EMBO/SourceData huggingface dataset:
Figure 2B) Log(odds enrichment) scores for charged GKs in the flanks of the 30 % strongest versus the 30 % weakest APRs in the E. coli cytoplasmic proteome. Whiskers indicate 95 % confidence intervals for no enrichment as determined through bootstrapping analysis.C) Percentual difference in non-synonymous mutation rate in E. coli for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.D) Percentual difference in non-synonymous mutation rate in mammalian species for GK versus background i.e. versus the corresponding residue in non-gatekeeper positions.
Figure 3B) Time-resolved Dynamic Light Scattering (DLS) analysis indicating average particle size over time for each of the peptides in the β-Gal peptide set (aggregating core sequence SVIIWSLGN).C) ThT binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.D) pFTAA binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.E) End-point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β-Gal peptide set. Bars depict mean concentration, whiskers indicate standard deviation (n=4). Statistical significance was determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.F) TEM images of the β-Gal peptide set after 3 days incubation. Top panels give an overview image of one square in a TEM grid, lower panel shows enlarged image (magnification 12000X). Scale bars indicate 12 µm (top row) or 1 µm (bottom row).H) Violin and boxplots showing the distribution of end-point solubilities across 11 aggregating cores, grouped by GK. The dotted line indicates a concentration of µM, corresponding to completely insoluble peptides.I) Solubility analysis of the in vitro solubility screen performed by Niwa et al (Niwa et al, 2009). Left panel shows combined box- and violin plots depicting the solubility distributions of proteins containing at least one strong APR (TANGO score > 70) not protected by any negative GKs (red) versus proteins where all strong APRs are flanked by at least one negative charge (blue). The right panel shows a control analysis, in which proteins were classified by the occurrence of at least one strong APR (TANGO score > 70) not flanked by any positive GKs (red) versus proteins where all strong APRs are flanked by at least one positive charge (blue). The upper boxplot whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Statistical significance was determined through Kruskal-Wallis test with a post-hoc Wilcoxon analysis for pairwise testing. P-values are indicated.
Figure 4D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.
Figure 6A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE.D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.
Figure 1A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines. Actin was used as a loading control.B Subcutaneous melanoma tumour growth in BL/6 WT mice (n=5) using B16F1 WT and XIAPKO (clone1 and 2).C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).D qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in B16F1 WT cells plotted vs B16F1-XIAPKO cells (clone1 and 2). Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr (control) or siXIAP and Western blot analysis of the corresponding cell lysates (bottom).F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells.G qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in HEK293T cells overexpressing myc-XIAP plotted vs HEK293T cells overexpressing myc-XIAPΔRING. Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.H Human cytokines array analysis of HEK293T cells transfected with myc-XIAP or myc-XIAPΔRING. Medium was changed after 16 h and the supernatant was collected after 48 h. Pre-spotted nitrocellulose membranes were incubated with the supernatants overnight at 4°C.
Figure 2A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments.B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue).C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.E IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA.F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.
Figure 4A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored. The numbers of melanomas with different intensities were plotted into the table as a percentage of the total. Specific staining intensity for XIAP and CD177, corresponding to relative amounts of infiltrated neutrophils, was arbitrarily set as the following: -, not expressed; +, low; ++, moderate; and +++, strong expression. Representative IHC pictures (right panel).B Representative IF (neutrophils staining) in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO tumours (Scale bar 50 µm) and quantification of Ly6G+ cells. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5). C B16F1 WT, XIAPKO (clone 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5). Mice were intra-peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells.D Representative IF (neutrophils) scale bar 50 µm and quantification of Ly6G+ cells in tumours from C. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (4 mice per genotype) was represented. Dots represent individual mice (n=4).
Figure 1Gene-trap-based genetic screen in haploid human cells for regulators of p38 kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38 population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).Human U2OS cells and cells deleted for ZAK (∆ZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting as indicated.Unique gene-trap insertions (dots) mapped to the genomic ZAK locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38 activation and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).U2OS/S-HA-ZAKβ cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.U2OS cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ were pre-treated with ZAK inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown material was used in a kinase assay, separated by SDS-PAGE and analyzed by autoradiography.U2OS and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.U2OS and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies. T1, TAK1; M2, MEKK2.
Figure 2U2OS cells stably expressing Strep-HA-ZAKβ or -ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody and counter-stained with DAPI. Data information: scale bars, 10 μm.U2OS cells stably expressing ZAKβ-GFP were treated with sorbitol (500 mM, 1 h), pre-extracted, fixed and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line. As in (B) except that Hela cells were transfected with ZAKbeta-GFP and immunostained with 53BP1 antibody. Data information: scale bars, 10 μm.Cells from (B) were imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.As in (D), except that cells were co-transfected with mCherry-FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers. Data information: scale bars, 10 μm.U2OS cells were transfected with constructs from (F) and imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.
Figure 3U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.
Figure 4Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow. Data information: scale bars, 20 μm.C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1. Data information: scale bars, 20 μm.16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.
Figure 5H&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm. Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm. Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.
Figure 4 Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect-size heat maps of six drugs (see titles of panels) targeting EGFR. It is evident that the different drugs showed different profiles but also that high MAP2K1 expression (blue/red gradient across cell lines) was consistently associated with drug resistance (dark blue/yellow gradient across cell lines; AUC: area under the curve; see main text and Appendix for details). See also Figure EV5.
Figure 5(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4.(C) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details).(D) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.(E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9.(F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.
Figure 6(A) Western Blot visualising MERTK expression in two negative (RKO, CC07) and two positive control cell lines (CaCo-2; HT55).(B) IHC staining of MERTK expression in RKO (negative control) and CaCo-2 (positive control) cells.(C) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists.(D) Kaplan-Meier plots showing worse overall, disease-free and recurrence-free survival of patients with high cytoplasmic/membranous expression of MERTK.
Figure 1(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i).(B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days.(C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii.(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).(D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i).(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.(F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).
Figure 2(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days.(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).
Figure 3(A) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).