EMBO/soda-nel huggingface dataset:
Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance (2022)
Figure 2

Figure 2. Endothelium-derived lactate feed pericytes.

A. Quantification of the mTFP/Venus fluorescence ratio of laconic (lactate sensor) showing concentration-dependent lactate uptake in HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM))

B. Representative images showing mTFP (blue) and Venus (yellow) fluorescence in control and lactate (20mM) treated HBVPs.

C. Schematic illustration for the lactate uptake assay with 2 well chamber. Cells were seeded on a glass bottom dish. BMECs and adenoviral laconic-infected HBVPs were separated by 2 well chamber. After attachment, the 2 well chamber was removed for cell migration.

D. Schematic illustration for the lactate uptake assay after chamber removal. At 24hr after chamber removal, the fluorescence of mTFP and Venus were observed in the middle of the wells (yellow area)

E. Representative stitched image showing mTFP (green), Venus (yellow), DAPI (blue) and ERG1/2/3 (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber.

F. Representative images (high magnification; 60x) showing mTFP (cyan), Venus (yellow) and VECAD (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact.

G. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs with endothelial contact (EC-PC contact) or HBVPs alone (PC only). (n=16 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)

H. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or GLUT1 (lower panel) siRNA treated BMECs. VECAD immunostaining (purple) was used for endothelial staining.

I. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or GLUT1 (siGLUT1) siRNA-treated BMECs. (n=12 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance (2022)
Figure 6

Figure 6. MCT5 in endothelial cells regulates lactate export into pericyte.

A. Violin plot showing the expression of Slc16a family members (monocarboxylate transporters) in brain endothelial cells from scRNAseq database (EndoDB; 3,755 endothelial cells)

B-D. Barplots showing the expression of B) MCT1 (Slc16a1), C) MCT5 (Slc16a4) and D) MCT8 (Slc16a2) across brain cells from the scRNAseq database (Brain cell atlas) (vSMC: venous smooth muscle cells, aaSMC: arteriolar SMCs, aSMC: arterial SMCs, MG: Microglia, FB1-2 : Fibroblast subtypes, OL : Oligodendrocytes, EC1-3 : EC subtypes, vEC : venous ECs, capiEC: capillary ECs, aEC: arterial ECs, AC : astrocytes) (1,100 endothelial cells, mean±SD)

E. Western blot analysis for siRNA efficiency of MCT1.

F. Quantification of 13C3-Lactate using LC-MS/MS in media of siCON or siSlc16a1 treated BMECs after 13C6-glucose treatment (12hr). (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)

G. Western blot analysis for siRNA efficiency of MCT12.

H. Quantification of 13C3-Lactate using LC-MS/MS in media of siCON or siSlc16a4 treated BMECs after 13C6-glucose treatment (12hr). (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)

I. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT1 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining.

J. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT1 (siMCT1) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)

K. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT5 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining.

L. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT5 (siMCT5) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)
Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance (2022)
Figure 7

Figure 7. MCT12 in pericytes regulates lactate import from ECs.

A. Violin plot showing the expression of MCT family members (monocarboxylate transporters) in brain pericytes from scRNAseq database (Brain cell atlas; 1,100 endothelial cells)

B. Barplot showing the expression of MCT12 across brain cells from the scRNAseq database (vSMC: venous smooth muscle cells, aaSMC: arteriolar SMCs, aSMC: arterial SMCs, MG: Microglia, FB1-2 : Fibroblast subtypes, OL : Oligodendrocytes, EC1-3 : EC subtypes, vEC : venous ECs, capiEC: capillary ECs, aEC: arterial ECs, AC : astrocytes) (1,100 endothelial cells, mean±SD)

C. Relative mRNA expression level of MCT12 in HBVPs (blue) and BMECs (orange). (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)

D. Western blot analysis showing expression level of MCT12 and β-actin in HBVPs and BMECs.

E. Western blot analysis for siRNA efficiency of MCT12.

F. Quantification of [13C3]-lactate using LC-MS/MS in siCON (blue) or siMCT12 (orange) treated HBVPs after 20mM [13C3]-Lactate treatment (2hr). (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)

G. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in control (upper panel) or MCT12 (lower panel) siRNA treated HBVPs in a 2 well chamber assay system with BMECs. VECAD (purple) immunostaining was used for endothelial staining.

H. Quantification of the mTFP/Venus fluorescence ratio of Laconic in control (blue) or MCT12 (orange) siRNA treated HBVPs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.

I. Isotope carbon tracing with [13C3]-lactate treatment (20mM, 24hr) in siCON or siMCT12-treated HBVPs showing the extent of downstream metabolites and incorporation rate of labeled carbons. (F-6-P : Fructose 6-phosphate, G-3-P : Glycerol-3-phosphate, Lac : Lactate, Cit : Isocitrate and aKG : α-ketoglutarate) (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.
Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance (2022)
Figure 8

Figure 8. Lactate administration rescues pericyte coverage and vascular leakage but not lethality of Glut1iECKO mice.

A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice.

B. Quantification of the CD13-positive pericytes coverage in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)

C. Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)

D. Quantification of CSF lactate level in WT and Glut1iECKO mice with/without lactate supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)

E. Kaplan-Meier survival curve of Glut1iECKO mice after tamoxifen injection at P60 with (orange) or without (blue) lactate administration. (n=8)
Phosphorylation of beta-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis (2021)
Figure 2

Figure 2. Phosphorylation of β-catenin Ser60 residue by Plk1

(A) Schematic diagram of the β-catenin domain structure and truncation mutants used in this study. The amino acid positions, truncation mutant names, and domain names are indicated.

(B) Bacterially produced full-length GST-β-catenin and truncation mutant proteins were purified and reacted with Flag-Plk1 WT produced from mammalian cells in the presence of [γ-32P]ATP. Incorporation of 32P into GST-β-catenin WT and truncation mutants was confirmed by SDS-PAGE followed by autoradiography.

(C) Bacterially produced β-catenin T1 WT, T3 WT, and corresponding alanine ("A") mutants were purified and reacted with Flag-Plk1 WT in the presence of [γ-32P]ATP. Incorporation of 32P onto each "A" mutant of β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.

(D) Bacterially produced full-length β-catenin WT and alanine ("A") substitution mutants were purified and reacted with Flag-Plk1 WT produced from mammalian cells in the presence of [γ-32P]ATP. Incorporation of 32P into each "A" mutant or full-length WT β-catenin was confirmed by SDS-PAGE followed by autoradiography analysis.

(E) The Ser60 residue of β-catenin is highly conserved among various species. Plk1 consensus phosphorylation sequence ([D/E/Q]-X-[pS/pT]-φ-X-[D/E/Q]) was shown. Ser60 is marked in red. X, any amino-acid residue; φ, hydrophobic residue.

(F) A phospho-specific antibody against the β-catenin p-S60 epitope was generated and validated. Asynchronously growing HEK293T cells transfected with either Flag-β-catenin WT or S60A mutant (S60A) were harvested and immunoblotted using an anti-β-catenin p-S60 specific antibody. To confirm the specificity, either non-phospho-Ser60 peptide (+ S60 pep) or phospho-S60 peptide (+ p-S60 pep) was added to antibody solution prior to membrane incubation.

(G) Asynchronously growing HEK293T cells co-transfected with Flag-β-catenin WT and either HA-Plk1 WT or control (vector only) were harvested and subjected to immunoblot analysis using the indicated antibodies. Asterisk, a nonspecific cross-reacting band.

(H) HeLa CCL2 cells were treated for 18 h with 200 ng/mL nocodazole followed by treatment with either DMSO (control) or BI 2536 for an additional 1 h. The resulting cells were lysed with 2× Laemmli SDS sample buffer and used for immunoblot analysis with the indicated antibodies.
Phosphorylation of beta-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis (2021)
Figure 3

Figure 3. Midbody localization of β-catenin p-S60 and Plk1 in telophase HeLa CCL2 cells

(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies after extraction using 0.2% Triton X-100. The resulting samples were observed using a confocal laser microscope (Zeiss LSM 700). Immunostaining with pre-immune serum in (A) represents the negative control for immunoreactivity. Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images.

The relative thickness of the neck was calculated as in Appendix Figure S6A.

(B) Intercentrosomal (centrosome-to-centrosome) linear signal profiles of β-catenin/Plk1 (left panel) and β-catenin p-S60/Plk1 (right panel) from the XY plane of the immunostained cells in (A).

(C) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies as described in (A), and the midbody localization of β-catenin/β-catenin p-S60 in late telophase cells was observed using a confocal laser microscope (Zeiss LSM 700). Scale bars, 10 μm.

All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments (A, B). All images show representative data of more than 50 late telophase cells obtained from three independent experiments (C).
Phosphorylation of beta-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis (2021)
Figure 4

Figure 4. β-Catenin p-S60 and Plk1 were surrounded by actomyosin contractile ring in early telophase cells.

(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies, and early telophase cells were observed using a super-resolution confocal laser scanning microscope (LSM-880 with Airyscan) (left panel). Scale bars, 10 μm. Linear signal profiles of β-catenin/Plk1/β-catenin p-S60/F-actin in the contractile ring region from the XY plane of immunostained early telophase cells (right panel). All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments. The value of the relative neck thickness is shown in the lower left corner of the merged images (A).

(B) Schematic diagram for the proposed localization of β-catenin/Plk1/β-catenin p-S60/F-actin in the contractile ring region from early telophase cells in (A).

(C) The width of the neck/β-catenin/β-catenin p-S60 from Fig.4A sample was measured (upper panel) and the relative width (%) was marked in the graph. A dot density graph with a box plot shows the relative width in early telophase cells. Approximately 60 cells from three independent experiments were measured. ***, p<0.001 (unpaired two-tailed t test) The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier.
Phosphorylation of beta-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis (2021)
Figure 5

Figure 5. Requirement of β-catenin Ser60 phosphorylation for cytokinesis

(A-C) HeLa CCL2 cells stably expressing control vector only (+vector), β-catenin WT (+WTR), S60A mutant (+S60AR), or S60D mutant (+S60DR) were generated using lentiviral infection and then infected with β-catenin knockdown lentivirus (shβ-catenin). To confirm successful depletion of endogenous β-catenin and the expression of exogenous genes, the resulting cells were immunoblotted with the indicated antibodies (A). Band intensities were quantified using the ImageJ program and normalized with α-Tubulin, and the relative values of band intensities are marked. Cytokinesis-defective cellular phenotypes of each stable cell line were observed under a microscope using immunostained samples (B) and counted (C). A dot density graph with a box plot shows the percentage (%) of cells with cytokinesis-defective phenotypes (C). The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Each symbol in the graph represents a percentage of more than 30 cells from three independent experiments. ***, p<0.001 (one-way ANOVA); Scale bars, 10 μm.

(D) HeLa CCL2 cells infected with either control luciferase (shGL) or β-catenin (shβ-catenin) knockdown virus were subjected to FACS analysis. ***, p<0.001; *, p<0.05; NS, not statistically significant (unpaired two-tailed t test). Error bars, S.D. from three independent experiments.

(E, F) HeLa CCL2 cells transiently transfected with either GFP-β-catenin WT or GFP-β-catenin S60A were monitored during cell cycle progression by live cell imaging (E). The subcellular localizations of GFP-tagged β-catenin WT or S60A mutant construct were confirmed by immunofluorescence with anti-GFP antibody. The cells were additionally stained for Plk1 (see Appendix Figure S5A). The specific localization of GFP-β-catenin WT at the midbody was observed under a confocal laser microscope with high magnification (E, left panel). Cytokinesis-defective cellular phenotypes in each transfected HeLa cell were monitored (E, right panel) and counted using video clips (F). mCherry-Histone H2B was co-transfected with each GFP-β-catenin construct to visualize chromosomes. All live cell images and video clips show representative data of more than 50 telophase cells in each experimental group. Arrowhead indicates GFP−β-catenin WT located at the midbody in telophase HeLa CCL2 cells. Scale bars, 10 μm.
c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 Innate Lymphoid Cells (2022)
Figure 1

Figure 1: Human ILC2s express high level of c-Maf.

(A) Gating strategy used to identify ILCs and ILC subsets in humans (hILCs).

(B) Representative histogram and summary of the percentage and of the gMFI of c-Maf expression in hILC subsets (ANOVA analysis: F(2,18)=305.4, ****p<0.0001).

(C, D) Gating strategy for cKitlow and cKithigh ILC2s (C) and expression level (percentage and gMFI) of c-Maf in cKitlow and cKithigh ILC2s (D) (paired T tests, ****p<0.0001).

Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.
c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 Innate Lymphoid Cells (2022)
Figure 3

Figure 3: c-Maf deletion in mouse and human ILC2s dampens the production of ILC2-associated cytokines.

(A) Schedule for the injections of Tamoxifen and PBS or IL-33 in Id2-CreERT2+ (Id2-Cre+) or Id2-CreERT2- (Id2-Cre-) c-Maffl/fl mice.

(B) Level of c-Maf (2way ANOVA, interaction: F(1,20)=26.07, ****p<0.0001), percentage of lung ILC2s among CD45+ cells and level of activation of lung ILC2s in Id2-Cre-Maffl/fl (c-Maf WT) and Id2-Cre+Maffl/fl (c-Maf KO) mice after treatment as indicated in (A). Bars represent mean ± SEM.

(C) Workflow after transduction of ILC2s with shRNA.

(D, E) Relative mRNA level of c-Maf (T test, ****p<0.0001) (D), representative histogram and summary of the percentages of c-Maf+ ILC2s transduced with a control (Luc-sh) or a c-Maf targeting shRNA (Maf-sh) (E). Isotype indicates staining with the isotype control of the anti-c-Maf Ab (T test, ****p<0.0001). Bars represent mean ± SEM.

(F) Following the schedule indicated in (A), ILC2s were sorted from mouse lungs and stimulated for 48h with IL-33. Concentration of the indicated cytokines in the supernatants was measured (Multiple Mann-Whitney tests). (T test, *p<0.05, **p<0.01, ***p<0.001) Bars represent mean ± SEM.

(G) Human ILC2 sorted from peripheral blood and in vitro expanded were transduced with Luc-sh or Maf-sh as in (C). hILC2s were then stimulated for 48h with IL-33 and the concentration of the indicated cytokines in the supernatants was measured (Multiple Mann-Whitney tests, *p<0.05, **p<0.01) Bars represent mean ± SEM.

Data information: Each dot represents data from one individual mouse or from one donor.
c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 Innate Lymphoid Cells (2022)
Figure 5

Figure 5: c-Maf regulates Th2-associated gene expression in ILC2s.

(A) Principal component analysis of c-Maf knocked-down (empty circles) and WT (filled circles) mILC2s, performed using all genes (n=13855) detected by mRNA sequencing. Numbers indicate mouse IDs.

(B) Volcano plot showing genes more expressed in mILC2s purified from Id2-CreERT2-c-Maffl/fl mice (dark red), genes more expressed in mILC2s purified from Id2-CreERT2+c-Maffl/fl mice (dark blue), and genes not significantly differentially expressed (grey). The horizontal dashed line depicts the significance threshold at BH-adj. p-value=0.02, while the vertical dashed lines depict an absolute log2(fold change) threshold=1.

(C) Over-representation analysis of genes defining Th2, Th1 and Th17 cells (see Supplementary Table EV2 for a list of genes included in each signature) either for genes upregulated or downregulated in c-Maf WT ILC2s versus c-Maf knocked-down ILC2s.

(D) Genomic location of the top matches (red rectangles) of the mouse c-Maf binding profile (shown at the top) and of the MARE sequence identified by (Ho et al, 1996). Motif search was performed within 2 types of regions (purple rectangles): 1) The promoters of IL-4, IL-5 and IL-13, and 2) within open chromatin regions identified in 2 replicates of lung ILC2s or lung Th2 cells (dark blue tracks). The ATAC sequencing data (shown as bigwig coverage tracks) was retrieved from GEO (GSE77695). For each match of the c-Maf binding motif, numbers in parentheses indicate the log-likelihood ratio score and the raw p-value calculated by the FIMO tool. The transcripts of each gene are shown at the bottom of each panel. Mouse genome = mm9 and Integrative Genomics Viewer = v.2.8.0 (Robinson et al, 2011).

(E) ChIP-qPCR analysis of c-Maf occupancy on IL-4, IL-5 and IL-13 promoters in untreated mouse and human ILC2s (IL-2 stimulation) and in mouse and human ILC2s treated with IL-33 (n=3 biological replicates).

(F) Dot plot showing over-represented Gene Ontology gene sets among genes upregulated in wildtype compared to c-Maf knocked-down ILC2s. The color scale depicts the adjusted p-value, while the size of the dots and x-axis depict the absolute number or the proportion of upregulated genes included in each gene set, respectively.

(G) Sum of z-scores per sample of all genes upregulated in ST2+ Th2 cells and memory CD8 T cells (see Supplementary Table EV2 for a list of genes). The central band indicates the median, the box encompasses the interquartile range, and the whiskers reach the values that are within a distance of 1.5 times the interquartile range (either from the lower or upper quartile). The heatmap shows the z-scores of memory CD8 T cell genes significantly downregulated in c-Maf knocked-down ILC2s.
c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 Innate Lymphoid Cells (2022)
Figure 6

Figure 6: c-Maf induction enables ILC2s to respond stronger upon re-challenge.

(A) Treatment schedule.

(B) Representative staining and percentage of IL-13+ (ANOVA: F(2,22)=11.37, p=0.0004) and IL-5+ (ANOVA: F(2,22)=18.05, p<0.0001) ILC2s after 3h in vitro stimulation of cell suspension obtained from the lungs of mice treated as in (A). Each dot represent sample from one individual mouse (*p<0.05, ***p<0.001, ****p<0.0001). Bars represent mean ± SEM.

(C) Representative histograms of c-Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c-Maf+ and GATA3+ ILC2s from adult PBMC or cord blood (n=5 different donors; T test, ****p<0.0001).

(D) ILC2 expression of the indicated markers. Each dot represent sample from one donor. Bars represent mean ± SEM.

(E, F) c-Maf quantification by qPCR (E) and cytokine concentration (F) in the supernatants of adult and cord blood ILC2s unstimulated, stimulated for 3h with PMA-Ionomycin (3h stim) and stimulated for an overnight with IL-33 and then 3h with PMA-Ionomycin (o.n. stim). ILC2s were sorted from 3 different donors. (E) Multiple T tests, unstimulated: p=0.000101; 3h stim: p=0.0036. (F) Multiple T tests, IL-13: p=0.022; IL-5: p=0.015 (*p<0.05). Bars represent mean ± SEM.