient variability (P = 0.040 and P=0.032 by quasi-binomial F-test). Sizes are not significantly different for other clusters (P > 0.15).

H Relative cluster sizes as for (G) but by individual patients. Cluster colors correspond to (D). by cluster. A quasi-multinomial F-test was used to test for differences in cluster frequencies between pre- and post-menopausal samples (P = 0.007).

I Bar plots showing percentage of cells expressing selected immune and endothelial cell markers for pre- and post-menopausal samples and the cell clusters identified in Fig 2D.
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 3

Figure 3 Changes in epithelial-associated fibroblasts in postmenopausal breast tissue.

A Microenvironment t-SNE map as in Fig 2D and G but separated by menopausal status and colored by expression (red=high expression, grey=undetectable) of selected fibroblast markers (upper panel). The dotted lines indicate the pericyte subsets. Bottom panel shows percentage of cells expressing the markers for the cell clusters identified in Fig 2D.

B Co-immunofluorescence staining of pre- versus post-menopausal tissue for E-cadherin (cyan), PDGFRβ (yellow) and F-actin (pink). DAPI is shown in grey. The arrowheads in (v) depict fibroblasts in direct contact with the myoepithelial layer. For 2D and 3D confocal imaging: n=13 premenopausal and n=10 postmenopausal specimens. Scale bars: wholemount and optical sections:100 μm (panels i-iv); enlargements, 30 μm (panel v).

C t-SNE plot of the integrated scRNA-seq profiles of fibroblasts from pre-and post-menopausal tissue (reclustered cells from cluster 1 in Fig 2D). Cell colors correspond to tissue specimens.

D Same t-SNE plot as in (C) showing 5 distinct clusters (clusters 3 and 5 were specific to one patient).

E KEGG pathways enriched in cluster 1 versus 2 from (D) above (Fisher's exact test).

F Heat map of same cells as (D) showing expression of the top 20 marker genes in each cluster. Color bars at the top of the plot show cluster membership (colors as in (D)) and pre- or post-menopausal status (blue and yellow, respectively).
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 4

Figure 4 Comparison of the single-cell transcriptomes of normal tissue, BRCA1+/- preneoplastic tissue and BRCA1-associated tumors.

A t-SNE map of combined scRNA-seq profiles of total cells isolated from pathologically normal preneoplastic tissue from BRCA1 mutation carriers (BRCA1; n=4) and non-BRCA premenopausal women (n=8) with no family history of breast cancer. Cell colors represent individual samples.

B Same t-SNE map as in (A) but colored by cluster (cluster resolution 0.12).

C Epithelial clusters were identified by EPCAM expression and the non-epithelial cells were re-clustered to reveal immune and stromal cell populations (cluster resolution 0.08). Lineage identity was determined by hierarchical clustering according to top marker genes (Appendix Fig S1C).

D t-SNE plot showing the combined single-cell transcriptomes of total tissue cells from BRCA1 preneoplastic tissue and TNBCs (n=4 for each), colored according to individual patients (B1 = preneoplastic BRCA1+/- tissue; TN-B1= BRCA1-associated TNBCs).

E Same t-SNE map as (D) but colored by cluster (cluster resolution 0.15).

F Expression of epithelial markers indicated on the combined t-SNE plot as in (D, E) for BRCA1 preneoplastic and BRCA1-associated tumor cells.

G Epithelial clusters were identified by EPCAM expression and non-epithelial cells are indicated by the dotted line.

H Same t-SNE map as in (D, E) but colored according to cancerous state: preneoplastic tissue (blue) and BRCA1-associated TNBCs (yellow).

I Box plots of signature expression scores for the 13 cell clusters in (D, E) according to human breast epithelial and stromal gene signatures. Cluster 1 corresponds to tumor cells, while clusters 6, 9 and 10 represent adjacent normal LP, basal and ML cells, respectively. Box plots show quartiles, minimum and maximum.
A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast (2021)
Figure 5

Figure 5 The altered microenvironment in BRCA1+/- preneoplastic tissue versus tumors

A Reclustered EPCAM-negative cells (excluding clusters 1, 6, 9 and 10 from Fig 4E) revealed immune/stromal cells in the microenvironment, identified using lineage markers.

B Combined t-SNE plot as in (A) of the single-cell transcriptomes of immune and stromal cells from preneoplastic tissue of BRCA1 mutation carriers (blue; preneo) and BRCA1-associated TNBCs (yellow; tumor).

C t-SNE plots showing expression profiles of cardinal markers of immune and stromal cells.

D Bar plots showing the percentage of cells expressing typical immune cell (including Treg) genes for clusters in (A) by preneoplastic vs tumor.

E Left panel, same t-SNE map as in (A) but separated into cells from preneoplastic (blue) and tumor specimens (yellow). Endothelial cell (endo), fibroblast cell (fibro) and pericytes (peri) clusters are marked. Right panel, proportion of clusters as in (A) according to tissue type.

F Relative cluster sizes as in (E) but by individual patient.

G Co-immunofluorescence of tissue stained for the epithelial marker (cytokeratin 19; yellow) and the tumor-associated macrophage marker CX3CR1 (magenta). DAPI is shown in white (n=2 preneoplastic samples; n=2 tumors). Scale bar, 100 μm.

H Heat map of top differentially expressed genes in the major immune/stromal cell clusters, identified in (A). BRCA1+/- preneoplastic cells, blue; BRCA1-associated tumor cells, yellow.