( J ) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells . Cells were incubated in serum starvation medium for 16 h , depleted of cholesterol by 1 . 5 % HPCD for 0 . 5 h , and then treated with serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate and 1 μM AI3d for 6 h . Next , cells were incubated in the same medium containing ShhN and 10 μM MβCD - cholesterol for an additional

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(J) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells. Cells were incubated in serum starvation medium for 16 h, depleted of cholesterol by 1.5% HPCD for 0.5 h, and then treated with serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 1 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 10 μM MβCD-cholesterol for an additional 16


Nucleosomal array oligomers are globular . A Nucleosomal array oligomers were stained with DAPI and examined using FM ( fluorescence microscopy ) as described in the Materials and Methods section . Shown are representative images obtained in 4 . 5 mM and 10mM MgCl2 . B Control FM images obtained in 0 , 1 and 2 . 5 mM MgCl2 .

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Nucleosomal array oligomers are globular.A Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 mM and 10mM MgCl2.B Control FM images obtained in 0, 1 and 2.5 mM MgCl2.


C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods . Shown in the left panels are representative images obtained in 4 . 5 mM , and 10 mM MgCl2 . Shown in the right panels are images of the interior of the oligomers ( white arrows , left panels ) after cropping and re - scaling .

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C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods. Shown in the left panels are representative images obtained in 4.5 mM, and 10 mM MgCl2. Shown in the right panels are images of the interior of the oligomers (white arrows, left panels) after cropping and re-scaling.


Figure 2Sedimentation velocity analysis of the salt - dependent assembly of nucleosomal array oligomers . A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2 . The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample ( see Materials and Methods ) . The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments . B Analysis of the same raw data as in panel ( A ) by the time derivative method to yield the sedimentation coefficient distribution , g ( s * ) .

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Figure 2Sedimentation velocity analysis of the salt-dependent assembly of nucleosomal array oligomers.A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2. The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample (see Materials and Methods). The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).


A , B SAXS profiles of the nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) and 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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A, B SAXS profiles of the nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


A 601 - nucleosomal arrays were incubated in 0 . 5 , 5 mM MgCl2 , 5 mM MgCl2 + MNase , and analyzed by the differential centrifugation assay to determine the fraction oligomeric . The amounts of DNA in the supernatant fraction were measured . Note that for the MNase - digested oligomers the supernatant fraction also includes the digested free linker DNA . Each value is the mean of three measurements and the error bars represent the standard deviation .

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A 601-nucleosomal arrays were incubated in 0.5, 5 mM MgCl2, 5 mM MgCl2 + MNase, and analyzed by the differential centrifugation assay to determine the fraction oligomeric. The amounts of DNA in the supernatant fraction were measured. Note that for the MNase-digested oligomers the supernatant fraction also includes the digested free linker DNA. Each value is the mean of three measurements and the error bars represent the standard deviation.


B Verification of complete MNase digestion . DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase , and then electrophoresed on agarose gel . The position of mono nucleosome is marked with a star symbol .

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B Verification of complete MNase digestion. DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase, and then electrophoresed on agarose gel. The position of mono nucleosome is marked with a star symbol.


C Nucleosomal array oligomers ( left ) or with MNase treatment ( right ) were stained with DAPI and examined using FM . Shown are representative images obtained . Note that the sizes of MNase - treated oligomers are much smaller than those of the control oligomers ( left and Fig . 1A ) .

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C Nucleosomal array oligomers (left) or with MNase treatment (right) were stained with DAPI and examined using FM. Shown are representative images obtained. Note that the sizes of MNase-treated oligomers are much smaller than those of the control oligomers (left and Fig. 1A).


A Oligomers assembled from H1 - nucleosomal arrays were stained with DAPI and examined using FM . Shown are representative images obtained in 4 mM and 5 mM MgCl2 . B Control FM images obtained in 0 , 1 and 3 mM MgCl2 .

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A Oligomers assembled from H1-nucleosomal arrays were stained with DAPI and examined using FM. Shown are representative images obtained in 4 mM and 5 mM MgCl2.B Control FM images obtained in 0, 1 and 3 mM MgCl2.


C H1 - nucleosomal array oligomers were negatively stained and visualized by TEM . Shown in the left panel is a representative image obtained in 4 mM MgCl2 . Shown in the right panel is an image of the interior of the oligomer ( white arrow , left panel ) after cropping and re - scaling .

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C H1-nucleosomal array oligomers were negatively stained and visualized by TEM. Shown in the left panel is a representative image obtained in 4 mM MgCl2. Shown in the right panel is an image of the interior of the oligomer (white arrow, left panel) after cropping and re-scaling.


Figure 6Sedimentation analysis of salt - dependent H1 - oligomer assembly . A Representative experiment showing the second moment sedimentation coefficients of the H1 - oligomers as a function of MgCl2 . The dashed line indicates the upper limit of measurable sedimentation coefficients ( ~ 106 S ) . The white symbol is intended to show that the sedimentation coefficient of the H1 - oligomers in 5 mM MgCl2 is beyond the detectable limit . B Analysis of the same raw data as in panel ( A ) by the time derivative method to yield the sedimentation coefficient distribution , g ( s * ) . C The g ( s * ) profiles in 2 . 5 mM , 3 mM , and 3 . 5 mM from panel ( B ) are re - plotted on a smaller scale .

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Figure 6Sedimentation analysis of salt-dependent H1-oligomer assembly.A Representative experiment showing the second moment sedimentation coefficients of the H1-oligomers as a function of MgCl2. The dashed line indicates the upper limit of measurable sedimentation coefficients (~106 S). The white symbol is intended to show that the sedimentation coefficient of the H1-oligomers in 5 mM MgCl2 is beyond the detectable limit.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).C The g(s*) profiles in 2.5 mM, 3 mM, and 3.5 mM from panel (B) are re-plotted on a smaller scale.


A , B SAXS profiles of the H1 - nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) and 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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A, B SAXS profiles of the H1-nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


C , D SAXS profiles of the native chicken chromatin in 0 ( TE ) , 0 . 5 , 1 mM MgCl2 ( C ) and 2 . 5 , 5 mM MgCl2 ( D ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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C, D SAXS profiles of the native chicken chromatin in 0 (TE), 0.5, 1 mM MgCl2 (C) and 2.5, 5 mM MgCl2 (D) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


A FMimages of chromatin structure in the nuclei with 0 mM ( left ) , 1 mM ( center ) and 5 mM MgCl2 ( right ) . Insets show the intensity line profiles between the two marked arrow heads in the images .

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A FMimages of chromatin structure in the nuclei with 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right). Insets show the intensity line profiles between the two marked arrow heads in the images.


B SAXS profiles of the isolated HeLa nuclei . SAXS profiles of the nuclei in 0 mM ( left ) , 1 mM ( center ) and 5 mM MgCl2 ( right ) are shown in blue . For comparison , the scattering curves of H1 - arrays from Figs . 7A and B were overlaid as red lines ( left , 0 mM ; center and right , 10 mM MgCl2 ) .

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B SAXS profiles of the isolated HeLa nuclei. SAXS profiles of the nuclei in 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right) are shown in blue. For comparison, the scattering curves of H1-arrays from Figs. 7A and B were overlaid as red lines (left, 0 mM; center and right, 10 mM MgCl2).


( b ) Flow cytometry - based stratification of cells in different cell cycle phases and in G0 ( quiescence , 2n DNA - and low RNA content ) , shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours , respectively . Cells were detached , fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis .

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(b) Flow cytometry-based stratification of cells in different cell cycle phases and in G0 (quiescence, 2n DNA- and low RNA content), shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours, respectively. Cells were detached, fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis.


( c ) Distribution of cell cycle phases in six cell lines exposed to quiescence - inducing conditions ( see panel a ) for 8 and 96 hours , respectively . Each stacked bar plot reports the fraction of cells in G0 , G1 , S and G2 / M phases , estimated using the assay depicted in panel b .

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(c) Distribution of cell cycle phases in six cell lines exposed to quiescence-inducing conditions (see panel a) for 8 and 96 hours, respectively. Each stacked bar plot reports the fraction of cells in G0, G1, S and G2/M phases, estimated using the assay depicted in panel b.


( d ) Cell confluence of adherent cell cultures in quiescence - inducing conditions ( i . e . serum starvation and glutamine limitation ) was monitored using time - lapse microscopy in a plate reader ( TECAN Spark 10M ) in 1 . 5 - hour intervals . Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach . Data points and shaded areas indicate mean ± standard deviation ( SD ) over three biological replicates . Approximately 9500 , 4500 , 19500 , 6700 , 19500 and 9500 cells / well were seeded for glutamine limitation and serum starvation of A549 , CCD1070Sk , HCT116 , HFL1 , MCF7 and SKOV3 cells , respectively .

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(d) Cell confluence of adherent cell cultures in quiescence-inducing conditions (i.e. serum starvation and glutamine limitation) was monitored using time-lapse microscopy in a plate reader (TECAN Spark 10M) in 1.5-hour intervals. Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach. Data points and shaded areas indicate mean ± standard deviation (SD) over three biological replicates. Approximately 9500, 4500, 19500, 6700, 19500 and 9500 cells/well were seeded for glutamine limitation and serum starvation of A549, CCD1070Sk, HCT116, HFL1, MCF7 and SKOV3 cells, respectively.


( b ) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium ( RPMI - 1640 with 5 % dialyzed FBS ) . Cell confluence as a proxy of cell numbers was monitored using automated time - lapse microscopy in a plate reader ( TECAN Spark 10M ) . Data points and shaded areas indicate mean ± SD over three biological replicates . ( c ) Growth phenotypic changes induced by TMZ treatment in quiescence - inducing conditions ( serum starvation ) in A549 and HCT116 cancer cells . Growth was monitored via cell confluence as described in panel b . At 96 hours , cells were stimulated to exit quiescence by replacing serum - free media with fresh growth medium without TMZ ( regrowth ) . Data points and shaded areas indicate mean ± SD over three biological replicates .

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(b) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium (RPMI-1640 with 5% dialyzed FBS). Cell confluence as a proxy of cell numbers was monitored using automated time-lapse microscopy in a plate reader (TECAN Spark 10M). Data points and shaded areas indicate mean ± SD over three biological replicates. (c) Growth phenotypic changes induced by TMZ treatment in quiescence-inducing conditions (serum starvation) in A549 and HCT116 cancer cells. Growth was monitored via cell confluence as described in panel b. At 96 hours, cells were stimulated to exit quiescence by replacing serum-free media with fresh growth medium without TMZ (regrowth). Data points and shaded areas indicate mean ± SD over three biological replicates.


( d ) Viability of quiescence - induced cells treated with TMZ , or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes : Hoechst 33342 ( all nuclei ) and propidium iodide ( dead cells ) . Data points and error bars indicate mean ± SD over three biological replicates .

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(d) Viability of quiescence-induced cells treated with TMZ, or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes: Hoechst 33342 (all nuclei) and propidium iodide (dead cells). Data points and error bars indicate mean ± SD over three biological replicates.


( e ) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ . Fold - changes were estimated relative to steady - state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates .

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(e) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ. Fold-changes were estimated relative to steady-state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates.


( f ) Volcano plot showing TMZ - induced metabolic changes ( 1 mM , 48 hours ) in A549 cells in serum starvation Shown are differences in log2 fold - change between TMZ treatment and serum starvation alone ( x - axis ) , and their statistical significance ( y - axis , adjusted p - value , t - test and Benjamini - Hochberg correction ) .

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(f) Volcano plot showing TMZ-induced metabolic changes (1 mM, 48 hours) in A549 cells in serum starvation Shown are differences in log2 fold-change between TMZ treatment and serum starvation alone (x-axis), and their statistical significance (y-axis, adjusted p-value, t-test and Benjamini-Hochberg correction).


( h ) Comparison of relative changes ( log2 fold - change ) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment ( 1 mM , y - axis ) against untreated quiescence - induced A549 cells ( x - axis ) . Fold - changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations ( Spearman , p < 0 . 01 ) with cell death at 96 hours are highlighted in blue and yellow , respectively .

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(h) Comparison of relative changes (log2 fold-change) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment (1 mM, y-axis) against untreated quiescence-induced A549 cells (x-axis). Fold-changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations (Spearman, p < 0.01) with cell death at 96 hours are highlighted in blue and yellow, respectively.


( i ) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence - inducing conditions ( serum starvation , left panels ) or full growth medium ( 5 % dFBS , right panels ) , treated with TMZ ( 0 . 4 or 1 mM ) , 5 - fluorouracil ( 5 - FU , 0 . 5 mM , positive control ) or medium ( untreated ) . Early apoptotic cells were detected using ApoTracker Green under co - staining with Hoechst 33342 ( nuclei ) and propidium iodide ( dead cells ) . Cell staining was continuously monitored in 3 - hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto . Data points and shaded areas indicate mean ± SD over three biological replicates . Approximately 3400 and 7500 cells / well were seeded of A549 and HCT116 cells , respectively .

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(i) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence-inducing conditions (serum starvation, left panels) or full growth medium (5% dFBS, right panels), treated with TMZ (0.4 or 1 mM), 5-fluorouracil (5-FU, 0.5 mM, positive control) or medium (untreated). Early apoptotic cells were detected using ApoTracker Green under co-staining with Hoechst 33342 (nuclei) and propidium iodide (dead cells). Cell staining was continuously monitored in 3-hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto. Data points and shaded areas indicate mean ± SD over three biological replicates. Approximately 3400 and 7500 cells/well were seeded of A549 and HCT116 cells, respectively.


( D and E ) qRT - PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence ( D ) or presence ( E ) of translation - blocking GFP MO . Bar charts show the average of three independent biological replicates . Error bars show standard deviation ( SD ) . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(D and E) qRT-PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence (D) or presence (E) of translation-blocking GFP MO. Bar charts show the average of three independent biological replicates. Error bars show standard deviation (SD). Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( B - E ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the codon effects measured by PACE ( y - axis ) . Each dot represents a single codon . Graphs for all codons ( B ) , codons for polar amino acids ( C ) , nonpolar amino acids ( D ) , and charged amino acids ( E ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

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(B-E) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the codon effects measured by PACE (y-axis). Each dot represents a single codon. Graphs for all codons (B), codons for polar amino acids (C), nonpolar amino acids (D), and charged amino acids (E) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


( F - I ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the tRNA levels ( y - axis , Each dot represents a single codon . Graphs for all codons ( F ) , codons for polar amino acids ( G ) , nonpolar amino acids ( H ) , and charged amino acids ( I ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

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(F-I) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the tRNA levels (y-axis, Each dot represents a single codon. Graphs for all codons (F), codons for polar amino acids (G), nonpolar amino acids (H), and charged amino acids (I) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


( B ) Charged tRNA levels in mock - injected and AnsB - overexpressing embryos at 6 hpf . Total and charged tRNAs were measured by qRT - PCR coupled to the OXOPAP assay . The relative charged tRNA level in mock - injected embryos were set to one . Data information bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(B) Charged tRNA levels in mock-injected and AnsB-overexpressing embryos at 6 hpf. Total and charged tRNAs were measured by qRT-PCR coupled to the OXOPAP assay. The relative charged tRNA level in mock-injected embryos were set to one. Data information bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( D ) Poly ( A ) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) .

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(D) Poly(A) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).


( F ) Poly ( A ) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf . ( G ) Quantification of the PAT assay at 6 hpf in ( F ) .

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(F) Poly(A) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf. (G) Quantification of the PAT assay at 6 hpf in (F).


( B ) Western blotting to detect the sfGFP levels at 6 hpf . Tubulin ( Tub ) was detected as a loading control .

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(B) Western blotting to detect the sfGFP levels at 6 hpf. Tubulin (Tub) was detected as a loading control.


( C ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos at 6 hpf relative to 2 hpf . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(C) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos at 6 hpf relative to 2 hpf. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( D ) Poly ( A ) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) .

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(D) Poly(A) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).


( G ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf . ( H ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos , MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc - tagged full - length ( full ) or RING domain deleted ( ∆ RING ) Znf598 . mRNA levels at 6 hpf relative to 2 hpf are shown . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(G) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf. (H) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos, MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc-tagged full-length (full) or RING domain deleted (∆RING) Znf598. mRNA levels at 6 hpf relative to 2 hpf are shown. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( J ) Results of PACE in MZznf598 zebrafish embryos . Black circles show the relative stability of the reporter mRNAs ( averages of two biological replicates ) in MZznf598 embryos . Gray circles show the relative stability of reporter mRNAs in wild - type embryos The stability of a codon - tag reporter with a UGA stop codon was set to one . Error bars represent maximum and minimum data points . The relative effect of each codon on mRNA stability in wild - type embryos is shown as a color gradient from red ( destabilizing ) to blue ( stabilizing ) .

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(J) Results of PACE in MZznf598 zebrafish embryos. Black circles show the relative stability of the reporter mRNAs (averages of two biological replicates) in MZznf598 embryos. Gray circles show the relative stability of reporter mRNAs in wild-type embryos The stability of a codon-tag reporter with a UGA stop codon was set to one. Error bars represent maximum and minimum data points. The relative effect of each codon on mRNA stability in wild-type embryos is shown as a color gradient from red (destabilizing) to blue (stabilizing).


( B ) Results of the tandem ORF assay with Lys AAG × 8 and Lys AAA × 8 sequences . ( C ) Results of the tandem ORF assay with Gly × 8 and Pro × 8 sequences . ( D ) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags . ( E ) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities . Data information : normalized Fluc activity with no insert was set to one . All experiments were repeated three times , and the average Fluc activity is shown as bar charts . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

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(B) Results of the tandem ORF assay with Lys AAG×8 and Lys AAA×8 sequences. (C) Results of the tandem ORF assay with Gly×8 and Pro×8 sequences. (D) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags. (E) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities. Data information: normalized Fluc activity with no insert was set to one. All experiments were repeated three times, and the average Fluc activity is shown as bar charts. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( B ) SEC trace and SDS - PAGE of purified NHE9 * ( residues 8 to and 574 out of 644 ) as depicted by schematic .

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(B) SEC trace and SDS-PAGE of purified NHE9* (residues 8 to and 574 out of 644) as depicted by schematic.


( C ) Activity of NHE9 * co - reconstituted with ATPase into liposomes to mimic the in vivo situation ( schematic ) . Representative ACMA fluorescence traces of liposome reconstituted NHE9 * ( blue ) , NHE9 * double - mutant N243A - D244A ( red ) , and rat fructose transporter GLUT5 ( black ) . ATP - driven H + pumping establishes a ΔpH ( 0 - 3 min ) . H + efflux is initiated by the addition of 40 mM NaCl , and subsequent addition of NH4Cl ( 4 min ) collapses the proton gradient .

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(C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient.


( D ) Michaelis - Menten kinetics for NHE9 * ( red ) , NHE9 ΔCTD ( green ) , N243A - D244A ( blue ) and rat GLUT5 ( black ) as detected by ACMA dequenching following substrate addition . In all experiments errors bars , s . e . m . ; n = 3 technical repeats . The apparent KM values are an average from n = 3 separate protein reconstitutions .

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(D) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions.


A ) Native mass spectrum of NHE9 * purified in the presence ( top panel ) or the absence of additional brain lipids ( bottom panel ) . In the presence of brain lipids , NHE9 * is detected as exclusively dimeric protein with poorly resolved peaks , indicating the presence of multiple lipid adducts . Without brain lipid addition , NHE9 * appears as sharp peaks , revealing several adducts of approximately 1 kDa each , as well as a minor monomer population .

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A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population.


B ) Thermal stabilization of purified dimeric NHE9 * - GFP by lipids . Normalized mean FSEC peak fluorescence before heating ( open bars ) , and after heating and centrifugation ( grey bars ) in presence of different lipids . Data presented are mean values ± data range of n = 4 experiments ( see Materials and Methods ) .

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B) Thermal stabilization of purified dimeric NHE9*-GFP by lipids. Normalized mean FSEC peak fluorescence before heating (open bars), and after heating and centrifugation (grey bars) in presence of different lipids. Data presented are mean values ± data range of n = 4 experiments (see Materials and Methods).


C ) Thermal shift stabilization of purified dimeric NHE9 * - GFP in the presence of PIP2 ( red ) compared to PIP2 - free ( black ) . Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations .

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C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations . ( F ) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts ; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained .

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E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. (F) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained.


E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations .

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E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


A . Expression heat map of netrin - 3 and netrin - 1 . Receptors of netrin - 1 and putative receptors of netrin - 3 : DCC , UNC5 - A , - B , - C , - D , Neogenin , APP , CD146 are presented . The color legend ( right - hand side ) describes all tumor types included in the heat map . Asterisks indicate the two main pathologies in which netrin - 3 expression is detectable . C . Percentage of cell lines positive for netrin - 1 and / or - 3 expression . Co - occurrence of expression was calculated using Fisher ' s exact test . P - value is indicated in the table .

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A. Expression heat map of netrin-3 and netrin-1. Receptors of netrin-1 and putative receptors of netrin-3: DCC, UNC5-A, -B, -C, -D, Neogenin, APP, CD146 are presented. The color legend (right-hand side) describes all tumor types included in the heat map. Asterisks indicate the two main pathologies in which netrin-3 expression is detectable. C. Percentage of cell lines positive for netrin-1 and/or -3 expression. Co-occurrence of expression was calculated using Fisher's exact test. P-value is indicated in the table.


B . Analysis of netrin - 1 and - 3 expression in tumor cell lines and ranked according to netrin - 1 expression

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B. Analysis of netrin-1 and -3 expression in tumor cell lines and ranked according to netrin-1 expression


B . Analysis of netrin - 1 and - 3 expression in tumor cell lines and ranked according to netrin - 1

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A . Quantification of netrin - 3 gene expression by q - RT - PCR in a panel of 181 human NB stages 1 , 2 , 3 , 4 and 4S . Number of cases is indicated on the graph . Error bars indicate s . e . m . Statistical treatment of the data was performed using a two - sided Student t - test .

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A. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human NB stages 1, 2, 3, 4 and 4S. Number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.


B . Quantification of netrin - 3 gene expression by q - RT - PCR in a panel of 181 human neuroblastoma patients , defined as low - and high - risk NB . The number of cases is indicated on the graph . Error bars indicate s . e . m . Statistical treatment of the data was performed using a two - sided Student t - test .

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B. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human neuroblastoma patients, defined as low- and high-risk NB. The number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.


C . Netrin - 3 high expression is a marker of poor prognosis in NB . 280 months overall Kaplan - Meier survival curves in a panel of 181 patients of all NB stages . The cohort was dichotomized base on netrin - 3 median expression ( in green and red ) . Statistical treatment of the data : Mantel - Cox ; p value is indicated below the graph .

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C. Netrin-3 high expression is a marker of poor prognosis in NB. 280 months overall Kaplan-Meier survival curves in a panel of 181 patients of all NB stages. The cohort was dichotomized base on netrin-3 median expression (in green and red). Statistical treatment of the data: Mantel-Cox; p value is indicated below the graph.


A . Gene Set Enrichment Analysis ( GSEA ) in high netrin - 3 NB patients . The cohort was dichotomized with the n = 50 lowest vs 50 highest : amplification the MYCN pathway ( p = 0 . 039 ; fdqr = 0 . 025 ) .

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A. Gene Set Enrichment Analysis (GSEA) in high netrin-3 NB patients. The cohort was dichotomized with the n=50 lowest vs 50 highest: amplification the MYCN pathway (p=0.039; fdqr=0.025).


C . Q - RT - PCR analysis of netrin - 3 gene expression in IMR32 ( n = 5 ) and IGRN91 ( n = 5 ) NB cell lines after MYCN silencing by siRNA ( U - test ) . Error bars indicate s . e . m .

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C. Q-RT-PCR analysis of netrin-3 gene expression in IMR32 (n=5) and IGRN91 (n=5) NB cell lines after MYCN silencing by siRNA (U-test). Error bars indicate s.e.m.


D . ChIP - seq analysis of MYCN binding , active enhancer epigenetic marks H3K27ac , H3K4me1 , and active promoter epigenetic mark H3K4me3 , on netrin - 3 locus ( red line ) . An enrichment of MYCN , associated with active enhancer marks , was detected in three different neuroblastoma cell lines NB - 1643 , COGN415 and LAN5 .

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D. ChIP-seq analysis of MYCN binding, active enhancer epigenetic marks H3K27ac, H3K4me1, and active promoter epigenetic mark H3K4me3, on netrin-3 locus (red line). An enrichment of MYCN, associated with active enhancer marks, was detected in three different neuroblastoma cell lines NB-1643, COGN415 and LAN5.


E . Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin - 3 ( n = 11 siScr , n = 9 siNetrin - 3 ; U - test ) . Error bars indicate s . e . m . F . Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin - 3 ( n = 5 siScr , n = 7 siNetrin - 3 ; U - test ) . Error bars indicate s . e . m .

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E. Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin-3 (n=11 siScr, n=9 siNetrin-3; U-test). Error bars indicate s.e.m. F. Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin-3 (n=5 siScr, n=7 siNetrin-3; U-test). Error bars indicate s.e.m.


A . Deep analysis of netrin - 3 gene expression in lung cancer cell lines ( n = 76 ; RNA - Seq ) .

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A. Deep analysis of netrin-3 gene expression in lung cancer cell lines (n=76; RNA-Seq).


B . Q - RT - PCR analysis of netrin - 1 and - 3 expression , in lung cancer cell Lines ( Error bars indicate SD , n = 3 ) .

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B. Q-RT-PCR analysis of netrin-1 and -3 expression, in lung cancer cell Lines (Error bars indicate SD, n=3).


C . Representative netrin - 3 and - 1 mRNA detection , using RNAscope on SCLC paraffin embedded tumor sections . Negative control DAPB , positive control PPiB . Each brown dot is a unique molecule of mRNA of each targeted gene ( n = 10 , scale bars 20µm ) . D . Quantification of netrin - 3 and - 1 expression in human SCLC paraffin embedded tumor sections ( High ≥ 50 % marked cells ; Low ≤ 5 % ) .

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C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm). D. Quantification of netrin-3 and -1 expression in human SCLC paraffin embedded tumor sections (High≥ 50% marked cells; Low≤ 5%).


C . Representative netrin - 3 and - 1 mRNA detection , using RNAscope on SCLC paraffin embedded tumor sections . Negative control DAPB , positive control PPiB . Each brown dot is a unique molecule of mRNA of each targeted gene ( n = 10 , scale bars 20µm ) .

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C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm).


A . ChIP - seq analysis of NeuroD1 and ASCL - 1 binding on the netrin - 3 gene promoter . A major binding site was detected ( red and blue peaks for NeuroD1 and ASCL1 respectively ) . Input controls are shown below ( black ) .

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A. ChIP-seq analysis of NeuroD1 and ASCL-1 binding on the netrin-3 gene promoter. A major binding site was detected (red and blue peaks for NeuroD1 and ASCL1 respectively). Input controls are shown below (black).


B . ChIP - seq analysis of NeuroD1 and ASCL - 1 chromatin enrichment , which was associated with broad H3K27ac - enriched regions at the netrin - 3 locus ( red line ) , indicating a transcriptionally active region in SCLC cell lines . ( rep = experimental replicates ) .

B. ChIP-seq analysis of NeuroD1 and ASCL-1 chromatin enrichment, which was associated with broad H3K27ac-enriched regions at the netrin-3 locus (red line), indicating a transcriptionally active region in SCLC cell lines. (rep= experimental replicates).


C . Q - RT - PCR analysis of netrin - 3 gene expression in NCI - H82 ( n = 6 ) and NCI - H69 ( n = 4 ) SCLC cell lines after Neurod - 1 and ASCL - 1 silencing by siRNAs ( U - test ) . Error bars indicate s . e . m .

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C. Q-RT-PCR analysis of netrin-3 gene expression in NCI-H82 (n=6) and NCI-H69 (n=4) SCLC cell lines after Neurod-1 and ASCL-1 silencing by siRNAs (U-test). Error bars indicate s.e.m.


A . Bio - layer interferometry analysis of netrin - 2L / receptor interactions . Other members of the netrin family were also tested : netrin - 1 , - 4 and - G2 . ( red : netrin - 2L ; bleu : netrin - 1 ; green netrin - 4 )

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A. Bio-layer interferometry analysis of netrin-2L/receptor interactions. Other members of the netrin family were also tested: netrin-1, -4 and -G2. (red: netrin-2L; bleu: netrin-1; green netrin-4)


C . NCI - H82 CRISPR / Cas9 were subjected to immunoblots . sGx , designates the number of guide RNAs , control cells ( sGc ) .

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C. NCI-H82 CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc).


D . Netrin - 3 silencing delays or inhibits SCLC engraftment in vivo . NMRI nude mice were subcutaneously engrafted with either NCI - H82 - CRIPSR / cas9 - NTN3 with three different guides RNAs ( sG1 p = 0 . 0022 ; sG2 p ≤ 0 . 001 ; and sG3 p ≤ 0 . 001 ) or control cells ( sGc ) n = 6 / group . Positive tumor engraftment was report when tumors reached 50 mm3 ( Mantel Cox ) .

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D. Netrin-3 silencing delays or inhibits SCLC engraftment in vivo. NMRI nude mice were subcutaneously engrafted with either NCI-H82-CRIPSR/cas9-NTN3 with three different guides RNAs (sG1 p=0.0022; sG2 p≤0.001; and sG3 p≤0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 50 mm3 (Mantel Cox).


E . NCI - H69 - CRISPR / Cas9 were subjected to immunoblots . sGx , designates the number of guide RNAs , control cells ( sGc )

E. NCI-H69-CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc)


F . NCI - H69 - CRISPR / Cas9 cells presented in E . were subcutaneously engrafted with either NCI - H69 - CRIPSR / cas9 - NTN3 with three different guide RNAs ( sG1 p = 0 . 017 ; sG2 p = 0 . 028 ; and sG3 p = 0 . 001 ) or control cells ( sGc ) n = 6 / group . Positive tumor engraftment was report when tumors reached 20 mm3 ( Mantel Cox ) .

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F. NCI-H69-CRISPR/Cas9 cells presented in E. were subcutaneously engrafted with either NCI-H69- CRIPSR/cas9-NTN3 with three different guide RNAs (sG1 p=0.017; sG2 p=0.028; and sG3 p=0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 20 mm3 (Mantel Cox).


B . Analysis of NP137 binding on netrin - 2L , netrin - 1 ; netrin - 4 or netrin - G2 by bio - layer interferometry assays . Kd rhNTN1 : 0 . 96 ± 0 . 30nM ; Kd for rchNTN2 : 1 . 53 ± 0 . 96nM

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B. Analysis of NP137 binding on netrin-2L, netrin-1; netrin-4 or netrin-G2 by bio-layer interferometry assays. Kd rhNTN1: 0.96 ± 0.30nM; Kd for rchNTN2: 1.53 ± 0.96nM


B . Analysis of NP137 binding on netrin - 2L , netrin - 1 ; netrin - 4 or netrin - G2 by bio - layer interferometry assays . Kd rhNTN1 : 0 . 96 ± 0 . 30nM ; Kd for rchNTN2 : 1 . 53 ± 0 .

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C . NP137 inhibits NTN1 / UNC5B or NTN - 2L / UNC5B interaction , detected by Elisa assays . Here the recombinant proteins are the murine forms ( Control IgG = mouse IgG2A , mNP137 = murine Fc , UNCB = rat UN5H2 ) bars indicate SD , n = 3 .

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C. NP137 inhibits NTN1/UNC5B or NTN-2L/UNC5B interaction, detected by Elisa assays. Here the recombinant proteins are the murine forms (Control IgG= mouse IgG2A, mNP137= murine Fc, UNCB=rat UN5H2) bars indicate SD, n=3.


D . NMRI nude mice were engrafted with NCI - H82 cells by subcutaneous injection of 2 million cells . When the mean tumor volume reached approximately 80 mm3 , animals were treated 3 x time / weekly by intra - abdominal injection of PBS ; NP001 ( Human IgG1 , isotype ) ; NP137 ( anti - netrin1 / 3 ) during 20 days . n = 10 animals / group ( * p < 0 . 05 , two - way Anova , Error bars indicate s . e . m . ) . E . NP137 enhances survival of mice engrafted with NCI - H82 cell line ( see D ) . Analysis of Kaplan - Meier survival curves of mice treated or not with NP137 . Mantel Cox test ; n = 10 animals / group .

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D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.). E. NP137 enhances survival of mice engrafted with NCI-H82 cell line (see D). Analysis of Kaplan-Meier survival curves of mice treated or not with NP137. Mantel Cox test; n=10 animals/group.


D . NMRI nude mice were engrafted with NCI - H82 cells by subcutaneous injection of 2 million cells . When the mean tumor volume reached approximately 80 mm3 , animals were treated 3 x time / weekly by intra - abdominal injection of PBS ; NP001 ( Human IgG1 , isotype ) ; NP137 ( anti - netrin1 / 3 ) during 20 days . n = 10 animals / group ( * p < 0 . 05 , two - way Anova , Error bars indicate s . e . m . ) .

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D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.).


F . Quantification of cell death ( cleaved PARP ) in H82 treated tumors in D . Tumors were analyzed 7 days after the first treatment ( ie : 400mm3 ; n = 3 / group . U - test , bars indicate SD ) .

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F. Quantification of cell death (cleaved PARP) in H82 treated tumors in D. Tumors were analyzed 7 days after the first treatment (ie: 400mm3; n=3/group. U-test, bars indicate SD).


( A ) ROS burst response in wild - type ( WT ) and map4k4 lines in response to 1 μM flg22 . Values are means ± SEM , n = 16 ( biological replicates ) . Results are representative of three independent experiments . Statistical analysis was performed using a one - way ANOVA with Tukey post - test , p < 0 . 01 . Data information Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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(A) ROS burst response in wild-type (WT) and map4k4 lines in response to 1 μM flg22. Values are means ± SEM, n = 16 (biological replicates). Results are representative of three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey post-test, p < 0.01. Data information Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( B ) qRT - PCR analysis of the expression of PTI marker gene FRK1 in wild - type and map4k4 - 1 seedlings 1 h after treatment with 1 μM flg22 . Bars represent means ± SD based on three biological replicates , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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(B) qRT-PCR analysis of the expression of PTI marker gene FRK1 in wild-type and map4k4-1 seedlings 1 h after treatment with 1 μM flg22. Bars represent means ± SD based on three biological replicates, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( D ) Heat map of gene expression response to flg22 in wild - type and map4k4 - 1 , red and blue indicate high and low expression levels .

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(D) Heat map of gene expression response to flg22 in wild-type and map4k4-1, red and blue indicate high and low expression levels.


( F ) qRT - PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour . Values are the means of three biological replicates ± SD , p < 0 . 01 . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different .

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(F) qRT-PCR analysis of 14 days old seedlings treated with H2O or 1 µM flg22 for 1 hour. Values are the means of three biological replicates ± SD, p < 0.01. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different.


( A ) left panel , map4k4 - 1 shows enhanced resistance to Pseudomonas syringae pv . tomato DC3000 . The growth of Pst DC3000 after inoculation in the indicated genotypes ( Col - 0 , white bars , map4k4 - 1 , green bars ) . Bacterial titers were determined 2 hours ( day 0 ) and 3 days after inoculation . Right panel , disease symptoms of wild - type and map4k4 - 1 at 3 days after inoculation . Values are the means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(A) left panel, map4k4-1 shows enhanced resistance to Pseudomonas syringae pv. tomato DC3000. The growth of Pst DC3000 after inoculation in the indicated genotypes (Col-0, white bars, map4k4-1, green bars). Bacterial titers were determined 2 hours (day 0) and 3 days after inoculation. Right panel, disease symptoms of wild-type and map4k4-1 at 3 days after inoculation. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( B ) map4k4 - 1 disease resistance phenotype was recovered to wild - type levels in two independent complementation lines , c867 and c872 . Bacterial titers were determined as described above . Values are the means ± SD , n = 6 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(B) map4k4-1 disease resistance phenotype was recovered to wild-type levels in two independent complementation lines, c867 and c872. Bacterial titers were determined as described above. Values are the means ± SD, n = 6 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( C ) qRT - PCR analysis of PR1 expression levels in Col - 0 , map4k4 - 1 and c867 after mock ( MgCl2 ) or Pst DC3000 inoculation , values are the means ± SD , n = 3 ( biological replicates ) , p < 0 . 01 . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

(C) qRT-PCR analysis of PR1 expression levels in Col-0, map4k4-1 and c867 after mock (MgCl2) or Pst DC3000 inoculation, values are the means ± SD, n = 3 (biological replicates), p < 0.01. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( D ) Flg22 - protection assay . Col - 0 , map4k4 - 1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation . The bacterial population was measured 3 days after infection . Values are the means ± SD , n = 6 . * * p < 0 . 01 , Results are representative of three independent experiments . Data information : Statistical analysis were performed using a one - way ANOVA with Newman - Keuls multiple comparison tests or t - tests . n . s . indicate non - significant .

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(D) Flg22-protection assay. Col-0, map4k4-1 and c867 were pretreated with H2O or flg22 1 day before Pst DC3000 inoculation. The bacterial population was measured 3 days after infection. Values are the means ± SD, n = 6. **p < 0.01, Results are representative of three independent experiments. Data information: Statistical analysis were performed using a one-way ANOVA with Newman-Keuls multiple comparison tests or t-tests. n.s. indicate non-significant.


( A ) Subcellular localization of MAP4K4 - GFP in Arabidopsis mesophyll protoplasts . MAP4K4 - GFP ( green ) partially co - localized with the plasma membrane dye FM4 - 64 ( red ) . 14 hours after transformation , photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4 - 64 ( < 3 min ) . Scale bars = 10 µm .

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(A) Subcellular localization of MAP4K4-GFP in Arabidopsis mesophyll protoplasts. MAP4K4-GFP (green) partially co-localized with the plasma membrane dye FM4-64 (red). 14 hours after transformation, photographs were taken when the plasma membrane of mesophyll protoplasts was stained by FM4-64 (< 3 min). Scale bars = 10 µm.


( B ) Auto - phosphorylation of recombinant HisMBP - MAP4K4 wild - type , no auto - phosphorylation in the kinase - inactive HisMBP - MAP4K4 - D139A protein . The phosphorylation level was determined by Pro - Q Diamond phosphoprotein gel stain ( upper panel ) , protein loading amounts were visualized by Coomassie G - 250 staining ( lower panel ) . The predicted molecular weight of HisMBP - MAP4K4 is 118 . 4 kDa .

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(B) Auto-phosphorylation of recombinant HisMBP-MAP4K4 wild-type, no auto-phosphorylation in the kinase-inactive HisMBP-MAP4K4-D139A protein. The phosphorylation level was determined by Pro-Q Diamond phosphoprotein gel stain (upper panel), protein loading amounts were visualized by Coomassie G-250 staining (lower panel). The predicted molecular weight of HisMBP-MAP4K4 is 118.4 kDa.


( C ) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts . BIK1 - nYFP and MAP4K4 - cYFP were co - expressed in protoplasts to show fluorescence of complemented YFP signal ( green ) , 14 h after transformation , the results were photographed by confocal laser scanning microscopy , scale bars = 10 µm . Chl indicates Chloroplast , BF indicate Bright field . nYFP and cYFP constructs were used as empty controls . MAP3K5 - K375M and MKK4 were used as control .

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(C) BiFC visualization of interaction between MAP4K4 and BIK1 in Arabidopsis mesophyll protoplasts. BIK1-nYFP and MAP4K4-cYFP were co-expressed in protoplasts to show fluorescence of complemented YFP signal (green), 14 h after transformation, the results were photographed by confocal laser scanning microscopy, scale bars = 10 µm. Chl indicates Chloroplast, BF indicate Bright field. nYFP and cYFP constructs were used as empty controls. MAP3K5-K375M and MKK4 were used as control.


( D ) MAP4K4 associates with BIK1 in vivo by Co - IP . BIK1 - HA and MAP4K4 - GFP were co - expressed in plants by crossing of 35S : : BIK1 - HA and 35S : : MAP4K4 - GFP transgenic lines . 14 days old plants were treated ( + ) with 1 µM flg22 or H2O ( - ) for 10 min . GFP trap beads were used for immunoprecipitation , and target proteins were detected by western blots using anti - GFP or anti - HA antibodies . The predicted molecular weight of BIK1 - HA , MAP4K4 - GFP is 49 . 3 kDa and 103 . 8 kDa respectively .

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(D) MAP4K4 associates with BIK1 in vivo by Co-IP. BIK1-HA and MAP4K4-GFP were co-expressed in plants by crossing of 35S::BIK1-HA and 35S::MAP4K4-GFP transgenic lines. 14 days old plants were treated (+) with 1 µM flg22 or H2O (-) for 10 min. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of BIK1-HA, MAP4K4-GFP is 49.3 kDa and 103.8 kDa respectively.


( A ) Decreased BIK1 abundance in map4k4 - 1 . 35S : : BIK1 - HA was introduced into map4k4 - 1 by crossing , sibling lines carrying either map4k4 - 1 or MAP4K4 genotype were isolated from F3 generation . BIK1 levels were detected by western blot analysis using anti - HA antibodies .

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(A) Decreased BIK1 abundance in map4k4-1. 35S::BIK1-HA was introduced into map4k4-1 by crossing, sibling lines carrying either map4k4-1 or MAP4K4 genotype were isolated from F3 generation. BIK1 levels were detected by western blot analysis using anti-HA antibodies.


( B ) MAP4K4 regulates BIK1 accumulation through the proteasome pathway . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with DMSO ( - ) or the proteasome inhibitor MG132 ( 50 µM ) for 5h , then the plants were collected for immunoblot analysis .

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(B) MAP4K4 regulates BIK1 accumulation through the proteasome pathway. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with DMSO (-) or the proteasome inhibitor MG132 (50 µM) for 5h, then the plants were collected for immunoblot analysis.


( C ) Flg22 - induced BIK1 phosphorylation assay . 10 days old 35S : : BIK1 - HA seedlings of WT or map4k4 - 1 were treated with H2O ( - ) or 1 µM flg22 for 15 min , BIK1 phosphorylation levels were detected by mobility - shift assay using anti - HA immunoblot .

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(C) Flg22-induced BIK1 phosphorylation assay. 10 days old 35S::BIK1-HA seedlings of WT or map4k4-1 were treated with H2O (-) or 1 µM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot.


( D , E and F ) Identification of phosphorylation sites by LC - MS / MS analysis . Five residues in GST - BIK1 - K105E were phosphorylated in vitro by HisMBP - MAP4K4 . The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue . In the spectrum , the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions . ( G ) S233 and T242 were required for BIK1 hyper - phosphorylation . BIK1 - HA , BIK1S233 - HA and BIK1S242 - HA mutants were expressed in Col - 0 protoplasts and treated with 100 nM flg22 for 15 min , BIK1 phosphorylation levels were detected by mobility - shift assay using anti - HA immunoblot . Each experiment was performed at least three times with similar results .

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(D, E and F) Identification of phosphorylation sites by LC-MS/MS analysis. Five residues in GST-BIK1-K105E were phosphorylated in vitro by HisMBP-MAP4K4. The peptide sequences are shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions. (G) S233 and T242 were required for BIK1 hyper-phosphorylation. BIK1-HA, BIK1S233-HA and BIK1S242-HA mutants were expressed in Col-0 protoplasts and treated with 100 nM flg22 for 15 min, BIK1 phosphorylation levels were detected by mobility-shift assay using anti-HA immunoblot. Each experiment was performed at least three times with similar results.


( A ) MAP4K4 associates with PP2C38 in vivo by Co - IP . MAP4K4 - GFP and PP2C38 - HA were co - expressed in protoplasts . GFP trap beads were used for immunoprecipitation , and target proteins were detected by western blots using anti - GFP or anti - HA antibodies . The predicted molecular weight of MAP4K4 - GFP and PP2C38 - HA is 103 . 8kDa and 48 . 1 kDa respectively .

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(A) MAP4K4 associates with PP2C38 in vivo by Co-IP. MAP4K4-GFP and PP2C38-HA were co-expressed in protoplasts. GFP trap beads were used for immunoprecipitation, and target proteins were detected by western blots using anti-GFP or anti-HA antibodies. The predicted molecular weight of MAP4K4-GFP and PP2C38-HA is 103.8kDa and 48.1 kDa respectively.


( B ) Identification of phosphorylation sites by LC - MS / MS analysis . In vitro phosphorylation site of HisMBP - PP2C38 by HisMBP - MAP4K4 . The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue . In the spectrum , the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions .

(B) Identification of phosphorylation sites by LC-MS/MS analysis. In vitro phosphorylation site of HisMBP-PP2C38 by HisMBP-MAP4K4. The peptide sequence is shown above the spectrum with the PH designating phosphorylated residue. In the spectrum, the matched fragment ions shows in red line for terminal series ions and in orange for immonium ions and modified ions.


( C ) S77 is required for flg22 - induced PP2C38 band shift . PP2C38 - HA and PP2C38 - S77A - HA were expressed in Col - 0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min . The phosphorylated PP2C38 was detected by western blot mobility shift assay .

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(C) S77 is required for flg22-induced PP2C38 band shift. PP2C38-HA and PP2C38-S77A-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min. The phosphorylated PP2C38 was detected by western blot mobility shift assay.


( D ) flg22 - induced PP2C38 band shift was compromised in map4k4 mutants . PP2C38 - HA were expressed in Col - 0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min .

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(D) flg22-induced PP2C38 band shift was compromised in map4k4 mutants. PP2C38-HA were expressed in Col-0 and map4k4 protoplasts and treated with 1µM flg22 for 30 min.


( E ) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity . HisMBP - MAP4K4 was incubated with HisMBP - PP2C38 or HisMBP - PP2C38D88N D289N ( phosphatase - inactive ) or alone , and the phosphorylation level of MAP4K4 was determined by Pro - Q Diamond phosphoprotein gel stain . All the experiments were performed three times with similar results .

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(E) PP2C38 dephosphorylates MAP4K4 by its phosphatase activity. HisMBP-MAP4K4 was incubated with HisMBP-PP2C38 or HisMBP-PP2C38D88N D289N (phosphatase-inactive) or alone, and the phosphorylation level of MAP4K4 was determined by Pro-Q Diamond phosphoprotein gel stain. All the experiments were performed three times with similar results.


( A ) BIK1 transgene restores flg22 - induced ROS burst in map4k4 - 1 . 35S : : BIK1 - HA WT or 35S : : BIK1 - HA map4k4 - 1 were generated by crossing as described before . ROS burst response in the indicated genotypes in response to 1 μM flg22 . Values are means ± SEM , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) BIK1 transgene restores flg22-induced ROS burst in map4k4-1. 35S::BIK1-HA WT or 35S::BIK1-HA map4k4-1 were generated by crossing as described before. ROS burst response in the indicated genotypes in response to 1 μM flg22. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


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(B) Morphological phenotype of 6-week-old of Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants.


( C ) ROS burst response in Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1 mutants . Values are means ± SEM , n = 16 . Results are representative of three independent experiments . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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(C) ROS burst response in Col-0, map4k4-1, bik1 and map4k4-1 bik1 mutants. Values are means ± SEM, n = 16. Results are representative of three independent experiments. Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


( D ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , bik1 and map4k4 - 1 bik1mutants by qRT - PCR analysis . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

(D) Relative expression level of PR1 in Col-0, map4k4-1, bik1 and map4k4-1 bik1mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


(E) Morphological phenotype of 6-week-old of Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants.


( F ) Relative expression level of PR1 in Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants by qRT - PCR analysis . Values are means ± SD , n = 3 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

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(F) Relative expression level of PR1 in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants by qRT-PCR analysis. Values are means ± SD, n = 3 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


( G ) ROS burst response in Col - 0 , map4k4 - 1 , map4k3 and map4k4 - 1 map4k3 mutants . Values are means ± SEM , n = 16 ( biological replicates ) . Data information : Different letters indicate statistical significant base on one - way ANOVA with Tukey post - test , samples sharing letters are not significantly different , p < 0 . 05 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) ROS burst response in Col-0, map4k4-1, map4k3 and map4k4-1 map4k3 mutants. Values are means ± SEM, n = 16 (biological replicates). Data information: Different letters indicate statistical significant base on one-way ANOVA with Tukey post-test, samples sharing letters are not significantly different, p < 0.05.


ACE2 - Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV . HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection . Luciferase activities in target cells were measured , and the percent neutralization was calculated .

ACE2-Ig potently neutralizes viruses pseudotyped with S glycoproteins from the SARS CoV and 2019 nCoV.HIVs pseudotyped with the S glycoprotein from CoVs were incubated with different fusion proteins for 1 h before infection. Luciferase activities in target cells were measured, and the percent neutralization was calculated.


Inhibition of cell fusion by ACE2 - Ig . Potent inhibition of cell fusion mediated by the SARS - CoV ( left ) or 2019 nCoV ( right ) spike ( S ) glycoprotein . Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2 - expressing cells . The activity of the reporter gene , β - gal , was measured as a correlate of fusion . The curves represent the best fit to the experimental data and were used for calculation of the IC50 . Bars indicate the S . E .

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Inhibition of cell fusion by ACE2-Ig.Potent inhibition of cell fusion mediated by the SARS-CoV (left) or 2019 nCoV (right) spike (S) glycoprotein. Cells expressing diffferent S glycoprotein were incubated with indicated fusion proteins and mixed with ACE2-expressing cells. The activity of the reporter gene, β-gal, was measured as a correlate of fusion. The curves represent the best fit to the experimental data and were used for calculation of the IC50. Bars indicate the S.E.


A , B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG - A for 16 h at 37 ° C . Anti - BDCA2 - mediated IFNα inhibition in whole - blood assays ( A ) . IFNα levels were detected using human IFNα ELISA . Graph depicts average of duplicate wells of one representative donor ( n = 10 ) . IC50 of 24F4A - mediated IFNα inhibition ( B ) in whole blood ( circles ) ( n = 10 ) and PBMC ( n = 18 ) from healthy human donors ( squares ) or SLE patients ( triangles ) ( n = 11 ) . Horizontal bar represents the mean IC50 for each sample type . Error bars represent SD of IC50 between donors .

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A, B Whole blood or PBMC were treated with increasing concentrations of 24F4A and stimulated with CpG-A for 16 h at 37°C. Anti-BDCA2-mediated IFNα inhibition in whole-blood assays (A). IFNα levels were detected using human IFNα ELISA. Graph depicts average of duplicate wells of one representative donor (n = 10). IC50 of 24F4A-mediated IFNα inhibition (B) in whole blood (circles) (n = 10) and PBMC (n = 18) from healthy human donors (squares) or SLE patients (triangles) (n = 11). Horizontal bar represents the mean IC50 for each sample type. Error bars represent SD of IC50 between donors.


C Anti - BDCA2 - mediated internalization in whole - blood assays . Whole blood was treated with increasing concentrations of 24F4A for 16 h . Mean fluorescence intensity ( MFI ) of BDCA2 was determined with a non - cross - blocking anti - BDCA2 mAb ( 2D6 ) . Shown is a representative plot of 10 experiments conducted .

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C Anti-BDCA2-mediated internalization in whole-blood assays. Whole blood was treated with increasing concentrations of 24F4A for 16 h. Mean fluorescence intensity (MFI) of BDCA2 was determined with a non-cross-blocking anti-BDCA2 mAb (2D6). Shown is a representative plot of 10 experiments conducted.


A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0 , 1 , 3 , 6 , 9 , and 24 h . FACS analysis was performed to determine BDCA2 levels on pDCs . Fluorescence minus one ( FMO ) represents background staining of BDCA2 . Shown is a representative plot of three independent experiments .

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A Whole blood was treated with the indicated concentrations of 24F4A or the IgG1 isotype control antibody for 0, 1, 3, 6, 9, and 24 h. FACS analysis was performed to determine BDCA2 levels on pDCs. Fluorescence minus one (FMO) represents background staining of BDCA2. Shown is a representative plot of three independent experiments.


B Isolated pDCs were treated with 10 μg / ml 24F4A - AF647 at 4 ° C or at 37 ° C and analyzed at the indicated time points . Subcellular localization of BDCA2 ( red ) compared to LAMP1 ( green ) was assessed using confocal microscopy . Phalloidin was used to delineate the cell membrane . Yellow represents co - localization of BDCA2 and LAMP1 . Shown is a representative image of four experiments conducted .

B Isolated pDCs were treated with 10 μg/ml 24F4A-AF647 at 4°C or at 37°C and analyzed at the indicated time points. Subcellular localization of BDCA2 (red) compared to LAMP1 (green) was assessed using confocal microscopy. Phalloidin was used to delineate the cell membrane. Yellow represents co-localization of BDCA2 and LAMP1. Shown is a representative image of four experiments conducted.


C , D Whole blood was either treated with 24F4A for 1 h at 37 ° C ( pre - incubation ) or left untreated . PBMC were isolated from each condition . PBMC from the untreated whole blood were subsequently treated with 10 μg / ml 24F4A , and cells from all conditions were stimulated with CpG - A for 16 h . Plot represents mean of duplicate wells . Flow cytometry was used to measure BDCA2 levels ( MFI ) . Shown is a representative donor of eight donors tested .

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C, D Whole blood was either treated with 24F4A for 1 h at 37°C (pre-incubation) or left untreated. PBMC were isolated from each condition. PBMC from the untreated whole blood were subsequently treated with 10 μg/ml 24F4A, and cells from all conditions were stimulated with CpG-A for 16 h. Plot represents mean of duplicate wells. Flow cytometry was used to measure BDCA2 levels (MFI). Shown is a representative donor of eight donors tested.


E Whole blood was pre - treated for 0 , 1 , 3 , 6 , and 9 h with 10 μg / ml of 24F4 or the isotype control and then stimulated with CpG - A for additional 16 h . IFNα levels were measured by ELISA . Shown is a representative plot of two independent experiments .

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E Whole blood was pre-treated for 0, 1, 3, 6, and 9 h with 10 μg/ml of 24F4 or the isotype control and then stimulated with CpG-A for additional 16 h. IFNα levels were measured by ELISA. Shown is a representative plot of two independent experiments.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . A - C Prior to in vivo dosing , baseline surface levels of BDCA2 for both the vehicle ( Ai ) and 1 mg / kg ( Bi ) animals ( red , dotted line ) were established by staining with fluorescently labeled 24F4A ( direct method ) . Maximal binding of 24F4A to BDCA2 was also established pre - dose in the vehicle ( Aii ) and 1 mg / kg ( Bii ) animals ( red , solid line ) by treating whole blood with 10 μg / ml of 24F4A at 4 ° C and then detecting bound 24F4A with a fluorescently labeled anti - human IgG1 ( indirect method ) . The direct method was used to stain whole blood from both the vehicle ( Aiii ) and 1 mg / kg 24F4A ( Biii ) animals 6 h post - dose ( red , dotted line ) . In a separate stain , the indirect method was used to detect bound 24F4A in the vehicle ( Aiv ) and 1 mg / kg ( Biv ) treated animals ( black , solid line ) . ( C ) Percent BDCA2 internalization relative to pre - dose BDCA2 levels 6 h post - dose with vehicle , 10 mg / kg , or 1 mg / kg 24F4A . Graph shows mean ± standard deviation for each group ( n = 3 ) .

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Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A-C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . D PK / PD relationship between 24F4Aserum concentrations ( red triangle , left axis ) and BDCA2 expression on pDCs ( black squares , right axis , normalized to pre - dose levels ) from the 1 mg / kg group ( i - iii ) . Serum 24F4A was measured by ELISA . ( iv ) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested .

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Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.D PK/PD relationship between 24F4Aserum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i-iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.


Cynomolgus monkeys were administered 24F4A ( 10 or 1 mg / kg ) or vehicle ( n = 3 for each dose group ) intravenously . Cynomolgus monkeys were bled at various time points , and flow cytometry was used to measure BDCA2 expression and receptor occupancy . PDCs were defined as CD20 − , CD14 − , CD123 + , and HLADR + . E Whole blood from vehicle - or 1 mg / kg 24F4A - treated monkeys was stimulated with CpG - A , and induction of IFN - I was measured by MxA bioassay at various time points pre - and post - treatment . Horizontal black lines represent the model - based estimates of the geometric mean of IFN - I in pre - and post - dose samples . Duplicate symbols represent independent replicates of the MxA bioassay for that time point . Statistical analysis was performed using a two - way mixed - effects analysis of variance ( ANOVA ) .

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Cynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).