EMBO/sd-character-level-ner huggingface dataset:
Figure 6Western blot analysis of cytoplasmic TDP-43 protein levels in healthy controls (C0, C1 and C2), isogenic P1 (Iso-P1) and mutant P1 (P1) motor neurons. Quantification of cytoplasmic TDP-43 levels, each dot represents an independent differentiation, ratio paired-t-test. Western blot analysis of nuclear TDP-43 and D quantification of nuclear TDP-43 levels, each dot represents an independent differentiation, ratio paired-t-test. Immunofluorescent analysis showing fluorescence intensity of pTDP-43 in motor neurons. Scale bar: 10µm and its perspective intensity plot in F. G Quantification of E, each dot represents one analysed cell, unpaired Mann-Whitney-test. Western blot analysis showing pTDP-43 levels in healthy controls, Iso-P1 and P1, quantification in I, each dot represents one analysed cell, unpaired Mann-Whitney test. axonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K) and number of stationary mitochondria (L) normalized to neurite length 100µm.
Figure 5A. H&amp;amp;E-stained sections of seminiferous tubules from testes of WT and Brca1ex2/ex2/Trp53bp1-/- male mice. Scale bar: 100 μm. B. Immunofluorescence on meiotic chromosomes from WT and Brca1ex2/ex2;Trp53bp1-/-spermatocytes. Spreads were stained for SYCP3 to indicate chromosome axes and extent of synapsis and were stained for SYCP3 to indicate chromosome axex body. Scale bar: 5 apsi C. Quantification of the number of γ-H2AX foci observed at the pachytene stage in the indicated genotypes. P value is Mann-Whitney, two-tailed. D. G2M checkpoint analysis in mouse B cells after IR treatment. Left, mitotic cells were identified by flow cytometry as having 4c DNA content (based on propidium iodide staining) and pSer10-H3+. Right, quantification of flow cytometry data, showing mitotic cells after IR as percentage of that seen in untreated cells. N≥2.
Enteroblast life and death decision is governed by opposing Notch and EGFR activity (A-E) Confocal images of representative midguts of the indicated genotypes using kluReDDM for tracing and age- and EB-specific transgene expression at day 7 after temperature shift.Figure 3(A-E) Confocal images of representative midguts of the indicated genotypes using kluReDDM for tracing and age- and EB-specific transgene expression at day 7 after temperature shift. ISC mitosis was monitored by using PH3 labelling (white) in all guts. (B-E) Accumulation and loss of klu+ cells by loss (B and E) and gain (C and D) of N and EGFR signalling. In (C), the accumulation of EC-committed EBs (klu+ cells) in midguts with constitutively activated EGFR (kluReDDM> EGFRact) is accompanied with a non-autonomous increase in ISC mitosis. Inset in (C) shows labelling with Dl (blue), revealing symmetric ISC mitoses Data information: Scale bar, 100µm for all images. (F) Quantification of the number of klu+ (EBs) in the indicated midguts (n= 21, 14, 5, 6, 8, 6, 4, 10, 12, 9, 13, 14). Data information: Note that impaired cell renewal reduces the intestinal width. **, P< 0.01; ****, P< 0.0001, one-way ANOVA with Bonferroni correction. (G) Quantification of apoptotic esg+ (TUNEL+ GFP+) cells in the indicated midguts (n= 28, 4, 7, 7, 16, 8, 8, 7, 10, 7, 20, 24). Data information: Note that impaired cell renewal reduces the intestinal width. **, P< 0.01; ****, P< 0.0001, one-way ANOVA with Bonferroni correction. Confocal images of representative midguts of the indicated genotypes Rescue of the loss of EBs induced by gain of Notch (H-RNAi) by expressing Debcl-RNAi. Data information: Scale bar, 100µm for all images. Confocal images of representative midguts of the indicated genotypes escue of the loss of EBs induced by loss of EGFR (EGFRDN) by expressing Debcl-RNAi. (J and K) Impact of gain of expression (J) and loss by RNAi expression (K) of lz using kluReDDM White arrowheads point to examples of mitosis labelled by PH3+. (L and M) Impact of gain of expression (L) or loss by RNAi expression (M) of klu (M) using kluReDDM. Data information: Scale bar, 100µm for all images. (N-R) Images of representative midguts of esgReDDM at day 7 after temperature shift of the indicated genotypes. PH3 labelling (white) detected rare mitoses in the mutant genotypes. Data information: Scale bar, 100µm for all images.
Elevated miR-21-3p level correlates with exacerbated inflammation in murine skin exposed to acute UV.Figure 5(A) RT-qPCR quantification of Il6, Il1b, Ptgs2 and Ccl5 levels in the epidermis of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Il6: Ppard +/+ no UV vs Ac-UV P = 7E-04, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.008; Il1b: Ppard +/+ no UV vs Ac-UV P = 0.010, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.016, Ppard -/- no UV vs Ac-UV P = 0.015; Ptgs2: Ppard +/+ no UV vs Ac-UV P = 0.028, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.028, Ppard -/- no UV vs Ac-UV P = 8E-04; Ccl5: Ppard +/+ no UV vs Ac-UV P = 0.002, Ppard +/+ Ac-UV vs Ppard -/- Ac-UV P = 0.014, Ppard -/- no UV vs Ac-UV P = 0.010; ns: non significant. N=4 animals per group, one representative experiment is shown out of three independent replicates. (B) Left panel: Immunostaining for infiltrating F4/80-positive macrophages in acutely irradiated (Ac-UV) and non-irradiated (no UV) Ppard +/+ and -/- miceskin sections. Magnification bar: 200m. Right panel: RT-qPCR quantification of relative levels of the macrophage marker Emr1 mRNA in total skin of acutely irradiated (Ac-UV; +) and non-irradiated (-) Ppard +/+ and -/- mice. Emr: Ppard +/+ no UV vs Ac UV P = 0.034; Ppard +/+ Ac UV vs Ppard -/- Ac UV P = 0.033. N=4 animals per group, one representative experiment is shown out of three independent replicates. (C) RT-qPCR quantification of relative levels of Il6, Il1b, Ptgs2 and Tnfa in mouseepidermis acutely irradiated (Ac-UV; +) and non-irradiated (-), treated with miR-21-3p inhibitor (+) or mismatched control (-) as indicated.
Pacs1 deletion causes a defect in cytosolic Ca2+ flux after antigen receptor stimulation (A) Pacs1+/+ and Pacs1−/− splenocytes were labeled with Indo-1 and stained for B220, CD21, and CD23 to identify FOB and MZB cells.Figure 2(A) Pacs1+/+ and Pacs1−/− splenocytes were labeled with Indo-1 and stained for B220, CD21, and CD23 to identify FOB and MZB cells. Fluorescence was measured for 30 seconds to establish a baseline and then cells were stimulated with the indicated amounts of anti-IgM (black arrowhead). Cytosolic Ca2+ flux was monitored with FACS analysis by measuring the violet:blue fluorescence emission ratio of Indo-1. Kinetic traces are displayed from five independent Pacs1+/+ and Pacs1−/− pairs and were normalized to baseline (Pacs1+/+ gray traces, Pacs1−/− pink traces). The mean Ca2+ flux for each genotype is overlaid in bold (­Pacs1+/+ black, Pacs1−/− red). (B) Maximum Ca2+ flux (peak height) at each anti-IgM concentration for FOB and MZB cells. Paired t test, ns = not significant, *P < 0.05, and ***P < 0.001. (C) Pacs1+/+ and Pacs1−/− FOB cells were labeled with Indo-1 and stimulated in Ca2+ free buffer with 5 mcg/ml anti-IgM (black arrowhead) to assess ER Ca2+ efflux. Then, 2 mM Ca2+ was added back to assess SOCE. Kinetic traces normalized to baseline from three independent Pacs1+/+ and Pacs1−/− pairs are shown with the mean Ca2+ flux overlaid in bold. (D) Maximum Ca2+ flux after stimulation under Ca2+ free conditions and after Ca2+ was added back. Two-tailed paired t test, ns = not significant, *P < 0.05.
Identification of ECHDC1 as breast cancer tumor suppressor gene.Figure 5(A) The CLIP-signature of ECHDC1 suggests that it was hypermethylated and down-expressed in all the breast cancer cell lines (n=24) in which it was identified as rCCS gene. (B) Breast cancer specific survival (BCSS) based on gene expression and methylation levels of ECHDC1 in breast cancer patient tumors in the combined Metabric and Oslo datasets (n=3885). Patients in the low GEXP category class have lower BCSS than those in the non-low GEXP group. Numbers above the x-axis line indicate the number of patients in each group, defined by the color code, at each time point. P-value from age-adjusted Cox-proportion hazard model. (C) Benign breast epithelial MCF10A and breast carcinoma BT-474 cells were embedded in 3D collagen as single cells or as spheroids, respectively, and the growth was followed for 5 days. Light micrographs show filamentous actin (phalloidin) and nuclei (Hoechst) in representative cell colonies. Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout. At 72 hours, MCF10A mock versus ECHDC1_sgRNA_1 and ECHDC1_sgRNA_2 P < 0.05; at 96 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.001; at 120 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.0001. Nuclei count relative to mock 0 hours. Error bars indicate mean ± SEM; n ≥ 10 colonies. Statistical significance was assessed with one-way ANOVA with Tukey's multiple comparison test. Scale bar 50 µm. (D) Metabolic pathway of propanoate metabolism. (E) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status (n=7 in both groups). Boxes represent the interquartile range, whiskers represent the range of the values and solid line within the box correspond to the median value. Outlier points indicates values not included between the whiskers. Statistical significance was assessed with Wilcoxon test.
Figure 3Immunofluorescence showing the Ago3 signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This panel arrangement applies also for panels B, H and I. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 106; Small: IntDen value < 106). Immunofluorescence showing the Vret signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). Western blotting was performed before and after Siwi KD using the anti-Ago3 antibody. Total BmN4 lysate (Total), supernatant (Sup) and pellet (Pellet) fractions upon centrifugation were used. Western blotting was performed using the anti-Ago3 antibody on Ago3 immunoisolated from Siwi-depleted cells. Ago3 immunoprecipitated was untreated (−) or treated with phosphatase (+) prior to western botting. Western blot showing the degree of phosphorylation of Flag-Ago3 WT and the 8SA mutant in Siwi-depleted cells (Siwi KD). Western blots showing the degree of phosphorylation of Flag-Ago3 WT and its DDH and KA mutants in Siwi-depleted cells (Siwi KD). The second panel from the top corresponds to the top panel with a longer exposure time. Siwi and β-Tub as a loading control were also detected also by western blotting. Western blotting was performed using anti-Ago3, anti-Vret and anti-Siwi antibodies on lysates of BmN4 cells before (Control) and after Siwi (Siwi KD) and Siwi/Vret (Siwi/Vret KD) depletion. β-Tub was detected as a loading control. Immunofluorescence showing the Flag-Ago3 signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Flag-Ago3 8SA and 8SE mutant signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.
Figure 6A Venn diagram showing the number of significantly downregulated genes upon YAP/TAZ knockdown in resistant SKMEL28 and WM3248 cells. B Gene ontology analysis of significantly downregulated genes upon YAP/TAZ knockdown in resistant SKMEL28 and WM3248 cells. The list of significantly downregulated genes (Fold change > 2.0 and p-value 0.05 by LPE test) were submitted to functional annotation chart analysis of DAVID, using Biological Process Gene Ontology database. Top 5 enriched gene ontology terms are shown. C Significantly enriched transcription motifs in promoters (from -800 to +200 of transcription start sites) of downregulated genes upon YAP/TAZ knockdown, predicted by TransFind software. D GSEA analysis of enriched gene sets downregulated in response to YAP/TAZ knockdown. E GSEA enrichment plots demonstrating downregulation of YAP, E2F1, EGFR and c-MYC pathway signatures after YAP/TAZ knockdown in resistant SKMEL28 and WM3248 cells. F A heatmap of top 10 E2F1, EGFR and c-MYC pathway signature genes on GSEA. Expression levels are shown in log2 scale. Red color indicates higher expression levels and blue color indicates lower expression levels than median.
CTD shortening leads to minor changes in Pol II pausing at steady state.Figure 3A- CTD shortening leads to a decreased Pol II occupancy in promoter-proximal region. Median coverage plot of PRO-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. B,C- Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp. D- Median coverage plot of TT-seq signal at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. E- Box plot showing productive transcription initiation rate in RPB1-52R and RPB1-25R cells for genes encoding major isoforms. p value=0.13 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. F- Box plot showing Pol II pausing duration in RPB1-52R and RPB1-25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. G- Median scaled coverage plot of TT-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. H- Pol II velocity remains unchanged upon CTD shortening. Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. I- Median coverage plot of TT-seq signal centered at PAS at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed. J - Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. K- Box plot showing distance from the polyadenylation site (PAS) to the transcription termination site (TTS) in RPB1-52R and RPB1-25R cells for 1958 genes encoding major isoforms. p value= 0.8 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.
Figure 8Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis. Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to D Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population. Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies. Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.
Fig.Figure 8A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE. B: Left panel: The PP2A inhibitor okadaic acid rescues the y-H2AX defect of the "Ubc9 DA" cells. "Ubc9 wt" or "Ubc9 DA" cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence microscopy was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images. C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on "Ubc9 DA" cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.
Figure 3CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (A-F) Cytokine secretion was measured by ELISA after incubating CD4+ (A,C,E) and CD8+ (B,D,F) T cells for 48 hrs in resting conditions (A,B) or stimulated with either anti-TCRβ (C,D) or anti-CD3/CD28 antibodies (E,F). CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR4KO mice by cell sorting. (G) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4+ and CD8+ T cells. CD4+ and CD8+ T cells were isolated from the spleens of WT, and EGR4KO mice by cell sorting. (H) Comparison of IFNγ levels in WT vs. EGR4-/- T cells. CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (I) Representative FACS plots of CD4+ T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL-17 antibodies. CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (J) Comparison of the number of Th1 polarized cells between WT, EGR1-/- and EGR4-/- CD4+ T cells. CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (K) CD4+ T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA.
AβOs bind to and impact hypothalamic neurons.Figure 3Representative immunocytochemistry images of mature hypothalamic neurons in culture exposed to vehicle (Veh) or AβOs (500 nM) for 3 h. Binding of AβOs was detected using anti-oligomer monoclonal antibody NU4 (red). Neurons were double-labeled using MAP-2 antibody (green). Images represent typical results from experiments with three independent hypothalamic cultures (three coverslips/experimental condition per independent experiment). Scale bar = 30 and 10 μm for main panels and insets, respectively. Representative immunocytochemistry image of mature hypothalamic culture exposed to AβOs (500 nM) and immunolabeled with anti-GFAP (green) and NU4 (red) antibodies. Insets show AβOs binding to neuronal dendrites, whereas no binding was detected to GFAP-positive cells. Representative DCFfluorescence images from hypothalamic neuronalcultures exposed to vehicle or AβOs (500 nM, 4 h). Insets show optical zoom images of the indicated areas. Scale bars = 100 and 50 μm for main panels and insets, respectively. Graph shows integrated DCFfluorescence intensities (relative units; see Materials and Methods) (n = 3 independent hypothalamic cultures; three wells/experimental condition per experiment; three images acquired per well). Bars represent means ± SEM. #P = 0.0604, one-sample t-test compared with a fixed value of 100 RUs. LDH activity (IU/l) in culture media of hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h). Representative immunofluorescence images of eIF2α-P in hypothalamic cultures exposed to vehicle or AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Scale bar = 30 μm. Graph represents integrated immunofluorescence intensities of eIF2α-P levels from three independent hypothalamic cultures (three coverslips/experimental condition per experiment, 20 images per coverslip). Bars represent means ± SEM. *P = 0.0489, one-way ANOVA followed by Bonferroni post hoc test comparing AβO-treated versus vehicle-treated cultures. Representative images of hypothalamic cultures exposed to AβOs (500 nM, 3 h) and double-labeled with NU4 (oligomer-sensitive) and eIF2α-P antibodies. Arrow points to a neuron presenting high levels of eIF2α-P in the absence of AβO binding. Nuclear staining (DAPI) is shown in blue. Scale bar = 30 μm. Representative images of hypothalamic neurons labeled with NU4 antibody exposed to AβOs (500 nM, 3 h) in the absence or presence of infliximab (1 μg/ml). Similar patterns of AβO binding were observed in both conditions. Scale bar = 20 μm.
Vasculotide has no effect on experimental liver metastases from a colon cancer cell line (HT29luc), nor on experimental lung metastases from a renal cancer cell line (SN12luc).Figure 3A-D An experiment where HT29luc tumor cells were implanted intravenously into YFP-SCID mice. (A) Kaplan-Meier survival analysis. (B) Quantification of total metastatic burden in the liver and lymphatics, at 35 days post-implantation (DPI), from whole-body bioluminescent imaging (C). (D) Photograph of typical liver metastases. E-G An experiment where SN12luc tumor cells were implanted intravenously into YFP-SCID mice. (E) Kaplan-Meier survival analysis. (F) Quantification of total metastatic burden at 50DPI, mostly within the lungs, from whole-body bioluminescent imaging (G).
Figure 5(A-D) DNA topology of the indicated minichromosomes of increasing DNA length (kb) before (26oC) and after inactivation of cohesin (35oC) in scc1-73 cells. In each case, the first 2D gel resolves the Lk topoisomers (Lk), the second 2D gel uncovers the knotted forms (Kn). Gel signals are denoted as in Fig 2. Bottom graphs compare the Pkn before and after the inactivation of cohesin (mean ± SD from three independent experiments). P-values (Student's t test): ns, p > 0.05. (E) Plot of Pkn values of minichromosomes of increasing DNA length (including YEp13) before and after inactivation of cohesin. P-values (Student's t test): **p < 0.01.