EMBO/SourceData huggingface dataset:
Figure 4Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).
Figure 5B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.
Figure 7A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F
Figure 1(A) Graph derived from two published data available in the PubMed GEO database (GSE6919 and GSE35988). The box charts depict the relative expression of ESM1 in benign, primary, and metastatic prostate cancer patients. The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers.(B) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines. P: parental cells, M: metastatic cells.(C) Cell invasion assays of 22Rv1-M and PC3-M cells transfected with either Scramble-shRNA or ESM1-shRNA. The data are shown as the relative fold change of invasive cells compared with the shScramble group. Scale bar: 100 μm.(D) Representative images of lungs, livers, intestines, and kidneys from mice 8 weeks after orthotopical injection of 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Scale bar: 0.5 cm. Tumors were removed and weighed. Quantitative summary of tumor luminescence in different metastases measured in photons/second are shown.(E) Formalin-fixed, paraffin-embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis (n=16 and n=49, respectively) were stained with an anti-ESM1 antibody. Tissue-bound ESM1 is shown in brown. Scale bar: 50 μm. Arrowheads indicate positively stained nucleus of cancer cells. Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis. The scores are calculated by intensity (score 1-3) x percentage (score 1-4) of stained cells. The line in the middle of the box is plotted at the median. The box extends from the 25th to 75th percentiles. Whiskers above and below the box indicate the 5th and 95th percentiles.
Figure 2(A) Immunoblotting analysis of ESM1 in supernatant (S), nucleus (N), and cytoplasm (C) was performed. Ponceau S, Lamin A/C, and α-tubulin were used as loading control in supernatant, nucleus, and cytoplasm, respectively. P: parental cells, M: metastatic cells.(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.(D) Elicitation of ESM1 overexpression using HA-tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1-P cells. Lamin A/C and α-tubulin were used as loading control in the nucleus and cytoplasm, respectively.(E) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant (S) of 22Rv1-P cells.(F) Left, representative images of 22Rv1-P cells in the transwell invasion assay. Right, statistic data of invasive ability of ESM1 expressing cells. Scale bar: 100 μm.(G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.
Figure 3(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody.(B) Representative images of tumor spheres. Spheres were cultured for 14 days before counting. Histogram shows the mean numbers of spheres cultured. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.(C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. **(D) Cells with or without ESM1-NLS overexpression were subcutaneously injected (10, 100, 1000 cells per mouse) into NOD/SCID mice. Tumor formation ability and tumor weight were analyzed. Scale bar: 1 cm. Bars are the mean ± SD of indicated (n) independent experiments.(E) Flow cytometry analysis of the ratio of CD44+ and CD133+ cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed.(F) qRT-PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. *
Figure 4(A) IPA analysis of RNA-seq data showing the 14 top-predicted upstream regulators of differentially expressed genes. Positive Z-scores (orange) represent activated upstream regulators, negative Z-scores (blue) represent inhibited upstream regulators.(E) The transcriptional activity of β-catenin in 22Rv1-P cells with different patterns of ESM1 overexpression. Bars are the mean ± SD of three independent experiments.(F) Manipulating ESM1 expression impacts transcriptional activity of NFκB. Bars are the mean ± SD of three independent experiments.
Figure 5(A) qRT-PCR analysis of the mRNA levels of the genes related to β-catenin downstream signaling pathway. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.(B) qRT-PCR analysis of the mRNA levels of NFκB targeting genes. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.(C) Immunoblotting analysis of 22Rv1-P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated.(D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.(E) Tumor spheres formed by 22Rv1-P cells stably expressing ESM1-NLS with either β-catenin or NFκB shRNAs. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.(F) Representative images and quantitative data comparing cell invasion of the indicated cells. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.
Figure 6(A) Nuclear and cytosolic extracts of 22Rv1-M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting.(B) Nuclear or cytosolic extracts of 22Rv1-M cells were immunoprecipitated with an ESM1 antibody.(C) Human recombinant His-ESM1 and GST-β-catenin protein were pull down with either Ni sepharose or Glutathione sepharose.(E) HEK293T cells were co-transfected with the indicated β-catenin-Flag and ESM1-HA constructs. Cell extracts were immunoprecipitated with Flag-M2 agarose beads. Light chain was labeled as l.c.(F) HEK293T cells were transfected with ESM1-HA constructs and pull-down was carried out with different purified GST-ARM fragments.(G) HEK293T cells were transfected with the indicated ESM1-GFP constructs. Cell extracts were pulled down with purified GST-ARM.
Figure 7(B) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM.(C) Cell extracts of 22Rv1-M transfected with either Scramble-shRNA or ESM1-shRNA were immunoprecipitated with an β-catenin antibody and blotted with anti-TCF4 and anti-BCL9 antibody.(E) Upper, Schematic diagram of ESM1-targeting short peptides. Lower, HEK293T cells were transfected with ESM1-HA constructs. Cell extracts were treated with the short peptides, pulled down with purified GST-ARM and blotted with anti-HA and anti-TCF4 antibody.(F) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM and blotted with an anti-TCF4 antibody.(G) ChIP-qPCR validation of β-catenin binding to the CCND1 and c-Myc promoter in 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Bars are the mean ± SD of three independent experiments.(H) Upper, Schematic diagram of ChIP-qPCR primers targeting regions upstream of the CCND1 and c-Myc transcription start sites (TSSs). Bar graphs show β-catenin and ESM1 binding at the CCND1 and c-Myc promoter regions. Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments.(I) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.(J) The effects of ESM1 point mutants on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.
Figure 4(B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.(D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.
Figure 1A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.B, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. Data information data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.E, Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA, in presence of dimethyl sulfoxide (DMSO) or 400 µM guanosine. GAPDH was used as a loading control.
Figure 2A, HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone (-) or the indicated concentration of MPA for 24 hrs. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.B, HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.C, HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence (-) or the presence (+) of the combination of 10 µM of the ATR inhibitor VE-821 (ATRi) and 10 µM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (-) or MPA 10 µM for additional 24 hrs. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.
Figure 3A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. Data information: , data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.B, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section.C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysatesHCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). D, Levels of HDM2, RPL5, RPL11, and the IP negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5 and RPL11 of at least two independent experiments is shown (lower right panel).HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). E, the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates. Data information: data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment.G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.
Figure 4A, HCT116 were treated as indicated for 24 hrs, subjected to propidium iodide staining and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments.B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells.C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means. D, Representative images from (C). E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm.F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.
Figure 5A, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels.B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.
Figure 6A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments.B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control.Statistical analysis of the immunostaining of γ-H2AX (C) in HCT116 cells treated Total nuclear intensities were obtained on >6000 (γ-H2AX) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.Statistical analysis of the immunostaining of 53BP1 (D) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.E, Representative images cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μmF, HCT116 cells treated were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test
Figure 1b. Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound-induced tumours (Pap) compared to fibroblasts from wild-type (WT) and inflamed (InvEE) back skin (n=3 per condition). PRSS35 is indicated in red as a top upregulated gene in CAFs.
Figure 2a. Immunofluorescent images of skin sections from PDGFRα-eGFP reporter mice with normal (WT, n=4), inflamed (InvEE, n=4), wounded (d14 post-wounding; wound, n=4), or tumour-associated (Papilloma, Pap d17 and d30 post-wounding, n=4 per condition) skin. Nuclei were stained with Dapi (4', 6-diamidino- 2- phenylindole; blue). Dotted line represents epidermal-dermal boundary. Scale bars: 50 μm. b. Quantification of the number of fibroblasts present in different skin conditions (n=4 mice per condition; ** p<0.01; *** p<0.001; **** p<0.0001; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M. c. Herovici staining of sections of normal (WT), inflamed (InvEE), wound, or tumour-associated (Papilloma) skin. Scale bars: 100 μm.