EMBO/soda-nel huggingface dataset:
Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C (2020)
Figure 4

Figure 4. Phosphorylation of T651 stabilizes the loop conformation of CENPC-CT

A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome.

B) Structure of the loop region harboring phosphorylated T651CENP-C. CENPC-CT is shown in magenta, except for the pink canonical CENPC motif. The cryo-EM density map is overlaid on the cartoon model. The main chain interactions forming a short β-sheet are indicated by black dotted lines.

C) Magnified view of the structure around phosphorylated T651CENP-C. Key residues in this region are shown as stick models. The side chains of R656CENP-C and R71H2A are situated in the vicinity of the phosphoryl group of T651CENP-C in the distance ranges of 3.5-4 Å and 3.5-4.5 Å, respectively.

D) All constructs analyzed in (E) were stably expressed in CENP-C knockout cells.

E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.

F) Model indicating the functional role of T651CENP-C phosphorylation in the CENP-A-CENP-C interaction. Primarily, the CENPC motif associates with CENP-A nucleosome. Phosphorylation of T651CENP-C by CDK1 facilitates the loop conformation in the CM upstream region through an intramolecular bridge with the side chain of R656CENP-C. Subsequently, the N-terminal α-helix is folded over to stabilize the CENP-C-CENP-A nucleosome interfaces mediated by the CENPC motif and CM downstream regions.
Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C (2020)
Figure 6

Figure 6. A model of the dynamic change of binding partners of the CENP-A nucleosome during the cell cycle progression

A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3).

B) A model of the dynamic change of binding partners of the CENP-A nucleosome during the cell cycle progression in chicken (upper) or human (bottom). During the interphase, chicken CENP-C without phosphorylation at T651 is dissociated from the CENP-A nucleosome. Then, CENP-N can bind to the RG loopCENP-A of the CENP-A nucleosome in interphase cells. In mitosis, the CENP-C C-terminal region is phosphorylated by CDK1, and this phosphorylation facilitates CENP-A-CENP-C interaction. The stable CENP-A-CENP-C interaction excludes CENP-N from the RG loop. This phospho-regulation by the CENP-C C-terminal region is conserved in chicken and human. In humans, the second CENP-A binding region (central domain, CD) might associate with the CENP-A nucleosome throughout the cell cycle. However, it is still unclear how CD is involved in formation of centromeric chromatin structure (bottom).

Table 1| Statistics for Cryo-EM Single Particle Image Analysis and Structure Refinement

CA-CCCT (4.5Å)

(EMDB-30239)

(PDB 7BY0)

asymmetric

CA-CCCT-CNNT

(EMDB-30241)

CA-CCpep-CNNT

(EMDB-30237)

(PDB 7BXT)

CA-CCCT (6.78Å)

(EMDB-30240)

CA-CCCT-CNNT

complex

CA-CCpep-CNNT

CA-CCCT

a) FSC, Fourier shell correlation

b) R.m.s. deviations, root-mean-square deviations

Expanded View Figure legends
Cancer of Unknown Primary (CUP): genetic evidence for a novel nosological entity? 
A case report. (2020)
Figure 3

Figure 3 - Genetic analysis of fifteen CUP metastases.

A Somatic mutations distribution: SNVs and InDels displayed by each metastasis (listed in the Y axis) were detected by Strelka2 tool (Kim et al., 2018). Brown traits represent single mutations. The number of fully shared (red), partially shared (orange) or private (yellow) mutations is indicated in the horizontal bar below. Five cancer-associated genes (TP53, ARID2, NTRK1, SMARCA4 and ZFHX3) mutated in all metastases are highlighted.

B Genetic similarity among metastases: the heatmap has been drawn according to the Jaccard index.

The similarity ranges from 58% to 82% among all pairs with the exception of L_08 due to the scarce number of mutations.

C Clonal composition analysis: representative nested view of metastasis L_01. The five sub-clones (represented in different colours) inferred by clustering similarities of the variant allele frequencies (VAFs) follow a linear pattern.

D Variant allele frequency of fully shared mutations present in copy neutral regions. The box plot represents the fluctuation of the VAF (in the Y axis) of single mutations (grey dots) in each metastasis; each box represents the upper and lower quartiles, while the central short black line within each box represents the median; whiskers indicate variability outside the upper and lower quartiles.

E Reconstruction of the phylogenetic tree linking the fifteen CUP metastases: the model was reconstructed by taking into account the maximum likelihood molecular evolutionary tree according to Phylip (Felsenstein, 2005) and the incremental number of mutations. 'Brooks' lengths are proportional to the amount of 'private' mutations.

EXPANDED VIEW LEGENDS
Very Low Carbohydrate Diet Enhances Human T Cell Immunity through Immunometabolic Reprogramming (2021)
Figure 1

Figure 1 Beta-hydroxybutyrate enhances human T cell immune capacity in vitro.

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Human pan T cell RNA was isolated and cell culture supernatant was sampled.

A mRNA expression of CD4+ cytokines Il2, IL4, IL8 and IL22 relative to endogenous controls, n=13/11/10/8 biological replicates.

B Protein expression of IL2, IL4, IL6 and IL8, analyzed in the supernatant of stimulated PBMCs, n=9/12/11/11 biological replicates.

C Th1/Th2 cell transcription factors Tbet and GATA3 quantified via RT-qPCR and flow cytometric ratio of Th1/Th2 cells following differentiation, n=6/6/4 biological replicates.

D mRNA expression of CD8+ cytokines IFNγ, PRF1, TNFα, GZMB and CTLA4 in stimulated human T cells relative to internal controls, n=15/14/10/14/9 biological replicates.

E Protein expression of IFNγ/TNFα in the supernatant of stimulated PBMCs and cell lysis-dependent calcein-fluorescence of isolated T cells, n=8/12/9 biological replicates.

F Foxp3 mRNA relative to internal control in human PBMCs and quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg) following 5 days of Treg differentiation with a representative Foxp3 histogram plot (right side), n=7/11 biological replicates.

G IL10 and TGFβ1 mRNA and protein expression of human Treg cells, n=8/9 (IL10), 5/9 (TGFβ1) biological replicates.

Data information: Data depicted as mean ± SEM (protein data) and box plots with median, twenty-fifth and seventy-fifth percentiles and range (all other). Dots indicating individual values. *p<0.05, **p<0.01, ***p<0.001, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Very Low Carbohydrate Diet Enhances Human T Cell Immunity through Immunometabolic Reprogramming (2021)
Figure 3

Figure 3 Beta-hydroxybutyrate increases ROS formation in human T cells and directs them to memory cell formation in vitro.

Human peripheral blood mononuclear cells (PBMCs) were cultivated for 48h in RPMI containing 80mg/dl glucose (NC) and supplemented with 10mM beta-hydroxybutyrate (BHB). T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Flow cytometry analyses were performed using the indicated dyes and antibodies identifying T cells and CD4+/CD8+ T cell subsets. For JC1 analysis, magnetic CD4+/CD8+ T cell separation was performed prior to staining.

A Quantification of cellular reactive oxygen species (ROS) using CellROX dye, indicated by mean fluorescence intensity (MFI) FITC in human pan/CD4+/CD8+ T cells, n=8/6/6 biological replicates.

B Mitochondrial superoxide production quantified through MitoSOX staining, depicted as MFI Phycoerythrin (PE) in human pan/CD4+/CD8+ T cells, n=8/14/14 biological replicates.

C Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.

D Quantification of mitochondrial mass using MitoTracker green, indicated by MFI FITC in human CD4+/CD8+ T cells +/- CD3/CD28 stimulation, n=5/6/6/6 biological replicates.

E Western Blot of mitochondrial oxidative phosphorylation proteins in pan/CD4+/CD8+ T cells as indicated, representative of at least three individual experiments.

F Quantification of cellular glutathione content using ThioTracker, indicated by MFI FITC in human CD4+/CD8+ T cells, n=5/6 biological replicates.

G Evaluation of mitochondrial membrane potential of human pan/CD4+/CD8+ T cells using quantification of JC1 fluorescence (MFI PE/ FITC), FCCP served as the negative control, n=8/7/7/7(FCCP) biological replicates.

H IL15 mRNA expression in stimulated pan T cells, n=6 biological replicates.

I T memory cell differentiation protocol: Following initial incubation and stimulation, CD4+/CD8+ T cells were isolated via magnetic cell separation and cultivated with 50 U/ml IL2 and 25 ng/ml IL15 for an additional 72 hours.

J IL15 and IL7R mRNA expression in CD4+/CD8+ T cells after memory T cell differentiation, n=7/6 biological replicates.

K Flow cytometric quantification of memory T cells: CD4+/CD8+ T cells were stained for CCR7 (PE+/-) and subsequently defined memory phenotype for CD45RO+ (Pacific Blue+) and CD45RA- (PerCP-) staining. Histogram plots depicting the exemplary change of CD45RO and CD45RA distribution for CD8+ T cells following memory cell differentiation (left side; NC=black, BHB=red). Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (right side), n=12/11/8/7 biological replicates.

Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Very Low Carbohydrate Diet Enhances Human T Cell Immunity through Immunometabolic Reprogramming (2021)
Figure 5

Figure 5 Ketogenic diet enhances human T cell immune capacity in vivo.

Healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to (T0) and post KD (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD4+/CD8+ T cells were separated via magnetic cell labeling.

A IL2, IL4, and CTLA4 mRNA expression in stimulated CD4+ T cells, n=17/18/17 individual human subjects.

B Tbet and GATA3 mRNA expression in CD4+ T cells, n=7 individual human subjects.

C Flow cytometric quantification of Th1/Th2 cells and respective ratio of Th1/Th2 cells, n=7 individual human subjects.

D IL10 mRNA expression and flow cytometric quantification of CD4+CD25+Foxp3+ regulatory T cells (Treg), n=19/9individual human subjects.

E IFNγ GZMB, PRF1 and CTLA4 mRNA expression in CD8+ T cells, n=16/18/17/17 individual human subjects.

F relative CD8+ cell lysis activity as measured by calcein-fluorescence of isolated T cells, n=4 individual experiments.

Data information: Data depicted as box plots with median, twenty-fifth and seventy-fifth percentiles and range. Dots indicating individual values. *p<0.05, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.
Very Low Carbohydrate Diet Enhances Human T Cell Immunity through Immunometabolic Reprogramming (2021)
Figure 6

Figure 6 Ketogenic diet primes human T cells to mitochondrial metabolism and memory cell development in vivo.

44 healthy volunteers conducted a three week KD with a limited carbohydrate consumption of <30g per day. Blood was taken and analyzed prior to starting the diet (T0) and again after three weeks of strict adherence to the diet (T1). PBMCs were isolated. T cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. Pan/CD4+/CD8+ T cells were separated via magnetic cell labeling. Mitochondrial metabolism was analyzed for each subpopulation using a Seahorse XF96 Analyzer.

(A, B) OCR, maximum respiration, spare respiratory capacity and basal respiration were measured in A CD4+ and B CD8+ T cells, n=14 individual experiments, each performed in three technical replicates.

C Quantification of cellular and mitochondrial ROS using CellROX/ MitoSOX, indicated by MFI FITC/ MFI PE in human Pan/CD4+/CD8+ T cells, n=9/10/10 (CellROX), 12/11/12 (MitoSOX) individual human subjects.

D Quantification of mitochondrial mass using MitoTracker green, indicated by MFI FITC in human CD4+/CD8+ T cells, n=11/8 individual human subjects.

E Western Blot of mitochondrial oxidative phosphorylation proteins in CD4+/CD8+ T cells as indicated, representative of five individual experiments.

F Confocal microscopy images of human CD4+/CD8+ T cells, stained with MitoTracker green, representative of two individual experiments.

G Flow cytometric quantification of memory T cells in vivo: CD4+/CD8+ T cells were stained for CCR7 (PE+/-) and subsequently defined memory phenotype for CD45RA- (PerCP-) and CD45RO+ (Pacific Blue+) staining, representative histogram plots (top), T0=grey, T1=purple. Fractions of CCR7+CD45RA-CD45RO+ central memory (CM) and CCR7-CD45RA-CD45RO+ effector memory (EM) CD4+/8+ T cells (bottom), n=8/7 (CM/EM) individual human subjects.

H TruCulture IFNγ, IL4, IL6, IL8, IL12 subunit p40, IL23 and TNFα protein quantification of LPS-stimulated whole blood samples, n=11/12/11/10/11/10/11 individual human subjects.

Data information: Data depicted as mean ± SEM (OCR) or as box plots with median, twenty-fifth and seventy-fifth percentiles and range (all others), crosses indicating mean (A/B). Dots indicating individual values. *p<0.05, **p<0.01, paired t-test/ Wilcoxon matched-pairs signed rank test, as appropriate.

Expanded View Figure Legends
Dual melanocortin-4 receptor and GLP-1 receptor agonism amplifies metabolic benefits in diet-induced obese mice. (2015)
Figure 4

Effect of liraglutide and RM-493 co-treatment on glucose metabolism in DIO mice

A-I Glucose metabolic parameters were assessed following 5 days of treatment of DIO male mice with vehicle (white), liraglutide (10 nmol/kg) (gray), RM-493 (3.6 μmol/kg) (black), or liraglutide (10 nmol/kg) and RM-493 (3.6 μmol/kg) (checkered). (A) Fasted blood glucose levels, (B, C) glucose tolerance, (D) fasted insulin levels, (E, F) glucose-induced insulin secretion, (G) HOMA-IR, (H) insulin sensitivity and (I) hepatic phosphorylation of AKT (p-AKTSer473) were analyzed. Compounds were administered by daily subcutaneous injections. To assess insulin-stimulated p-AKTSer473, insulin (n = 5) or saline (n = 3) was injected 10 min prior to liver sampling. Data represent means ± SEM;n = 8 in (A–H); *P < 0.05, **P < 0.01, ***P < 0.001.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 1

Figure 1: ER stress-induced reduction of protein synthesis is preserved in PERK-deficient neurons

A Schematic overview of timeline and experimental setup.

B WB showing depletion of PERK in Perk KO astrocytes and neurons.

C Schematic representation of the core events of the PERK signalling pathway.

D-I eIF2α phosphorylation in WT and Perk KO astrocytes (N=3) and neurons (N=6) ± 20-24 hours of TM-induced ER stress. Representative WB (D, G), quantification of absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (E, H) and the ER stress-induced p-eIf2α response (F, I).

J-O Protein synthesis in WT and Perk KO astrocytes (N=5) and neurons (N=5) ± 20-24 hours of TM-induced ER stress. Representative images obtained by high-content microscopy showing puromycinilated proteins detected by immunofluorescence (J, M), quantification of puromycin intensity as measure for de novo protein synthesis (K, N) and the ER stress-induced protein synthesis response (L, O). Astrocytic soma (GFAP, white), dendrites (MAP2, white), nuclei ((Δ)Cre, red), de novo synthesized proteins (Puromycin, green). Scale bar: 25 μm.

Data information: (E, H, K, N) Data are normalised to untreated WT. (F, I, L, O) Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: ** P<0.01, *** P<0.001, **** P<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, H, K , N); Students t-test (F, I); Nested t-test (L, O). Source data is available online. See also Figure EV3 and Appendix Figure S1.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 2

Figure 2: The ER stress-induced ATF4 response is rescaled in PERK-deficient neurons

A-F ATF4 expression in WT and Perk KO astrocytes (N=3) and neurons (N=9) ± 20-24 hours of TM-induced ER stress. (A, D) Representative confocal images of ATF4 immunofluorescence. Astrocytic soma (GFAP, cyan), dendrites (MAP2, cyan), nuclei ((Δ)Cre, green), ATF4 (magenta). Scale bar: 10 μm. (B, E) Quantification of absolute nuclear ATF4 intensity. Data are normalised to untreated WT. (C, F) Quantification of the ER stress-induced ATF4 response. Data are related to untreated cells of the same genotype. Baseline level (without ER stress) is depicted by a dashed line.

G-J (G, I) Venn diagram showing the number of significantly upregulated ATF4 target genes in WT (grey and blue) and Perk KO (green and blue) astrocytes (G, N=3) and neurons (I, N=3) upon 20-24 hours of TM-induced ER stress determined by mRNA-seq. Percentage: fraction of upregulated ATF4 target genes in WT cells that are also upregulated in Perk KO cells. (H, N=3; J, N=3) Log2 fold change of ATF4 target genes that are significantly upregulated in both WT and Perk KO cells (blue in Venn diagram).

Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant p-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (B, E); Nested t-test (C, F). Source data is available online. See also Figure EV3 and Appendix Figure S2.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 3

Figure 3: Preservation of ER stress-induced translational control is a conserved neuron-specific feature

A-L Protein synthesis and ATF4 expression in mouse (A-F) and human (G-L) astrocytes (A-C and G-I, ) and neurons (D-F and J-L) ± 20-24 hours of TM-induced ER stress ± PERK-i (Pi). (A, D, G, J) Representative images obtained by high-content microscopy. Astrocytic soma (GFAP/Phalloidin, white), dendrites (MAP2, white), nuclei (ΔCre, red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. (B, N=5; E, N=5; H, N=3; K, N=3) Quantification of puromycin intensity as measure for de novo protein synthesis. (C, N=5; F, N=7; I, N=4; L, N=3) Quantification of absolute nuclear ATF4 intensity.

Data information: Data are normalised to untreated WT and presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *** p<0.001, **** p<0.0001. Statistical analysis: Two-way ANOVA with Tukey's post hoc test. Source data is available online. See also Figure EV4.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress (2022)
Figure 4

Figure 4: HRI mediates part of the ER stress-induced translational control in PERK-deficient neurons

A Oxidative stress related processes identified by GO analysis comparing TM-treated Perk KO and WT neurons (N=3).

B Oxidative stress related processes are only enriched in TM-treated Perk KO neurons. Source data ((adjusted) p-values, gene IDs and gene names) is available online.

C Schematic representation of the method to quantify ROS accumulation by nuclear CellROX green intensity.

D-G ROS accumulation in WT and Perk KO neurons (N=3) and astrocytes (N=3) ± 20-24 hours of TM-induced ER stress. (D, F) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), astrocytic soma (Phalloidin, white), nuclei ((Δ)Cre, red), ROS (CellROX green, green). Scale bar: 25 μm. (E, G) Quantification of CellROX green intensity.

H mRNA expression of Eif2ak1 (HRI) in HRI KD neurons determined by RT-qPCR, normalised to WT transduced with shRNA control (represented by a dashed line) (N=3).

I Quantification of the increase in nuclear ATF4 intensity in WT neurons ± HRI KD ± 20 hours of sodium arsenite (Ars) treatment (N=3). Representative images are shown in Appendix Figure S5.

J The remaining ATF4 response (%) upon HRI KD, related to ATF4 response of WT transduced with shRNA control (100%, represented by a dashed line) (N=3). Calculated from data in (I): [[(KD_Ars+-KD_Ars-)/KD_Ars-]/[(WT_Ars+-WT_Ars-)/WT_Ars-]]*100.

K-M eIF2α phosphorylation in HRI KD WT and Perk KO neurons (N=3) ± 20-24 hours of TM-induced ER stress. Representative WB (K), quantification of the ER stress-induced p-eIf2α response (L) and absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (M).

N-S Protein synthesis (N=3) and ATF4 expression (N=4) in HRI KD WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress. Representative images obtained by (N) confocal and (Q) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, green/red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis response (O) and puromycin intensity as measure for de novo protein synthesis (P). Quantification of the ER stress-induced ATF4 response (R) and absolute nuclear ATF4 intensity (S).

Data information: (E, G, M, P, S) Data are normalised to untreated WT. (L, O, R) Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, G, I, L, O, R); One sample t-test with a hypothetical value of 100 (H,J); Students t-test (M); Nested t-test (P, S). Source data is available online. See also Figure EV5 and Appendix Figure S2 and S4.