( K and L ) Quantification of osteoclast number per bone surface ( N . Oc / BS ) and percentage of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test was used for statistical analysis .

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(K and L) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis.


( A ) TRAP staining of hPBMCs treated with human RANKL ( 50 ng / mL ) or dimemorfan for 12 days . Scale bars , 500 μm .

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(A) TRAP staining of hPBMCs treated with human RANKL (50 ng/mL) or dimemorfan for 12 days. Scale bars, 500 μm.


( B ) Western blot analysis of NFATc1 expression during a 7 - day induction of hPBMCs to osteoclasts .

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(B) Western blot analysis of NFATc1 expression during a 7-day induction of hPBMCs to osteoclasts.


( C ) hPBMCs were induced to differentiate into osteoclasts for 7 days , and the relative mRNA levels of marker genes were evaluated by RT - qPCR ( n = 6 biological replicates ) . Data information : All results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test ( C ) was used for statistical analysis .

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(C) hPBMCs were induced to differentiate into osteoclasts for 7 days, and the relative mRNA levels of marker genes were evaluated by RT-qPCR (n=6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C) was used for statistical analysis.


Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes ( D ) Binding is reduced in Delta ∆ β1 - 2 , but not to the Serrate equivalent using liposomes composed of PC : PS : PE - fluoroscein ( 80 : 15 : 5 ) . Notch binding to Drosophila Delta NE3 ( E ) WT and ∆ β1 - 2 ( Delta ∆ β1 - 2 ) variants both bind to Notch , unlike variant with F204A substitution in DSL domain . Comparisons were performed with a two - tailed unpaired t - test . Values are shown as scattered data points with the dark lines representing the means . ns , no significant difference , * * P < 0 . 01 ; * * * P < 0 . 0001 .

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Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes (D) Binding is reduced in Delta∆β1-2, but not to the Serrate equivalent using liposomes composed of PC:PS:PE-fluoroscein (80:15:5). Notch binding to Drosophila Delta NE3 (E) WT and ∆β1-2 (Delta∆β1-2) variants both bind to Notch, unlike variant with F204A substitution in DSL domain. Comparisons were performed with a two-tailed unpaired t-test. Values are shown as scattered data points with the dark lines representing the means. ns, no significant difference, **P < 0.01; ***P < 0.0001.


Adult wings from Dl ∆ β1 - 2 flies . No defects are detected in wings from Dl ∆ β1 - 2 / + ( B ) , Homozygous Dl ∆ β1 - 2 / Dl ∆ β1 - 2 have extra vein tissue near L5 and uneven L2 veins ( arrowheads ; C ) .

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Adult wings from Dl∆β1-2 flies. No defects are detected in wings from Dl∆β1-2/+ (B), Homozygous Dl∆β1-2/ Dl∆β1-2 have extra vein tissue near L5 and uneven L2 veins (arrowheads; C).


Microchaetae are arranged in rows on the thorax of control ( yw ) flies ; these become disordered and more dense in Dl ∆ β1 - 2 / Dl ∆ β1 - 2 . White rectangle indicates area scored for E Number of microchaetes per central area ( white rectangle in D ) in the indicated genotypes .

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Microchaetae are arranged in rows on the thorax of control (yw) flies; these become disordered and more dense in Dl∆β1-2/Dl∆β1-2. White rectangle indicates area scored for E Number of microchaetes per central area (white rectangle in D) in the indicated genotypes.


Representative images of adult females wings in combinations of Dl ∆ β1 - 2 with loss of function Delta alleles . In combinations with Dlrev10 ( A ) , Dl ∆ Exon2 ( B ) , or Df ( 3R ) DlFx3 ( C ) , vein thickening is strongly enhanced ( right panels ) compared to heterozygous mutants alone ( left panels ) . Vertical square brackets indicate the regions used for vein thickness quantification . Quantification of wing - vein thickness in females of the indicated genotypes . Representative images of adult female wings demonstrate that Dl ∆ β1 - 2 rescues the wing notching phenotype , caused by a Notch loss of function allele ( N55e11 ) . Horizontal square bracket indicates the L5 vein " delta " at the intersection with the wing margin analyzed in EV2E . Quantification of wing notching in females of the indicated genotypes , Dl ∆ β1 - 2 rescues notching in a similar manner to Dlrev10 .

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Representative images of adult females wings in combinations of Dl∆β1-2 with loss of function Delta alleles. In combinations with Dlrev10 (A), Dl∆Exon2 (B), or Df(3R)DlFx3 (C), vein thickening is strongly enhanced (right panels) compared to heterozygous mutants alone (left panels). Vertical square brackets indicate the regions used for vein thickness quantification. Quantification of wing-vein thickness in females of the indicated genotypes. Representative images of adult female wings demonstrate that Dl∆β1-2 rescues the wing notching phenotype, caused by a Notch loss of function allele (N55e11). Horizontal square bracket indicates the L5 vein "delta" at the intersection with the wing margin analyzed in EV2E. Quantification of wing notching in females of the indicated genotypes, Dl∆β1-2 rescues notching in a similar manner to Dlrev10.


Equatorial region of eye imaginal discs where E ( spl ) mδ0 . 5 expression ( green ) becomes restricted to a single photoreceptor in each cluster ( magenta ) , as detected in control and Dl ∆ β1 - 2 / + discs ( top panels ) . In Df ( 3R ) DlFx3 / + and Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 discs ( bottom panels ) E ( spl ) mδ0 . 5 expression is reduced ( Df ( 3R ) DlFx3 / + ) or absent from several clusters ( Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 ) indicative of reduced Notch signalling . Scale bars correspond to 10 μm .

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Equatorial region of eye imaginal discs where E(spl)mδ0.5 expression (green) becomes restricted to a single photoreceptor in each cluster (magenta), as detected in control and Dl∆β1-2/+ discs (top panels). In Df(3R)DlFx3 /+ and Dl∆β1-2/Df(3R)DlFx3 discs (bottom panels) E(spl)mδ0.5 expression is reduced (Df(3R)DlFx3 /+) or absent from several clusters (Dl∆β1-2/Df(3R)DlFx3) indicative of reduced Notch signalling. Scale bars correspond to 10 μm.


Proportion of photoreceptors clusters that fail to express the E ( spl ) mδ0 . 5 reporter in the indicated genotypes . ns , no significant difference , * * * P < 0 . 0001 ( one - way ANOVA ) . On the violin plot , dashed line represents the median and the dotted lines show the quartiles .

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Proportion of photoreceptors clusters that fail to express the E(spl)mδ0.5 reporter in the indicated genotypes. ns, no significant difference, *** P<0.0001 (one-way ANOVA). On the violin plot, dashed line represents the median and the dotted lines show the quartiles.


Apical view of wing imaginal disc with homozygous Dl ∆ β1 - 2 clones ( GFP negative ) stained for Dl ( red ) and Cadherin ( blue ) . ( A ' ) Z - projection of apical layers spanning a Dl ∆ β1 - 2 clone ( GFP negative ) located at the DV boundary . No change in apical localisation of Dl ( red ) and Cadherin ( blue ) is detected . Panels on the right show the Dl ( grayscale ) apical localisation on the wing imaginal disc .

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Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc.


Uptake assay at t = 0 . After exposure to extracellular anti - Dl antibody , Dl protein ( red ) is detected at similar levels apical to Cadherin ( blue ) in wild - type ( GFP ) and Dl ∆ β1 - 2 mutant tissue ( GFP negative ) . ( B ' ) Cross - sectional view of B , Dl protein ( red ) is present apically relative to Cadherin ( Blue ) which marks adherens junctions . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively . Uptake assay after 30 minutes , internalised anti - Dl ( red ) enters the endocytic route and in the cross sectional view ( C ' ) can similarly be detected as puncta along the cell axis in wild - type ( GFP ) and homozygous Dl ∆ β1 - 2 ( GFP negative ) tissue . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively .

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Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively. Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.


Average piRNA precursor abundance relative to EV in EV and ints - 11 RNAi samples , normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs ( Chen et al . , 2013 ) . Error bars represent the standard error of the mean ( 5 replicates ) .

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Average piRNA precursor abundance relative to EV in EV and ints-11 RNAi samples, normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs (Chen et al., 2013). Error bars represent the standard error of the mean (5 replicates).


Distributions of piRNA precursor length in ints - 11 RNAi - treated nematodes compared to empty vector - treated nematodes , obtained from deeply sequencing one pair of libraries ( inserts sequenced up to 75nt ) . Distributions of total sequences ( E ) and total read counts ( F ) are shown .

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Distributions of piRNA precursor length in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes, obtained from deeply sequencing one pair of libraries (inserts sequenced up to 75nt). Distributions of total sequences (E) and total read counts (F) are shown.


Co - localization between INTS - 6 : : eGFP and mCherry : : PRDE - 1 in germ cells . PRDE - 1 and INTS - 6 co - localize in early stages of meiosis ( 1 , 2 ) and up to mid - pachytene ( 3 ) , where an additional INTS - 6 focus appears ( 3 ) . When PRDE - 1 foci are about to disappear , co - localization is lost ( 4 ) , and the additional INTS - 6 focus remains present well after the loss of PRDE - 1 foci ( 5 ) .

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Co-localization between INTS-6::eGFP and mCherry::PRDE-1 in germ cells. PRDE-1 and INTS-6 co-localize in early stages of meiosis (1,2) and up to mid-pachytene (3), where an additional INTS-6 focus appears (3). When PRDE-1 foci are about to disappear, co-localization is lost (4), and the additional INTS-6 focus remains present well after the loss of PRDE-1 foci (5).


Length distributions of nucleoplasmic and chromatin - bound piRNA precursors in N2 empty vector , N2 ints - 11 RNAi , tfiis empty vector , and tfiis ints - 11 RNAi nematodes . Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows . The length distributions were calculated from two merged replicates ( see Appendix Figure S2 for the length distributions in each replicate ) .

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Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).


Signal of 5 ′ P small RNA 5 ′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs , after removal of reads corresponding to mature 21U - RNAs ( see Methods ) . The signal is normalized to counts per million of non - structural mapped reads . A peak of 5 ′ P ends centered at + 38 from piRNA TSSs is observed .

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Signal of 5′ P small RNA 5′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs, after removal of reads corresponding to mature 21U-RNAs (see Methods). The signal is normalized to counts per million of non-structural mapped reads. A peak of 5′ P ends centered at +38 from piRNA TSSs is observed.


Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength ( top panel ) . Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength ( bottom panel ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range . The data shown here corresponds to one of two N2 EV replicates , see Appendix Figure S6 for this analysis across replicates and genotypes .

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Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength (top panel). Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength (bottom panel). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. The data shown here corresponds to one of two N2 EV replicates, see Appendix Figure S6 for this analysis across replicates and genotypes.


Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif - dependent piRNAs upon ints - 11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates . N2 empty vector was used as a baseline for fold change calculation . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range .

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Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-dependent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.


Average normalized CAGE signal ( transcripts per million ) 2 nt upstream of motif - dependent and motif - independent piRNAs ( excluding undetected loci ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile

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Average normalized CAGE signal (transcripts per million) 2 nt upstream of motif-dependent and motif-independent piRNAs (excluding undetected loci). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range


Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif - independent piRNAs upon ints - 11 RNAi knockdown in an N2 and a tfiis mutant background . N2 empty vector was used as a baseline for fold change calculation . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile

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Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-independent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range


A 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 ,

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A 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


A 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 ) .

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A 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).


A - 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 ) .

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A- 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).


A 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 ) .

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A 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 ) .

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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).


B 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 ) .

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B 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).


A 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 ) .

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A 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 ) .

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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).


D . ( 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 .

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D. (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 .

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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 .

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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.


B . 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 )

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B. 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 )

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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)


All 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 .

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All 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 .

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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.


H . 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 . .

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H. 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..


D . 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 ) .

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D. 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 ) .

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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).


MSCs 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 .

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MSCs 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 .

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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 .

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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.


Hierarchical 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 .

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Hierarchical 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 .

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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 .

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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

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ELISA results depict an up-regulated expression of CXCL-6 after 24h of LPS treatment


ELISA results depict a significantly increased IL - 8 expression after 6 and 24h of LPS treatment .

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ELISA 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 .

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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.


The 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 .

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The 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 .

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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.


Representative 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 .

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Representative 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 .

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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 .

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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


Representative clinical pictures of murine wounds at 0 , 3 , 5 , 7 and 10 days after wounding . Enhanced wound healing in LPS primed MSCs group as opposed to all other groups . Statistical analysis of 20 wound areas per group at the indicated time points , expressed as percentage of the initial wound size ( day 0 ) , for PBS control , non - primed MSCs , LPS primed MSCs , non - primed scrambled siRNA - treated ( Scr ) MSCs , LPS primed Scr MSCs and LPS primed TLR4 silenced MSCs . Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments . Statistical analysis was performed using one way ANOVA .

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Representative clinical pictures of murine wounds at 0, 3, 5, 7 and 10 days after wounding. Enhanced wound healing in LPS primed MSCs group as opposed to all other groups. Statistical analysis of 20 wound areas per group at the indicated time points, expressed as percentage of the initial wound size (day 0), for PBS control, non-primed MSCs, LPS primed MSCs, non-primed scrambled siRNA-treated (Scr) MSCs, LPS primed Scr MSCs and LPS primed TLR4 silenced MSCs. Results are mean ± SD of five biological replicates representing 1 of 3 independent experiments. Statistical analysis was performed using one way ANOVA.


Representative 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 .

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Representative 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.


D . 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 ) .

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D. 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).


B . 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 .

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B. 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.


A . 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 .

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A. 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 .

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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 .

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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.


A . 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 ) .

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A. 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 .

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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 .

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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.


B . 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 .

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B. 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 .

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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.


( 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 .

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(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.


( 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 .

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(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 ) .

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(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 .

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(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 )

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(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)


( E ) Representative transport current recordings from cells expressing L448A or L448T ( GltPh A360 ) or WT EAAT1 upon voltage jumps to ˗ 140 mV before and after superfusion with 1 mM l - glutamate . Cells were intracellularly dialyzed with a K + - free solution without permeating anions to isolate K + - independent transport currents . Transient capacitive currents during the first 5 ms after a voltage jump were blanked .

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(E) Representative transport current recordings from cells expressing L448A or L448T (GltPh A360) or WT EAAT1 upon voltage jumps to ˗140 mV before and after superfusion with 1 mM l-glutamate. Cells were intracellularly dialyzed with a K+-free solution without permeating anions to isolate K+-independent transport currents. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.


( F ) Current - voltage relationship for net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for choline - based pipette solutions . Values are given as mean ± SD with indicated numbers of experiments .

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(F) Current-voltage relationship for net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for choline-based pipette solutions. Values are given as mean ± SD with indicated numbers of experiments.


( G ) Time - dependent changes in L448A EAAT1 ( GltPh A360 ) uptake currents during two repetitive glutamate applications and subsequent removals . Current amplitudes were measured at the end of voltage jumps to - 140mV and plotted versus the time . Glutamate application is indicated by green bars . Values are given as mean ± SD of the last 100 data points in the current trace .

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(G) Time-dependent changes in L448A EAAT1 (GltPh A360) uptake currents during two repetitive glutamate applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus the time. Glutamate application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.


( H ) Net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for different pipette solutions , Values are given as mean ± SD with indicated numbers of experiments ( * * * , p < 0 . 001 Student ' s t - test ) .

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(H) Net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for different pipette solutions, Values are given as mean ± SD with indicated numbers of experiments (***, p<0.001 Student's t-test).


( E ) Representative transport current recordings from a cell expressing R478A EAAT2 ( GltPh R397 ) in the absence of internal K + . Transport currents were elicited by voltage jumps to - 140mV before and after superfusion with 5 mM l - serine . Transient capacitive currents during the first 5 ms after a voltage jump were blanked .

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(E) Representative transport current recordings from a cell expressing R478A EAAT2 (GltPh R397) in the absence of internal K+. Transport currents were elicited by voltage jumps to -140mV before and after superfusion with 5 mM l-serine. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.


( F ) Time - dependent changes in steady - state R478A EAAT2 ( GltPh R397 ) uptake currents during two consecutive serine applications and subsequent removals . Current amplitudes were measured at the end of voltage jumps to - 140mV and plotted versus time . Serine application is indicated by green bars . Values are given as mean ± SD of the last 100 data points in the current trace .

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(F) Time-dependent changes in steady-state R478A EAAT2 (GltPh R397) uptake currents during two consecutive serine applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus time. Serine application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.


( G ) Current - voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l - serine for different intracellular solutions . Values are given as mean ± SD for the indicated number of experiments .

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(G) Current-voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.


( H ) Mean net transport currents for WT and R478A EAAT2 ( GltPh R397 ) induced by 5 mM l - serine for different intracellular solutions . Values are given as mean ± SD for the indicated number of experiments .

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(H) Mean net transport currents for WT and R478A EAAT2 (GltPh R397) induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.


( A ) Family pedigree . Variant segregation analysis of PSMC3 . Electropherogram of a part of intron 10 of PSMC3 encompassing the identified variation ( c . [ 1127 + 337A > G ] ; [ 1127 + 337A > G ] , p . [ ( Ser376Arg15 * ) ] ; [ ( Ser376Arg15 * ) ] ) in the affected individuals , their unaffected parents and siblings . The variation was found at the homozygous state in the affected individuals ( II . 2 , II . 4 , II . 7 ) , at the heterozygous state in the parents ( I . 1 , I . 2 , I . 3 , I . 4 , I . 5 , I . 6 ) and was either at the heterozygous state ( II . 5 ) or absent in the unaffected siblings ( II . 1 , II . 3 , II . 6 ) .

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(A) Family pedigree. Variant segregation analysis of PSMC3. Electropherogram of a part of intron 10 of PSMC3 encompassing the identified variation (c.[1127+337A>G];[1127+337A>G], p.[(Ser376Arg15*)];[(Ser376Arg15*)]) in the affected individuals, their unaffected parents and siblings. The variation was found at the homozygous state in the affected individuals (II.2, II.4, II.7), at the heterozygous state in the parents (I.1, I.2, I.3, I.4, I.5, I.6) and was either at the heterozygous state (II.5) or absent in the unaffected siblings (II.1, II.3, II.6).


( B ) Face ( up ) and profile ( down ) photographs for patients II . 4 ( a : 8 yo , b : 16 yo ) , II . 2 ( c : 6 yo , d : 14 yo ) and II . 7 ( e : 1 yo , f : 7 yo ) over time . One can observe prominent supraciliay arches , synophris , sunken cheeks , short philtrum and retrusion in the malar region .

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(B) Face (up) and profile (down) photographs for patients II.4 (a: 8 yo, b: 16 yo), II.2 (c: 6 yo, d: 14 yo) and II.7 (e: 1 yo, f: 7 yo) over time. One can observe prominent supraciliay arches, synophris, sunken cheeks, short philtrum and retrusion in the malar region.


( C ) Sub cutaneous calcifications found only on knees ( g : 9 yo and h : 16 yo ) on elbows ( i : 9 yo ) of patient II . 4 . White hair were present only on the legs of the 3 patients as illustrated for patient II . 4 ( j : 16 yo ) .

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(C) Sub cutaneous calcifications found only on knees (g: 9 yo and h: 16 yo) on elbows (i: 9 yo) of patient II.4. White hair were present only on the legs of the 3 patients as illustrated for patient II.4 (j: 16 yo).


( D ) Temporal bone CT - scan from patient II . 7 ( left column ) and a control ( right column ) showing malformation of the semicircular canal . The left ear is shown on the upper panels while de right ear on the lower panels .

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(D) Temporal bone CT-scan from patient II.7 (left column) and a control (right column) showing malformation of the semicircular canal. The left ear is shown on the upper panels while de right ear on the lower panels.


( E ) Amplification of the cDNA fragment between exons 9 - 10 and 11 of PSMC3 showing the abnormally spliced RNA fragment . One band at 180bp representing the normal allele is seen for the control and two bands for the individual II . 4 ( pathologic allele at 300bp ) .

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(E) Amplification of the cDNA fragment between exons 9-10 and 11 of PSMC3 showing the abnormally spliced RNA fragment. One band at 180bp representing the normal allele is seen for the control and two bands for the individual II.4 (pathologic allele at 300bp).


( A ) Anti - ubiquitin western blot in control and patient fibroblasts and total amount of proteins loaded ( stainfree ) showing increased ubiquitination in patient cells ( lane 4 ) .

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(A) Anti-ubiquitin western blot in control and patient fibroblasts and total amount of proteins loaded (stainfree) showing increased ubiquitination in patient cells (lane 4).