EMBO/SourceData huggingface dataset:
Figure 1A Activity of an NF-κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/mL LPS, 400 μM nickel chloride, or 25, 50 or 100 μM platinum(II) chloride and platinum(IV) chloride (n=3 independent biological replicates). B As per panel (A) but secreted IL-8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/mL LPS, 200 μM nickel chloride, 100 μM platinum(II) chloride or 100 μM platinum(IV) chloride (n=4 independent biological replicates). Data Information: For all panels actual individual data from each experiment are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2-way ANOVA: in A) hTLR4 compared to null2 cells; in B) agonist treatment compared to non-treated (nil); ns, not significant ; **, P<.01; ; ****, P<.0001 (Dunnett's test, A, B
Figure 2A IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/mL LPS, 200 μM nickel chloride, 2 μg/ml HMGB1, 100 μM platinum(II) chloride, 100 μM platinum(IV) chloride or 25 μM cisplatin (n=3 or 4 independent biological replicates). C IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test).
Figure 3A- HEI-OC1 cells containing a Tlr4 deletion (Tlr4-/-) were compared to HEI-OC1 non-targeting (NT) control cells and assessed for cell viability (A), AnnexinV/propidium iodide staining (B), following cisplatin treatment at the indicated concentrations (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2-way (A, B) *, P<.05; **, P<.0001(Bonferroni test).
Figure 4A IL-6 secretion in HEI-OC1 cells treated with 100 pg/mL LPS or 20μM cisplatin (n=3 or 4 independent biological replicates). B IL-6 secretion in TLR4-/- cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20μM cisplatin (n=6 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).C Il6 and Tlr4 transcript levels in HEI-OC1 cells following treatment with 20μM cisplatin for the indicated times (n=3 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).
Figure 5B Hair cell viability in larval zebrafish pre-treated with control-, tlr4ba- and/or tlr4bb-targeting morpholino oligonucleotides (MO) and subsequently treated with 15 μM cisplatin. Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons to control morpholino (B, except as indicated in blue) were assessed by one-way ANOVA. *, P<.05; **, P<.01; ****, P<.0001 (Tukey test).
Figure 6A IL-6 secretion in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 10 ng/mL LPS or 20μM cisplatin (n=5-7 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by 2-way (A) or one-way *, P<.05 ; ****, P<.0001(Dunnett's test).B ROS generation in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 20μM cisplatin (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by one-way (B) ANOVA. ; **, P<.01 P<.0001(Dunnett's test).
Figure 3D. (Top) Relative enrichment of the three microRNAs across different cell types. Quantitative expression of microRNAs in primary hippocampal neurons, primary astrocytes and primary microglia. miR-146a-5p is significantly enriched in microglia, while miR-148a-3p and miR-181a-5p are significantly enriched in neurons. N = 5/group, Two-way ANOVA, Tukey's multiple comparisons test, *P<0.05, **P<0.01. Bars and error bar indicates mean ± sem. (Bottom) miRNA expression in different cell types of mouse brain stem. The data was retrieved from Hoye et al. 2017.E. Overexpression of microRNAs in relevant cell types for 48 hours followed by genome wide RNA-seq analysis. miR-181a-5p was overexpressed in primary hippocampal neurons, while miR-146a-5p was overexpressed in microglia culture. Immortalized microglial cell line was used for this purpose. Given that miR-148a-3p was highly enriched in neurons (D), primary hippocampal neurons were treated with the corresponding miR-148a-3p mimic. PCA plot shows that the mimic and control treated samples cluster distinguishingly separate from one another. Volcano plot displays the genes significantly deregulated in mimic treated samples compared to control samples (FDR <0.05). Red color indicates the up-regulated genes while the blue color represents the genes those were down-regulated.H. qPCR analysis confirms the over-expression of pro-inflammation related genes (IL-1ß, IL-6, TNF-alpha) due to overexpression of miR-146a-5p. Expression of anti-inflammatory gene, IL-10 was downregulated in mimic treated cells compared to the controls. Unpaired t-tests, two-tailed, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Bars and error bar indicates mean ± sem. Number of biological replicates: 5-6/group.
Figure 4B. Functional mature synapses were quantified via co-localizations of pre- (Synatophysin 1) and the post-synaptic (PSD-95) markers and compared between 3-miR-mix and control groups. Scale bar: 10 μm. Two independent methods (SynQuant and Colocalization) were used for quantification. 3-miR-mix reduced the number of functional synapses compared to controls (n = 24-30 images)C. Dendrite labeling and quantification. Dendritic spines were stained with Dil. Scale bar: 10 μm. Spine density and total spine length are substantially reduced in 3-miR mix treated primary neurons compared to those treated with scramble RNA (n = 49-97 images)
Figure 5All data have been adjusted for age, gender and other latent covariates for downstream eigenvalue calculation. A. Eigen value showing the increased expression of the 3-microRNA signature in blood plasma samples of age matched MCI (n = 23) patients compared to controls (n = 27). B. Expression of 3-microRNA signature is increased in PAXgene blood samples of MCI patients (n=71) compared to controls (n=65) from the DELCODE cohort.E. Eigen value showing the expression the 3-microRNA signature in MCI patients for those the follow up diagnostics data assessed 2 years after were available. 15% of these MCI patients developed Alzheimer´s disease (AD), while the rest 85% patients remained with MCI (stable MCI). The boxplot depicts the increased expression levels of 3-miR signature in patients who converted to AD (n=8) compared to those that had stable MCI (n=47). F. Increased expression of 3-microRNA signature in cerebrospinal fluid (CSF) of MCI patients (n = 9) compared to controls (n = 26). Wilcoxon-rank test, P value is given on the corresponding panel. In the boxplots in (A, B, E, F), the center line indicates the median, while the upper and lower lines represent the 75th and 25th percentiles, respectively. The whiskers represent the smallest and largest values in the 1.5× interquartile range.
Figure 6H. Escape latency during the water maze training comparing 7 months old wild type mice (WT-control, n = 17, male: 9, female: 8) and APPPS-21 mice (APP-control, n = 8, male: 6, female: 2) injected with scramble control oligonucleotides and APPPS1-21 mice injected with microRNA inhibitors (APP miR-inhibtor mix, n = 12, male: 8, female: 4). Bars and error bars indicate mean ± sem..
Figure 7D. qPCR assay for several synaptic genes (LRKK2, Cadm3 and Slc6a11) confirms reinstatement of gene expression with anti-miR-mix (n = 5-7, Kruskal-wallis test).G. qPCR data shows rescue of AFF2/FMR2 and Hivep3 expression in APP/PS1 mice treated with inhibitor cocktail (n = 6-7, Kruskal-wallis test).
Figure 1MSCs were exposed to increasing LPS concentrations, thereafter washed and co-cultured with PMA activated neutrophils. Incubation of neutrophils with PMA alone served as positive control and DMSO treated neutrophils as negative control. Scale bars: 50μm.Quantification of NET bound elastase indicative of NET formation. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.Representative microphotographs of murine wound sections immunostained for Ly6G (neutrophils, green) and DNA-histone (displayed as expulsed streaks in red indicative of NETs). Wounds injected with LPS primed MSCs (upper row, outer right panel) depict enhanced NET formation (magnified inset) and increased expression of activation markers like neutrophil elastase (NE, lower panel, outer left panel) compared to wounds injected with non-primed MSC (middle panels) or PBS injected wounds (outer left panels). Murine skin wounds treated with PBS injections served as control. Scale bar: 10μm (upper row) and 50μm (lower row). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets.
Figure 2Hierarchical clustering analysis of RNAseq expression profile from MSCs stimulated with LPS for 6 and 24 h. The heat-map shows the gene expression profile of non-primed MSCs and MSCs which were LPS primed for 6 and 24h. Red color represents upregulation, while blue color depicts down regulation in gene expression, each data point represents FPKM in log2 value.Validation of the selected genes uncovered from global RNAseq analyses by qRT-PCR analyses displays up-regulation of CXCL-6 after LPS treatment.Validation of the selected genes uncovered from global RNAseq analyses by qRT-PCR analyses displays up-regulation of , IL-8 and IL-1β after LPS treatment.ELISA results depict an up-regulated expression of CXCL-6 after 24h of LPS treatmentELISA results depict a significantly increased IL-8 expression after 6 and 24h of LPS treatment.Western blot analysis showed up-regulation of IL-1β expression upon LPS priming of LPS primed MSCs. Actin served as control. Densitometry graph shows significant increase after 6h LPS stimulation compared to non-primed MSCs. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, n=3 biological replicates.
Figure 3The upper scheme depicts the experimental design. Representative microphotographs show that injection of LPS primed MSCs into wounds increased the expression of CXCL6, IL-8 and IL-1β. Graphs display numbers of double positive cells for h-β2M + CXCL6, h-β2M + IL-8 and h-β2M + IL-1β, respectively. MSCs injected wounds served as controls. Statistical analysis was performed using unpaired t-test, values are represented as mean ± SEM, six biological replicates. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Es, eschar on wound margin; wm, wound margin; scale bars: 50µm.Results show that LPS primed MSCs injected into wounds provoke increased expression of MIP/KC in endogenous neutrophils. MIP/KC represents functional homologues of IL-8 in mice and are known as neutrophil chemoattractant. Similar results were found for IL-1β, which is a strong chemoattractant for neutrophil recruitment and bacterial clearance. In addition, LIX which shares 63% amino acid sequence identity with human GCP-2/CXCL6, a chemoattractant for neutrophils. The graphs display numbers of double positive cells for Ly6G MIP/KC, IL-1β and LIX, respectively. PBS and non-primed MSCs injected wounds served as controls. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates. Scale bars: 50µm.
Figure 4Representative microphotographs of TLR4 silenced and non-silenced MSCs primed with LPS and co-cultured with neutrophils. Immunostaining was performed with an antibody detecting NETs (DNA-histone, red). Of note, TLR4 silenced MSCs upon LPS priming cannot mount any adaptive response and fail to enhance NET formation (lower row, outer right panel) as compared to non-silenced, LPS primed MSCs co-cultured with PMA activated neutrophils (lower row, outer left panel). Neutrophils and DMSO served as a negative control (upper row, outer left panel), while neutrophils activated by the protein C kinase activator PMA served as a positive control depicting enhanced NET formation (upper row, middle panel). Co-culture with MSCs suppressed enhanced NET formation (upper row, outer left panel) induced by PMA. Scale bars: 50μm.Quantitative assessment of NET bound elastase employing a specific ELISA in identical experimental setting as described in Figure 1. Similarly, to immunostaining, a significant reduction of NET bound elastase was observed in TLR4 silenced LPS primed MSCs when co-cultured with activated neutrophils as opposed to high NET bound elastase in non-silenced LPS primed MSCs co-cultured with activated neutrophils. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.High resolution scanning electron microscope analysis depicting enhanced NET formation (red arrows) expulsed from neutrophils (blue arrows) in the presence of LPS primed MSC co-cultured with PMA activated neutrophils (middle row, outer left panel) as compared to reduced NETs in non-primed MSCs co-cultured with activated neutrophils (middle row, outer right panel). Of note, TLR4 silenced LPS primed MSCs failed to activate neutrophils and NETs (lower row, outer left panel). TLR4 silenced and non-primed MSCs display reduced NETs in co-cultures with activated neutrophils (lower row, outer right panel), suggesting that PMA activation shares TLR4 signaling components. PMA activated neutrophils alone served as positive control with highly enhanced NETs (upper row, outer right panel) and DMSO treated neutrophils as negative controls (upper row, outer left panel). Red stars indicate neutrophil derived granules. Scale bars: 0.1µm
Figure 6Representative photomicrographs of confocal microscopy of sections from differently injected day 1 wounds stained for Ly6G+ neutrophils (green) and F4/80+ macrophages (red). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 1 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs Double stained cells indicate phagocytic engulfment of neutrophils by macrophages. To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bar: 100µm.Quantification of Ly6G and F4/80 double positive cells on sections of differently injected day 1 wounds. Wounds were injected as described in (a). Double positive cells were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.Quantification of Ly6G+ neutrophils and F4/80+ macrophages on sections of differently injected day 1 wounds. Wounds were injected as described in (a). Single positive cells were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.Representative photomicrographs of confocal microscopy of sections from differently injected day 3 wounds double-stained for TGFβ-1 (red) and for F4/80+ macrophages (green). Nuclei are stained with DAPI (blue). Double staining was performed for sections of day 5 wounds injected with PBS (control), non-primed MSCs, LPS primed MSCs and LPS primed TLR4-silenced MSCs. Scale bar: 50µm. Representative photomicrographs of sections of day 5 wounds immunostained for CD31 (indicative of endothelial cells and newly formed vessels) and for α-SMA (indicative of myofibroblasts differentiation) after injection of LPS primed MSCs, non-primed MSCs, LPS primed TLR4-silenced MSCs or PBS (middle and lower panel). To facilitate comparison, areas inside the rectangles are shown at 5x magnification in the insets. Scale bars: 50µm.Double positive macrophages stained for TGFβ-1 and F4/80 were counted and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.Quantitative analysis of CD31 positive endothelial cells in sections of wounds injected with PBS, non-primed MSCs, LPS primed MSCs and LPS primed TLR4 silenced LPS Cell counting was performed on immunostained wound sections and statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, six biological replicates.Western blot analysis of lysates from day 5 wounds (left panel) and the corresponding densitometric analysis (right panel) depicts enhanced α-SMA protein expression in wounds injected with LPS primed MSCs as opposed to the respective control groups. Actin served as loading control. Statistical analysis was performed using one way ANOVA, values are represented as mean ± SEM, three biological replicates.
Figure 1D. Selected 2D class averages obtained by cryo-electron microscopy of the yeast Smc5/6 hexamer. Representative classes with dimers of Smc5/6 hexamers are displayed (left images). Emerging details are indicated schematically (right panel).
Figure 2B. Limited proteolysis of a purified preparation of the Nse5/Nse6 complex by trypsin. Samples taken at different time points were analyzed by SDS-Page and Coomassie staining. Selected stable fragments identified by mass spectrometry are indicated.
Figure 3A. Pulldown ('pd') assays using immobilized Smc5/Smc6-Twin-Strep ('TS') hexamers and soluble input material ('in') of Nse5/6 (left), of Nse5/Nse6(177-C) (middle) and of Nse6(1-179)-CPD-His (right). Control pulldowns ('-') were performed by omitting the pre-binding of Smc5/Smc6-Twin-Strep to the beads. Fractions were analysed by SDS-Page and Coomassie staining.B. Salt stability of Smc5/Smc6-Twin-Strep interactions with Nse5/6 and Nse6(1-179). Immobilized Smc5/Smc6-Twin-Strep was mixed with Nse5/6 or Nse6(1-179)-CPD-His ('in'). Beads were washed with buffers containing the indicated salt concentrations. Bead fractions were analysed by SDS-Page and Coomassie staining. Pulldown efficiencies were estimated from the intensity of Coomassie gel bands.D. Analysis of spore viability by yeast tetrad dissection. Diploid strains heterozygous for alleles of Nse6 (wt, 86-C, 177-C and 179-C) were sporulated. Isolated spores were grown on YPD plates. Viable clones were tested for the marker cassette conferring resistance to G418 (marked by circles in green colours). Dead spores were marked by circles in red colours.
Figure 4A. ATP hydrolysis rates (given per Smc5/6 complex) were measured by an enzyme-coupled assay in the absence and presence of plasmidDNA (conc. 1.25 nM) for the Smc5/6 hexamer (conc. 150 nM) and the octamer (conc. 150 nM) with increasing concentrations of ATP. The curves were fitted to Michaelis Menten equation and Km and kcat values were determined. Please note that the hexamer without plasmidDNA shows cooperative behaviour, thus the Michalis-Menten kinetics is not formally applicable (marked by asterisk). Assays were performed in biological triplicates, mean values are shown with error bars indicating standard deviations. For bar graphs individual data points are also displayed. B. Same as in (A) using 40bpDNA (annealed 40 bp oligo DNA) (conc. 1 µM) instead of plasmidDNA. Note that the data points and curves for samples without DNA (hexamer -DNA; octamer -DNA) are identical to (A). D. Pulldown experiments with Smc5/6 complexes and circular plasmid (2.8 kbp). DNA is retained after high salt washes (1 M NaCl) only in the presence of both Nse5/6 and ATP. The graph on the right shows a quantification (mean values and standard deviations) of the amount of co-purified DNA from technical triplicates. Individual data points are also displayed.F. Salt-stable DNA association requires ATP binding and Smc5/6 head-engagement, but not ATP hydrolysis. Same experimental setup as in (D) using mutant versions of the Smc5/6 hexamer as well as the non-hydrolyzable analogue of ATP, ATPγS, as indicated.
Figure 5B. Cross-linking of engineered variants of the Smc5/6 hexamer. As in (a) using Smc5 and Smc6 cysteine mutants. Schemes indicate the location of engineered cysteines and their expected ability to cross-link in a rod-like and a ring-like conformation. High-molecular weight species were analysed by SDS-Page and Coomassie staining. Wild-type hexamer ('wt') is included as cross-linking control. Species occurring only in the presence of engineered cysteines are labelled by coloured arrowheads. Cross-linking efficiencies were calculated from the intensity of Coomassie-stained bands by comparing the band of the corresponding cross-linked species to the bands of unmodified Smc5 and Smc6. Numbers below the gel quantify the percentage of cross-linked protein species in the displayed gel. Comparable numbers were obtained in at least one additional independent experiment.E. Effects of 40bpDNA and plasmidDNA on head engagement (as judged by E-Cys cross-linking) of the Smc5/6 complex with wildtype (wt) or hydrolysis-deficient (EQ) heads, in both the presence or absence of the Nse5/6 complex. Cross-linking efficiencies were calculated from the intensity of Coomassie-stained bands by comparing the band of the corresponding cross-linked species to the bands of unmodified Smc5 and Smc6. Numbers below the gel quantify the percentage of cross-linked protein species in the displayed gel.
Figure 3(D) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 (n=3) or WT EAAT2 (n=3). Cells were intracellularly dialyzed with a 115mM KGluc-based solution and held at 0 mV.
Figure 4(B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3.(C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3).(D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane.(E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)