EMBO/SourceData huggingface dataset:
Figure 5B. Tumor volume quantification measured by MRI analysis of Tie2-GFP (n=6) or Tie2-IFNα mice (n=9) at the indicated time points post-transplant, one additional Tie2-IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs' test and not further analyzed; mean values are shown; error bars=S.E.M.; p=0.051, by Mann-Whitney test.C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm.D. Percentage of mice with peritoneal carcinomatosis (defined by the presence of multiple intra-peritoneal lesions) determined by MRI analysis from the same mice described in B. Note that while intrahepatic number of lesions remained similar between time points, extrahepatic tumor spreading was significantly reduced in Tie2-IFNα mice. P-values were calculated by Fisher's exact test.E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice at distal (upper panels) or proximal (bottom panels) areas to CRC liver metastasis, 30 days post HSPC transplant. Note that TEMs were identified as GFP+ cells and by the concomitant expression of different levels of the myeloid cell marker CD11b+ as highlighted in the inset (merge panels, white arrows); the dashed line identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm; insets depict the corresponding areas identified by dashed squares in the merge panels, magnified 1.5-folds.F, G. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2-GFP (n=4) or Tie2-IFNα (n=4) mice euthanized 30 days post-transplant. The basal expression of Irf7 estimated in liver of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed. Mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.
Figure 6A. IHL and TEM characterization in the liver of patients described in Appendix Fig S6A. Liver samples from surgical resections (n=9) were weighted and IHLs were isolated, counted and normalized over tissue weight (left panel). Flow cytometry analysis revealed that TEMs, identified as 7AAD-/CD45+/CD11b+/CD14+/Tie2+ cells, are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions (right panel). Control=hepatic hemangioma; Distal=distal to CRC liver metastasis (>1 cm from the lesion); Proximal=proximal to CRC liver metastasis (<1 cm from the lesion); p-values were calculated by Wilcoxon matched pairs Test.B-C. Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal (>1 cm; left panels) or proximal (<1 cm; right panels) sites from the CRC liver lesion. C. Higher magnification of the panels identified by a rectangle in panel B. Negative control (bottom panels) is obtained by omitting primary anti-TIE2 antibodies. TIE2immunostaining, indicates that sites that are distal from the lesion contain only TIE2+ cells with an apparent endothelial morphology (characterized by an elongated appearance, arrows) while sites that are proximal to the lesion also contain additional TIE2+ cells with an apparent monocyte-like morphology (characterized by a round appearance, arrowheads). The dashed lines identifies the metastasis margin; ✱=CRC metastatic area. Scale bars of upper panels=50μm; middle and bottom panels=20 μm.
Figure 1Cell height was measured using AFM for HeLa-FUCCI in the indicated phase of mitosis. Below: representative images of synchronised HeLa-FUCCIRepresentative images of synchronised HeLa stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 15 µm).Nucleus height was measured by immunofluorescence using Hoechst stainingRepresentative images of synchronised MEFs stained with an anti-geminin to confirm the indicated phase of mitosisNucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.Nucleus height was measured by immunofluorescence using lamin A/C staining.Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.Corresponding percentage of HeLa-FUCCI in mitosis.Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions. Corresponding percentage of EdU positive cells.
Figure 2Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels.TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5).AP1 activity was assessed in the indicated conditions using AP1 reporter assay.Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.
Figure 3Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity.Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol).Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP.Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition.Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2.Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.
Figure 4Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio).Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH.Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor.Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm. Corresponding quantification of p-Jun Ser63 nuclear intensity.
Figure 1A) Representative immunofluorescence images showing localization of Myc-tagged M18BP1-1 truncations schematized in (B) to metaphase sperm centromeres in M18BP1-depleted extract. Truncations analyzed are indicated on the left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X. B) Schematic of M18BP1-1 truncations used to define the metaphase targeting domain in (A) and (C). C) Quantification of the average Myc intensity at centromeres from (A). Values are normalized to centromeric signal of full-length M18BP1-1. Graph shows the mean ± SEM of at least three experiments. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.
Figure 2B) Representative Western blot showing disruption of the interaction between M18BP1-1 and CENP-C by mutation of the SANTA domain by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Flag-M18BP1-1WT or Flag-M18BP1-1SANTA. The M18BP1-1 species added to each reaction and the cell cycle state of the extract is indicated above. The immunoblotted species is indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.C) Representative images showing that mutation of the SANTA domain in M18BP1-1 causes decreased localization to sperm centromeres. Metaphase extracts were depleted of endogenous M18BP1 and either mock-depleted or depleted of CENP-C. The extracts were then complemented with WT or SANTA mutant M18BP1-1. M18BP1-1 species and depletion conditions are indicated at left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X. D) Quantification of the average Flag-M18BP1-1 intensity at centromeres from (C). Values are normalized to centromeric signal of M18BP1-1WT in mock-depleted extract. Graph shows the mean ± SEM of three experiments. Significance determined by Welch's unpaired two-tailed t-test, ** p<0.005.
Figure 3A) A representative autoradiograph from a GST-pulldown of CENP-C to map the CENP-C binding domain of M18BP1-1. Radiolabeled Myc-M18BP1-1 truncations (amino acids indicated at the top) were mixed with recombinant GST-CENP-C1191-1400. Material bound to glutathione agarose was resolved by SDS-PAGE and visualized by autoradiography C) Quantification of (A). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161-580, the CENP-C binding domain.D) Representative Western blot showing metaphase-specific binding of M18BP1-1161-580 to CENP-C by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Myc-M18BP1-1 or Myc-M18BP1-1161-580. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated above. Immunoblotted species indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.E) Representative immunofluorescence images showing Myc-M18BP1-1161-580 localization at sperm centromeres in extract depleted of endogenous M18BP1. In addition, extract was mock-depleted or immunodepleted of CENP-C (indicated at left). Cell cycle state indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets magnified 3X. F) Quantification of (E). Graph shows mean Myc-M18BP1-1161-580 intensity at centromeres ± SEM of two independent experiments normalized to the mock-depleted, metaphase condition.
Figure 4A, B) Metaphase binding of M18BP1-1 to CENP-C requires phosphorylation. Either full-length Myc-M18BP1-1 (A) or Myc-M18BP1-1161-580 (B) was immunoprecipitated from metaphase extract. Half of the immunoprecipitate was treated with λ-protein phosphatase (+ LPPase) to assess whether binding to CENP-C required phosphorylation. Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control. Immunoblotted species is indicated at left.C) Cdk inhibition with flavopiridol prevents M18BP1-1161-580 localization at metaphase centromeres. Representative images showing Myc-M18BP1-1161-580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors. Inhibitor treatment is indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.E) Quantification of immunofluorescence experiments examining localization of M18BP1-1161-580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1. Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.
Figure 5A) Representative immunofluorescence images showing M18BP1-1 mutant localization. M18BP1-depleted metaphase extract was supplemented with in vitro translated full-length Flag-M18BP1-1 mutants and sperm chromatin. In addition, extract was either mock-depleted or CENP-C depleted to assess CENP-C dependent metaphase localization. M18BP1-1 species and CENP-C depletion status are indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.B) Representative Western blot showing co-immunoprecipitation of CENP-C with phosphorylation site mutants of M18BP1-1. M18BP1-depleted metaphase or interphase extract was supplemented with in vitro translated Flag-M18BP1-1 mutants. M18BP1-1 mutation and cell cycle state indicated above, immunoblotted species indicated at right. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.C) Quantification of (A). Flag-M18BP1-1 mutant centromere intensity normalized to WT localization in the mock-depleted condition. Error bars represent SEM from three independent experiments.
Figure 6A) Representative immunofluorescence images showing incorporation of exogenous Myc-CENP-A at centromeres. Sperm nuclei were incubated in M18BP1-depleted interphase Xenopus egg extracts complemented with the indicated M18BP1-1 protein. Extracts were supplemented with RNA encoding Myc-CENP-A to track new CENP-A assembly and in vitro translated HJURP. Scale bar, 10 μm. Insets are magnified 3X. B) Quantification of Myc-CENP-A loading at Xenopus sperm centromeres in (A). Values are normalized to the centromere signals in mock-depleted extract. Dashed lines indicate the Myc-CENP-A assembly signal observed upon M18BP1 depletion (bottom) and Flag-M18BP1-1WT add-back (top) as points of reference for mutant rescue. Graph shows the mean ± SEM of four independent experiments. C) Interphase Mis18 complex formation is unaffected by M18BP1-1 mutations that prevent binding to CENP-C. Extract depleted of endogenous M18BP1 was supplemented with Myc-Mis18α, Myc-Mis18β, and Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated at the top. Co-immunoprecipitation of Myc-Mis18α/β was assessed by α-Myc immunoblot following Flag precipitation.D) HJURP association with M18BP1-1 is unaffected by mutations that prevent CENP-C binding. Extract depleted of endogenous M18BP1 was supplemented with Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract are indicated at the top. Coimmunoprecipitation of Flag-M18BP1-1 was assessed by α-Flag immunoblot following HJURP precipitation.
Figure 7A) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP-AID-M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1. Centromeric localization is indicated by localization with ACA (α-centromere autoantibody serum), early G1 cell cycle state is indicated by midbody staining in the tubulin channel. M18BP1 species indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X.B) Representative immunofluorescence images showing localization of full-length Flag-M18BP1-1 (mutant species indicated at left) without (left panels) and with (right panels) fusion to an SV40 NLS to sperm centromeres in interphase extract depleted of endogenous M18BP1. Immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X. Quantification of immunofluorescence intensity at centromeres as total integrated fluorescence signal normalized to wild type levels. The values shown on each panel are represented as a percentage of wild-type Flag-M18BP1-1 ± SEM from three independent experiments.C) Quantification of Myc-CENP-A assembly in M18BP1-depleted extract complemented with the indicated M18BP1-1 species. Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1-1. Error bars show SEM of at least three independent experiments.D) Representative Western blot of CENP-A assembly reactions in (B). Efficient M18BP1 depletion is indicated by comparing lanes 1 and 10. Add-back of wild-type or mutant M18BP1-1 is near endogenous levels.
Figure 1b) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.d) Boxplots illustrating key proteins that differ between patients with MPX and controls (p-values and fdr for corresponding contrast MPX vs Control are provided in brackets): TTR (p-value = 9E-10, fdr = 2E-7), LBP (p-value = 2E-4, fdr = 2E-3), APOC1 (p-value = 5E-8, fdr = 3E-6), and C9 (p-value = 2E-8, fdr = 2E-6). Here, as usual, the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.e) Correlation between MPX severity (NSkin lesions) and protein expression (y-axis). One MPX patient had an unclear additional skin condition (not a pure case of MPX) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome; however, the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity, the log2(1 + NLesions / 15) was used. Here NLesions is the number of lesions. R2 shows squared correlation coefficient. MPX patients are colored orange, control patients green.
Figure 2a) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX, COVID-19, and controls.b) Scatterplot of log fold-change (logFC) for contrast MPX vs control (C1, x-axis) and logFC for contrast COVID-19 vs control (C2, y- axis). Only the central part of the cloud is shown here. Three truncated dots (APOC1, CRP, and SAA1) are shown in the lower left and upper right corner. Differentially abundant (Regulated; 'Reg') proteins are color coded, with the red color corresponding to 37 proteins differentially abundant in both MPX vs control (C1) and COVID-19 vs control (C2), the orange color corresponding to proteins specifically changed in MPX vs control (C1) only (16 proteins), and the green color corresponding to proteins responding to both MPX vs control (C1) and MPX vs COVID-19 (C3) (3 proteins). There are no intersections between COVID-19 vs control (C2) and MPX vs COVID-19 (C3). The blue color corresponds to proteins responding to MPX vs COVID-19 (C3), but not in MPX vs control (C1) (11 proteins), and the pink color to proteins responding to COVID-19 vs control (C2) only (19 proteins). The red dotted line shows a linear regression through the red dots, i.e. proteins differentially abundant in MPX vs control (C1) and COVID-19 vs control (C2). Note that orange and pink points have the same direction of regulation in both MPXV vs control (C1) and COVID-19 vs control (C2). Only green and blue dots (except three proteins: ADIPOQ, GPLD1, and IGHV1-2) have opposite directions in C1 and in C2.d) Differentially regulated proteins of patients with MPX and COVID-19 (Volcano Plot); α ≤ 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins. The chosen significance level ensured that fdr for this contrast was below 22%.e) Key proteins that differ between patients with MPX and COVID-19 (Boxplots) (p-values and fdr for corresponding contrast MPX vs COVID-19 are provided in brackets): LCP1 (p-value = 3E-4, fdr = 3E-2), LDHB (p-value = 2E-3, fdr = 6E-2), CFHR1 (p-value = 2E-4, fdr = 3E-2). Here the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.f) Top 8 proteins of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6) (top) or COVID-19 (n=10) and MPX (n=6) cases (bottom). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.
Figure 3b) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls (Heatmap); p < 0.05 with Mann-Whitney U test with FDR-based multiple testing correction.c) Key proteins that differ between patients with MPX and controls, and COVID-19 (Boxplots). Dashed blue lines indicate the lowest detected peptide concentration from calibration curves. Statistics based on Mann-Whitney U test with multiple testing corrections. For boxplots, median is indicated by a solid line, hinges show the 25th and 75th percentiles, whiskers show values that, at maximum, are within 1.5 times the interquartile range.d) Top 15 peptides of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.