EMBO/soda-nel huggingface dataset:
Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress (2013)
f4

(a) Inhibition of ROCK1 kinase activity prevents metabolic stress-mediated phosphorylation of Beclin1. HeLa cells left untreated or treated with a ROCK1 inhibitor, Y27632 (10 μM), were starved of glucose (4 h) as indicated, and endogenous Beclin1 was immunoprecipitated and blotted against phospho-Ser/Thr antibody. Whole cell lysates were run as inputs for Beclin1 and β-actin. (b) Phosphorylation of Beclin1 by ROCK1. Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632, using recombinant His-Beclin1 (left) or Flag-Beclin1 immunopurified from transfected HeLa cells (right). Proteins were resolved by SDS–PAGE; phosphorylated protein was visualized with autoradiography, and Beclin1 by Coomassie staining. (c) Identification of ROCK1-mediated phosphorylation domains in Beclin1. The indicated Flag-Beclin1 fragments were purified from HeLa cells and used as substrates for in vitro ROCK1 kinase assay. 32P-autoradiogram (centre) analysed phosphorylation and western blotting (WB; right) determined protein levels. Schematic representation of Beclin1 domain structure and deletion constructs are shown (left). (d) Identification of the phosphorylation site on Beclin1. Representation of point mutations in the different domains of Beclin1 are shown (left). In vitro kinase assay using Flag-Beclin1 WT and mutants (T38A and T119A), immunoprecipitated from transfected HeLa cells, as substrate and recombinant ROCK1 was performed. Phosphorylation was detected by 32P-autoradiogram, and Flag-Beclin1 levels were examined by WB. (e) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by WB against phospho-T119 (Beclin) antibody and Beclin1. Input for Beclin1 was run on 7.5% gel and immunoblotted.
Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress (2013)
f5

(a) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl-2. HeLa cells, untreated or treated with 10 μM Y27632 (top panel), were cultured in control or glucose-free (4 h) medium, lysed and cell extracts prepared. Endogenous Bcl-2 was immunoprecipitated with cross-linked Bcl-2 agarose and resulting eluted immune complexes were resolved by SDS–PAGE and blotted against indicated antibodies. EJ shCont. and shROCK1#1 cells (bottom panel) were grown in control or starvation media for 4 h and Bcl-2 IP was performed as above. Inputs were rerun to confirm ROCK1 knockdown. (b) HeLa cells were transfected with full-length constructs of Flag-Beclin WT, Flag-Beclin T119A or Flag-Beclin T119E. Flag-Beclin WT cells were then treated with Y27632 for 8 h. Post transfection and treatment with Y27632, cells were starved in HBSS medium for 4 h, after which cells were collected and whole cell lysates prepared. IP with Flag-agarose was performed for 2 h and proteins bound to beads were eluted and used for western blotting. (c) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by western blotting against Bcl-2 and Beclin1. Inputs and IP were run on separate gels and same exposure is shown. (d) HeLa cells with a transient knockdown using siCont. and siBeclin1–3′-untranslated region for 30 h were further left untreated or treated with 10 μM Y27632. Cells were then incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS–PAGE and were analysed for Beclin1, LC3 and β-actin. (e) Endogenous LC3 staining in WT- or T119A-Beclin1-transfected cells. HeLa cells transfected with Flag-Beclin WT (±Y27632) and T119A were cultured in HBSS media for 6 h, fixed with cold acetone and endogenous LC3 immunofluorescence was performed. Representative images are shown; scale bar, 20 μm. Arrows indicate punctate LC3 staining. Graph represents mean±s.d., n=25, LC3 puncta/cell.
Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress (2013)
f6

(a) Genomic structure of ROCK1 (exon 4–6 only) with targeted allele, Flp-deleted allele and Cre-deleted allele. (b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane. (c) Mice were fed or starved for 16 h, and heart samples were prepared in lysis buffer. Resulting proteins were resolved by SDS–PAGE and blotted against ROCK1, LC3, autophagy marker p62 and β-actin. (d) ROCK1-deficient hearts or control hearts from animals fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures. Arrows indicate autophagic vacuoles. Scale bar, 500 nm. (e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.
Telomeric C-circles localize at the Nuclear Pore Complexes in Saccharomyces cerevisiae (2022)
Figure 1

Figure 1- Production of telomeric circles during senescence and survivor formation in S. cerevisiae.

A Schematic of the RCA assay. The Φ29 polymerase produces long telomeric single strand concatemers of the circular DNA in a self-primed reaction.

B Schematic of the senescence assay. Senescence assays were performed starting from the spores isolated from the heterozygous telomerase-negative diploids. The spore colonies were inoculated at OD600=0.01 in 15 ml liquid YPD medium. Every 24 hours, the cell density was measured and a new culture was restarted at OD600=0.01. Samples were collected at each time-point for further analyses.

C Upper panel: senescence curve of a representative est2∆ clone. DNA samples were prepared at the indicated time points to analyze telomere length (middle panel) and t-circles production (lower panel and graph) during senescence and cell immortalization. Telomeres were analyzed by Southern blot of the XhoI cut DNA hybridized with a TG1-3 probe. The presence of t-circles was monitored by RCA and analyzed by TG1-3 probed dot blot. The graph shows signal intensities of the dots measured with the Fiji software (arbitrary units). Similar results were obtained with at least 16 other est2∆ clones.

D RCA products were resolved by denaturing agarose gel electrophoresis, transferred on a membrane and hybridized with a telomeric TG1-3 probe.

E) Dot blot of the RCA product either treated or not with the Exonuclease VII that degrades linear single-stranded DNA in both the 3' to 5' and 5' to 3' directions (upper panel). The membrane was hybridized with a telomeric TG1-3 probe. The signal intensities of the dots are shown (lower graph).
Telomeric C-circles localize at the Nuclear Pore Complexes in Saccharomyces cerevisiae (2022)
Figure 3

Figure 3- C-circles bind to the NPCs.

A Senescence curves of four est2∆ and six est2∆ NIC96-RFP independent clones used to generate type II survivors for C-circles co-IP experiments. The time when the cells were collected for the Co-IP is indicated by an arrow. The results are shown for one of the 3 independent experiments with similar outcomes.

B Telomere length and recombination were analyzed by TG1-3 probed Southern blot of XhoI-digested DNA prepared from samples of the replicative senescence. Two representative clones are shown.

C Detection of the t-circles co-immunoprecipitated with Nic96-RFP from the extracts obtained from several type II survivors. The DNA co-immunoprecipitated with Nic96-RFP was amplified by RCA assay, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to the growth curves in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Nic96-RFP IP that was evaluated by western blot with anti-mCherry antibody.

D Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of ADA2 deletion. Dot signal intensities were quantified using the Fiji software and were calculated as the percentage of INPUT that was immunoprecipitated normalized to est2∆ signals. Relative signals were calculated by setting the mean of est2∆ signals from each independent experiment to 1. Means and s.e.m. of the six independent co-IPs experiments each with 2-6 clones per genotype are shown. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,000007), est2∆ NIC96-RFP versus est2∆ ada2∆ NIC96-RFP (p=0,00012).***P<0,0001.

E Quantification of the RCA product after co-IP of the C-circles with Nic96-RFP showing the effect of SUS1 deletion. Quantification was done as in D. The graph shows means and s.e.m. of the three independent co-IPs with 2-5 clones per genotype. Outliers were removed using ROUT robust outlier test. The p-values from one-way ANOVA were were adjusted using the Bonferroni's correction for multiple comparisons. est2∆ versus est2∆ NIC96-RFP (p=0,002), est2∆ versus est2∆ sus1∆ NIC96-RFP (p= 0,0006). **P<0,001, ***P<0,0001.
Telomeric C-circles localize at the Nuclear Pore Complexes in Saccharomyces cerevisiae (2022)
Figure 4

Figure 4- C-circles interact with SAGA/TREX2.

A Senescence curves of two est2∆ and two est2∆ ADA2-Myc clones used to generate type II survivors for Co-IP-RCA. This experiment is representative out of the 4 independent experiments performed during this study.

B Telomere length and recombination were analyzed by TG1-3-probed Southern blot of XhoI-digested DNA prepared from samples of the replicative senescence.

C Detection of the C-circles co-immunoprecipitated with Ada2-Myc in type II survivors. The DNA co-immunoprecipitated with Ada2-Myc was amplified by RCA, loaded on a dot blot and detected with a telomeric TG1-3 probe. The numbers above the blot refer to clones in (A) and correspond to type II survivors produced in independent cultures. The lower panel shows the efficiency of Ada2-Myc IP analyzed by western blot with anti-Myc antibody.

D Quantification of the dot blot signals in Ada2-Myc co-IP. Dot signals were quantified as in Fig 3D. The graph shows means and s.e.m. from n= 3 independent co-IP experiments each performed with 3-4 independent clones per genotype. The p-value is obtained for the Mann-Whitney two-tailed test (***p= 0. 00000005).
Telomeric C-circles localize at the Nuclear Pore Complexes in Saccharomyces cerevisiae (2022)
Figure 5

Figure 5- Inactivation of SAGA/TREX-2 affect type II telomere recombination.

A Mean senescence profiles of the est2∆ (n=7) and est2∆ sus1∆ (n=8) clones isolated from the same heterozygous diploid. The error bars are SDs.

B Mean senescence profiles of the est2∆ (n=10) and est2∆ sac3∆ (n=10) clones isolated from one heterozygous diploid. The error bars are SDs.

C Duration of the short telomere-induced crisis of the clones analyzed in A). The crisis period was determined as the number of days the cell population stayed arrested without notable increase in cell density. The average crisis period is plotted for each genotype. est2∆ ada2∆ data are from (Churikov et al 2016) and are shown for comparison. The error bars are SD of n independent clones. est2∆ (n=17), est2∆ ada2∆ (n=11), est2∆ sus1∆ (n=8) and est2∆ sac3∆ (n=10). The p values are from two-tailed Student's tests. p=0,000087 for ada2∆, p=0,047 for sus1∆, p=0,049 for ada2∆.*P<0,05, ***P<0,0001

D Frequencies of the telomerase-independent survivor types formed by the clones shown in A.
Telomeric C-circles localize at the Nuclear Pore Complexes in Saccharomyces cerevisiae (2022)
Figure 6

Figure 6- The nuclear diffusion barrier promotes type II recombination.

A Mean senescence profiles of the est2∆ (n=10) and est2∆ shs1∆ (n=16) clones. The error bars are SDs.

B Mean senescence profiles of the est1∆ (n=6) and est1∆ bud6∆ (n=7) clones. Est1, similarly to Est2, is essential for telomere maintenance. est1∆ has been used in this experiment instead of est2∆ because EST2 and BUD6 genes are linked on chromosome XII. The error bars are SDs.

C Duration of the short telomere-induced crisis for the clones analysed in A) and B). The crisis period was determined as the number of days the cell population stayed arrested without notable increase in cell density. The average crisis period is plotted for each genotype. The error bars are SDs of n independent clones: est2∆ (n=10), est2∆ shs1∆ (n=16), est1∆ (n=6) and est1∆ bud6∆ (n=7) . P values are from two-tailed Student's tests (*p=0.014).

D Frequencies of the survivor types formed by the clones used in the senescence assays shown in A and B.
Nucleosome dynamics of human iPSC during neural differentiation (2019)
Figure 3

Figure 3: The relationship of positioned nucleosomes at the TSS and gene expression.

A, B Nucleosome maps at and surrounding, the TSS region of the NANOG and GRIA1 genes in pl-iPSC (upper, black) and in NPC cells (lower, grey). The TSS is indicated by a dashed line at chr 12: 7,941,991 for NANOG and chr5: 152,870,105 for GRIA1. * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene.

C Global frequency distribution of nucleosome distributions at TSS of protein-coding genes. Alignment of nucleosome maps centred on TSS using the non-redundant list of 66,047 mapped human protein-coding gene TSS. The features corresponding to the nucleosome free region (NFR) and -1 to +2 nucleosomes are marked.

D Genome-wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene. The number of nucleosomes observed are plotted for both pl-iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl-iPSC (n=3,833) or NPC (n=2,082). If positioned nucleosomes are a strong predictor of gene activity, high numbers of nucleosomes would be expected for pluripotent-specific genes in pl-iPSC, but not NPC stages, and for NPC specific genes in NPC, but not pl-iPSC stages. No correlation between gene activity and positioned nucleosomes was observed.
Nucleosome dynamics of human iPSC during neural differentiation (2019)
Figure 4

Figure 4: Nucleosome patterning associated with the RE1 NRSF/REST binding motif.

A The average distribution of nucleosomes centred on the RE1 site (n=871), spanning 2 Kb of flanking genomic sequence.

B, C Aligned pair-end sequence read data were stratified into two size classes, (B) 122-137 bp and (C) 162-188 bp for pl-iPSC and NPC. Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site (n=871). Peaks correspond to the NRSF/REST complex, and nucleosomes in B and C respectively.

D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp. RE1 sites were clustered based on the 122-137 bp mid-point sequence read values (NRSF/REST protein complex) from pl-iPSC, shown as a frequency distribution in upper left panel. The frequency distribution data for the same sites was plotted using 122-137 bp data from NPC (upper right panel) and 162-188 bp data (nucleosomes; bottom panels) for the strongest (solid line) and weakest (dotted line) clusters.

E Schematic of nucleosome patterning at the RE1 site in pl-iPSC and NPC. Nucleosomes are represented by filled black circles.
Nucleosome dynamics of human iPSC during neural differentiation (2019)
Figure 5

Figure 5: Nucleosome patterning associated with the CTCF binding sites

A Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp centred on the CTCF binding site (n=9516). This shows a distinctive peak corresponding to the CTCF protein complex, which is present in pl-iPSC, but reduced in NPC.

B Average frequency distribution for sequence read mid-point data centred on the CTCF binding site in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC cells. This shows that the pattern of nucleosome positioning is independent of the amount CTCF protein complex. Although positioned nucleosomes are retained flanking CTCF sites, their positions are shifted closer to the CTCF site and their spacing is altered.

C Average frequency distribution for sequence read mid-point data centred on the CTCF site for pl-iPSC (162-188 bp) and K562 (total MNase-seq data) cells. Positioned nucleosomes are retained flanking CTCF sites in K562 cells, but with shifted positions and altered spacing.

D Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp for pl-iPSC and NPC cells centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. A distinctive peak corresponding to the CTCF protein complex is present in the NPC state, but reduced to background levels in pl-iPSC.

E Average frequency distribution for sequence read mid-point data centred on the CTCF binding sites in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. Nucleosome arrays are present in both cell states, independent of the CTCF protein complex. Nucleosomes in the CTCF sites in pl-iPSC tend to position closer to the CTCF site than in NPC. The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed.

F Schematic of nucleosome patterning at the CTCF site in pl-iPSC and NPC. Nucleosome arrays are present in both cell states in the absences of CTCF complex. When CTCF is present nucleosomes reposition to move a small distance from the CTCF binding site and increase their spacing. Nucleosomes are represented by filled black circles.

EV Figure legends
Store-Operated Calcium Entry (SOCE) Controls Innate and Adaptive Immune Cell Function in IBD (2022)
Figure 1

Figure 1. Deletion of SOCE in CD4+ T cells suppresses the induction of colitis in mice.

(A-E) Naïve CD4+ T cells were isolated from the spleen and LNs of WT, Stim2fl/fl Cd4Cre (S2), Orai1fl/fl Cd4Cre (O1) and Stim1fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17 and iTreg cells. (A) Fura-2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca2+ traces (left) and peak Ca2+ levels (right). Data are from the mean ± SEM of 4 mice per group. (B) Induction of colitis in Rag1-/- mice by adoptive transfer of naive CD4+ T cells from wildtype, Stim2fl/fl Cd4Cre, Orai1fl/fl Cd4Cre and Stim1fl/fl Cd4Cre mice. Weight curves of recipient Rag1-/- mice following injection of naive CD4+ T cells. Data are from two independent experiments with 8 recipient mice per group. (C) H&E stains of colon tissues and histological inflammation scores of Rag1-/- mice 8 weeks after T cell transfer. Data are from one representative experiment with 5 mice per group. (D-E) Representative flow cytometry plots and frequencies of donor CD4+ T cells producing IFNγ, IL-17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1-/- mice. Data are from 2 independent experiments with 3-8 mice per group. Statistical analyses by unpaired student's t-test: ***p<0.001 **p<0.01 *p<0.05.
Store-Operated Calcium Entry (SOCE) Controls Innate and Adaptive Immune Cell Function in IBD (2022)
Figure 2

Figure 2. Inhibition of SOCE reduces pro-inflammatory cytokine production by human lamina propria mononuclear cells (LPMC) in a dose-dependent manner.

(A,B) Ca2+ influx in human lamina propria CD4+ and CD8+ T cells from 3 IBD patient samples analyzed by flow cytometry. (A) Experimental design. (B) Ca2+ influx rates in T cells pre-incubated for 4 h in the presence of 15-1000 nM BTP2, which was present until data acquisition. SOCE was induced by stimulation with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG) in Ca2+ free buffer followed by the addition of 2 mM Ca2+. Bar graphs show the mean ± SEM of Ca2+ influx rates after Ca2+ readdition from 1 experiment (n=3 IBD patients). Statistical difference were calculated using a RM one-way ANOVA test: ****p<0.0001 ***p<0.001 **p<0.01 *p<0.05.

(C-G) Analysis of LPMC from 5 CD patients. (C) Experimental design for mass cytometry. (D) viSNE plots of CD45+CD3+ LPMCs stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 hours in the presence of 15-1000 nM BTP2. viSNE plots are colored according to the expression level of IL-2 (blue: low, red: high) and are representative of one CD patient. (E) Dose-response curves of the frequencies of cytokine producing CD4+ and CD8+ T cells after PMA/ionomycin stimulation for 4h in the presence of increasing doses of BTP2. The frequencies of cytokine producing cells were normalized to control samples treated with PMA/ionomycin alone. Bar graphs show the mean ± SEM of 5 CD patient samples. (F) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45+CD3+ LPMCs stimulated for 4h with PMA/ionomycin in the presence of BTP2; data are normalized to PMA/ionomycin treatment alone. Statistical significance was calculated using a one-tailed Wilcoxon matched-pairs signed rank test, *p< 0.05. (G) Cytokine production by CD19+HLA-DR+ B cells and CD11c+ dendritic cells in the presence of increasing doses (15-1,000 nM) of BTP2. Left: Dose-response curves showing frequencies of cytokine-producing cells after 4h stimulation with PMA/ionomycin ex vivo. Data are normalized to control samples stimulated with PMA/ionomycin in the absence of BTP2. Error bars represent SEM obtained from 1 experiment (n=5 CD patients). Right: heatmaps showing the median fold change of cytokines and surface markers in CD19+HLA-DR+ B cells and CD11c+ dendritic cells treated as described for the left panel. Significant fold changes in expression levels between samples were calculated using a one-tailed Wilcoxon matched-pairs signed rank test, *p < 0.05.
Store-Operated Calcium Entry (SOCE) Controls Innate and Adaptive Immune Cell Function in IBD (2022)
Figure 3

Figure 3. Global mass cytometric analysis of the immune cell composition in the colonic lamina propria of therapy refractory IBD patients.

(A) Experimental setup for mass cytometric assays. Non-inflamed: n = 4, CD: n =6, UC: n =6.

(B) FlowSOM plot of merged FCS files from LPMCs of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4h ex vivo. Colors of the t-SNE plot represent 20 clusters of distinct CD45+ LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters.

(C) FlowSOM map of merged FCS files from CD45+ cells of non-inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients.

(D) viSNE plots of one exemplary non-inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high).

(E) Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure (*padj< 0.05, **padj < 0.01, ***padj< 0.001). Boxes extend from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median from 1 experiment (non-inflamed n = 4, CD: n =6, UC: n =6 patients).

(F) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45+ LPMCs that were isolated from non-inflamed, UC and CD samples and activated for 4h with PMA/ionomycin ex vivo. Statistical significance was calculated using an unpaired t test with FDR adjustment to 1% using the Benjamini, Krieger and Yekutieli procedure; *padj < 0.05, **padj< 0.01, ***padj < 0.001.