Figure 3B ) Time - resolved Dynamic Light Scattering ( DLS ) analysis indicating average particle size over time for each of the peptides in the β - Gal peptide set ( aggregating core sequence SVIIWSLGN ) . C ) ThT binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats . D ) pFTAA binding kinetics of the β - Gal peptide set . Background - corrected fluorescence over time is shown for 3 independent repeats . E ) End - point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β - Gal peptide set . Bars depict mean concentration , whiskers indicate standard deviation ( n = 4 ) . Statistical significance was determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated . F ) TEM images of the β - Gal peptide set after 3 days incubation . Top panels give an overview image of one square in a TEM grid , lower panel shows enlarged image ( magnification 12000X ) . Scale bars indicate 12 µm ( top row ) or 1 µm ( bottom row ) . H ) Violin and boxplots showing the distribution of end - point solubilities across 11 aggregating cores , grouped by GK . The dotted line indicates a concentration of µM , corresponding to completely insoluble peptides . I ) Solubility analysis of the in vitro solubility screen performed by Niwa et al ( Niwa et al , 2009 ) . Left panel shows combined box - and violin plots depicting the solubility distributions of proteins containing at least one strong APR ( TANGO score > 70 ) not protected by any negative GKs ( red ) versus proteins where all strong APRs are flanked by at least one negative charge ( blue ) . The right panel shows a control analysis , in which proteins were classified by the occurrence of at least one strong APR ( TANGO score > 70 ) not flanked by any positive GKs ( red ) versus proteins where all strong APRs are flanked by at least one positive charge ( blue ) . The upper boxplot whiskers indicate the largest value no further than 1 . 5 times the Inter - Quartile Range ( IQR ) from the upper hinge , the lower whiskers show the smallest value at least 1 . 5 * IQR from the lower hinge . Notches extend 1 . 58 * IQR / sqrt ( n ) from either side of the median and represent a 95 % confidence interval for the median value . Statistical significance was determined through Kruskal - Wallis test with a post - hoc Wilcoxon analysis for pairwise testing . P - values are indicated .

O B-PANEL_START 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 B-PANEL_START 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-PANEL_START 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-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 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 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 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

Figure 3B) Time-resolved Dynamic Light Scattering (DLS) analysis indicating average particle size over time for each of the peptides in the β-Gal peptide set (aggregating core sequence SVIIWSLGN).C) ThT binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.D) pFTAA binding kinetics of the β-Gal peptide set. Background-corrected fluorescence over time is shown for 3 independent repeats.E) End-point solubility analysis quantifying the concentration of peptide remaining in the supernatant following ultracentrifugation for the β-Gal peptide set. Bars depict mean concentration, whiskers indicate standard deviation (n=4). Statistical significance was determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.F) TEM images of the β-Gal peptide set after 3 days incubation. Top panels give an overview image of one square in a TEM grid, lower panel shows enlarged image (magnification 12000X). Scale bars indicate 12 µm (top row) or 1 µm (bottom row).H) Violin and boxplots showing the distribution of end-point solubilities across 11 aggregating cores, grouped by GK. The dotted line indicates a concentration of µM, corresponding to completely insoluble peptides.I) Solubility analysis of the in vitro solubility screen performed by Niwa et al (Niwa et al, 2009). Left panel shows combined box- and violin plots depicting the solubility distributions of proteins containing at least one strong APR (TANGO score > 70) not protected by any negative GKs (red) versus proteins where all strong APRs are flanked by at least one negative charge (blue). The right panel shows a control analysis, in which proteins were classified by the occurrence of at least one strong APR (TANGO score > 70) not flanked by any positive GKs (red) versus proteins where all strong APRs are flanked by at least one positive charge (blue). The upper boxplot whiskers indicate the largest value no further than 1.5 times the Inter-Quartile Range (IQR) from the upper hinge, the lower whiskers show the smallest value at least 1.5 * IQR from the lower hinge. Notches extend 1.58 * IQR / sqrt(n) from either side of the median and represent a 95% confidence interval for the median value. Statistical significance was determined through Kruskal-Wallis test with a post-hoc Wilcoxon analysis for pairwise testing. P-values are indicated.


Figure 4D ) Time - resolved Dynamic Light Scattering ( DLS ) analysis showing average particle radius over time for the β - Gal peptides flanked by both natural and non - natural GKs , at increasing NaCl concentrations ( indicated in µM ) . Structures of the amino acids flanking the aggregating core are indicated on the right - hand side , the sequence of the aggregating core is indicated below .

O B-PANEL_START 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 O O O O

Figure 4D) Time-resolved Dynamic Light Scattering (DLS) analysis showing average particle radius over time for the β-Gal peptides flanked by both natural and non-natural GKs, at increasing NaCl concentrations (indicated in µM). Structures of the amino acids flanking the aggregating core are indicated on the right-hand side, the sequence of the aggregating core is indicated below.


Figure 6A ) DnaK binding to cellulose - bound peptide array . Sequences of the aggregating cores are indicated in the top of the panel , the GKs flanking them in each instance are indicated above the blots . C ) Representative Western blot of in vitro translation analysis of folding - incompetent model constructs . Bands show Total ( T ) and Soluble ( S ) fractions after centrifugation at 21 , 000 g for 30 mins . " + Dnak " indicates addition of either DnaK mix - containing DnaK , DnaJ and GrpE . D ) Bar plots showing mean solubility as determined through quantification of western blot in ( C ) , whiskers indicate standard deviation ( n = 4 ) . Statistically significant differences between the - DnaK and + DnaK conditions were determined through two - way ANOVA with Bonferroni ' s correction for multiple comparisons . P - values for significant differences are indicated . E ) Representative western blot of the solubility analysis of GK variants upon overexpression in E . coli K12 . Bands show Soluble ( Sol ) and Pellet fractions after centrifugation at 17 , 100 g for 15 mins . " - DnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE . F ) Barplots showing mean solubility as determined through quantification of western blot bands in ( E ) , whiskers indicate standard deviation ( n = 7 , except for the AA construct , where n = 4 ) . Statistical significance was determined through two - way ANOVA followed by Tukey ' s multiple comparisons test . P - values are indicated . G ) Structured Illumination Microsopy ( SIM ) images of E . coli after three hours of expression of one of the GK variants . GFP fluorescence is shown in green . " ΔDnaK " indicates expression in E . coli K12 DnaK knockout line , " + Dnak " indicates co - expression of pKJE7 , encoding DnaK , DnaJ and GrpE .

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Figure 6A) DnaK binding to cellulose-bound peptide array. Sequences of the aggregating cores are indicated in the top of the panel, the GKs flanking them in each instance are indicated above the blots.C) Representative Western blot of in vitro translation analysis of folding-incompetent model constructs. Bands show Total (T) and Soluble (S) fractions after centrifugation at 21,000 g for 30 mins. "+ Dnak" indicates addition of either DnaK mix - containing DnaK, DnaJ and GrpE.D) Bar plots showing mean solubility as determined through quantification of western blot in (C), whiskers indicate standard deviation (n=4). Statistically significant differences between the -DnaK and +DnaK conditions were determined through two-way ANOVA with Bonferroni's correction for multiple comparisons. P-values for significant differences are indicated.E) Representative western blot of the solubility analysis of GK variants upon overexpression in E. coli K12. Bands show Soluble (Sol) and Pellet fractions after centrifugation at 17,100 g for 15 mins. "-DnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.F) Barplots showing mean solubility as determined through quantification of western blot bands in (E), whiskers indicate standard deviation (n=7, except for the AA construct, where n=4). Statistical significance was determined through two-way ANOVA followed by Tukey's multiple comparisons test. P-values are indicated.G) Structured Illumination Microsopy (SIM) images of E. coli after three hours of expression of one of the GK variants. GFP fluorescence is shown in green. "ΔDnaK" indicates expression in E. coli K12 DnaK knockout line, "+ Dnak" indicates co-expression of pKJE7, encoding DnaK, DnaJ and GrpE.


Figure 1A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines . Actin was used as a loading control . B Subcutaneous melanoma tumour growth in BL / 6 WT mice ( n = 5 ) using B16F1 WT and XIAPKO ( clone1 and 2 ) . C Representative H & E ( scale bar 50 µm ) and fluorescent images of cl - Caspase 3 staining in B16 WT and XIAPKO tumours . Nuclei were stained with DAPI ( blue ) . Scale bar 100 µm . Quantification thereof ( right panel ) . The average of cl - Caspase 3 positive cells from 5 randomly selected areas of the tumour ( 5 mice per genotype ) was represented . Dots represent individual mice ( n = 5 ) . D qRT - PCR analysis ( PrimePCRTM Assay ) of different cyto - / chemokines in B16F1 WT cells plotted vs B16F1 - XIAPKO cells ( clone1 and 2 ) . Samples that are at least 2 - fold up - regulated are shown in red , samples that are at least 2 - fold down - regulated are shown in green . E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr ( control ) or siXIAP and Western blot analysis of the corresponding cell lysates ( bottom ) . F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells . G qRT - PCR analysis ( PrimePCRTM Assay ) of different cyto - / chemokines in HEK293T cells overexpressing myc - XIAP plotted vs HEK293T cells overexpressing myc - XIAPΔRING . Samples that are at least 2 - fold up - regulated are shown in red , samples that are at least 2 - fold down - regulated are shown in green . H Human cytokines array analysis of HEK293T cells transfected with myc - XIAP or myc - XIAPΔRING . Medium was changed after 16 h and the supernatant was collected after 48 h . Pre - spotted nitrocellulose membranes were incubated with the supernatants overnight at 4 ° C .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START 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 O O O O O O O O O O O O O O O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START 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 B-PANEL_START 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

Figure 1A Western blot analysis of B16F1 WT and two independent XIAPKO cell lines. Actin was used as a loading control.B Subcutaneous melanoma tumour growth in BL/6 WT mice (n=5) using B16F1 WT and XIAPKO (clone1 and 2).C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).D qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in B16F1 WT cells plotted vs B16F1-XIAPKO cells (clone1 and 2). Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.E IL8 measurement in the supernatant of the indicated cell lines after 48 h transfection with siScr (control) or siXIAP and Western blot analysis of the corresponding cell lysates (bottom).F IL8 measurement in the supernatant of the indicated cell lines after 24 h of seeding the cells.G qRT-PCR analysis (PrimePCRTM Assay) of different cyto-/chemokines in HEK293T cells overexpressing myc-XIAP plotted vs HEK293T cells overexpressing myc-XIAPΔRING. Samples that are at least 2-fold up-regulated are shown in red, samples that are at least 2-fold down-regulated are shown in green.H Human cytokines array analysis of HEK293T cells transfected with myc-XIAP or myc-XIAPΔRING. Medium was changed after 16 h and the supernatant was collected after 48 h. Pre-spotted nitrocellulose membranes were incubated with the supernatants overnight at 4°C.


Figure 2A Western blot analysis of cell lysates of BLM and BLM - XIAPKO cells ( input ) and XIAP - IP . Data are representative of two experiments . B Confocal microscopic analysis of transfected HCT116 cells with GFP - XIAP for 16 h and stained for TAB1 or RIPK2 ( red ) . Nuclei were stained with DAPI ( blue ) . C Cell - free ubiquitylation assay of recombinant GST - RIPK2 by Flag - XIAP after addition of respective E1 / E2 and recombinant ubiquitin . Poly - ubiquitylated RIPK2 and XIAP were detected by Western blotting . D Western blot analysis of cell lysates of BLM , BLM - XIAPKO , SK - Mel28 , SK - Mel28 - XIAPKO , B16 , and B16 - XIAPKO cells ( input ) and RIPK2 - IP . Poly - ubiquitin is detected using anti - ubiquitin antibody . E IL8 measurement in the supernatant of transfected BLM ( left panel ) or SK - Mel28 ( right panel ) cells with siScr ( control ) , siXIAP , siTAB1 or siRIPK2 after 48 h . Western blot analysis of the respective cell lysates of BLM cells ( left bottom ) or SK - Mel28 cells ( right bottom ) transfected with the indicated siRNA . F IL8 measurement in the supernatant of BLM or SK - Mel28 treated with 2 µM d89 ( BIR2 antagonist ) or DMSO as a negative control for 24 h . G IL8 measurement in the supernatant of BLM or SK - Mel28 treated with 20 µM birinapant ( Bir ) , 2 µM BV6 ( both BIR3 antagonist ) or DMSO as a negative control for 24 h .

O B-PANEL_START 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-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START 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-PANEL_START 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

Figure 2A Western blot analysis of cell lysates of BLM and BLM-XIAPKO cells (input) and XIAP-IP. Data are representative of two experiments.B Confocal microscopic analysis of transfected HCT116 cells with GFP-XIAP for 16 h and stained for TAB1 or RIPK2 (red). Nuclei were stained with DAPI (blue).C Cell-free ubiquitylation assay of recombinant GST-RIPK2 by Flag-XIAP after addition of respective E1/E2 and recombinant ubiquitin. Poly-ubiquitylated RIPK2 and XIAP were detected by Western blotting.D Western blot analysis of cell lysates of BLM, BLM-XIAPKO, SK-Mel28, SK-Mel28-XIAPKO, B16, and B16-XIAPKO cells (input) and RIPK2-IP. Poly-ubiquitin is detected using anti-ubiquitin antibody.E IL8 measurement in the supernatant of transfected BLM (left panel) or SK-Mel28 (right panel) cells with siScr (control), siXIAP, siTAB1 or siRIPK2 after 48 h. Western blot analysis of the respective cell lysates of BLM cells (left bottom) or SK-Mel28 cells (right bottom) transfected with the indicated siRNA.F IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 2 µM d89 (BIR2 antagonist) or DMSO as a negative control for 24 h.G IL8 measurement in the supernatant of BLM or SK-Mel28 treated with 20 µM birinapant (Bir), 2 µM BV6 (both BIR3 antagonist) or DMSO as a negative control for 24 h.


Figure 4A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored . The numbers of melanomas with different intensities were plotted into the table as a percentage of the total . Specific staining intensity for XIAP and CD177 , corresponding to relative amounts of infiltrated neutrophils , was arbitrarily set as the following : - , not expressed ; + , low ; + + , moderate ; and + + + , strong expression . Representative IHC pictures ( right panel ) . B Representative IF ( neutrophils staining ) in B16F1 WT , XIAPKO , TAB1KO or RIPK2KO tumours ( Scale bar 50 µm ) and quantification of Ly6G + cells . The sum of Ly6G - positive cells from 5 randomly selected areas of the tumour ( 5 mice per genotype ) was represented . Dots represent individual mice ( n = 5 ) . C B16F1 WT , XIAPKO ( clone 2 ) subcutaneous melanoma tumour growth in BL / 6 WT mice ( n = 5 ) . Mice were intra - peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells . D Representative IF ( neutrophils ) scale bar 50 µm and quantification of Ly6G + cells in tumours from C . The sum of Ly6G - positive cells from 5 randomly selected areas of the tumour ( 4 mice per genotype ) was represented . Dots represent individual mice ( n = 4 ) .

O B-PANEL_START 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 O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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 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 B-PANEL_START 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

Figure 4A Intensities of CD177 and XIAP immunostainings in tumour sections from 55 patients were visually scored. The numbers of melanomas with different intensities were plotted into the table as a percentage of the total. Specific staining intensity for XIAP and CD177, corresponding to relative amounts of infiltrated neutrophils, was arbitrarily set as the following: -, not expressed; +, low; ++, moderate; and +++, strong expression. Representative IHC pictures (right panel).B Representative IF (neutrophils staining) in B16F1 WT, XIAPKO, TAB1KO or RIPK2KO tumours (Scale bar 50 µm) and quantification of Ly6G+ cells. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5). C B16F1 WT, XIAPKO (clone 2) subcutaneous melanoma tumour growth in BL/6 WT mice (n=5). Mice were intra-peritoneal injected with IgG antibody as a negative control or Ly6G antibody 1 day prior to the subcutaneous injection of the B16F1 WT or XIAPKO cells.D Representative IF (neutrophils) scale bar 50 µm and quantification of Ly6G+ cells in tumours from C. The sum of Ly6G-positive cells from 5 randomly selected areas of the tumour (4 mice per genotype) was represented. Dots represent individual mice (n=4).


Figure 1Gene - trap - based genetic screen in haploid human cells for regulators of p38 kinase activation ( p38 Thr180 / Tyr182 phosphorylation ) in response to hyperosmotic shock ( 500 mM Sorbitol , 1 h ) . Per gene ( dots ) , the frequency of gene - trap insertions in the ' high ' phospho - p38 population divided by the frequency of insertions in the ' low ' population is plotted as mutation index ( y - axis ) against the total number of insertions assigned to the gene ( x - axis ) . Significant negative and positive regulators are colored in orange and blue , respectively ( two - sided Fisher ' s exact test , false discovery rate - corrected , p ≤ 0 . 05 ) . Human U2OS cells and cells deleted for ZAK ( ∆ ZAK ) were treated with anisomycin ( 1 h ) or 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting as indicated . Unique gene - trap insertions ( dots ) mapped to the genomic ZAK locus ( x - axis ) identified in the low ( blue ) and high ( orange ) channel of two individual haploid genetic screens for stress - induced p38 activation and Anisomycin ( Brockmann et al . , 2017 ) ) . The total number of identified insertions was similar for each channel within the individual screens . For visual purposes , insertion dots were spread on the y - axis and exons in the ZAK gene body schematic have been scaled up ( compared to introns ) . U2OS / S - HA - ZAKβ cells were induced for expression with doxycycline ( DOX ) and subjected to the indicated drugs and treatments ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . Sorb . , sorbitol ; Ani . , anisomycin ; Ars . , arsenite . U2OS cells stably expressing WT and kinase - dead ( KD ) versions of S - HA - ZAKβ were pre - treated with ZAK inhibitor ( ZAKi - 0 . 5 h ) and 500 mM sorbitol ( 1 h ) as indicated . Whole Cell Extracts ( WCE ) were analyzed by immunoblotting , and strep - pulldown material was used in a kinase assay , separated by SDS - PAGE and analyzed by autoradiography . U2OS and ∆ ZAK cells rescued with S - HA - ZAKβ , were DOX - induced and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies . U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and treated with sorbitol ( 500 mM , 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies . T1 , TAK1 ; M2 , MEKK2 .

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Figure 1Gene-trap-based genetic screen in haploid human cells for regulators of p38 kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38 population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).Human U2OS cells and cells deleted for ZAK (∆ZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting as indicated.Unique gene-trap insertions (dots) mapped to the genomic ZAK locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38 activation and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).U2OS/S-HA-ZAKβ cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.U2OS cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ were pre-treated with ZAK inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown material was used in a kinase assay, separated by SDS-PAGE and analyzed by autoradiography.U2OS and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.U2OS and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies. T1, TAK1; M2, MEKK2.


Figure 2U2OS cells stably expressing Strep - HA - ZAKβ or - ZAKα were treated with sorbitol ( 500 mM , 1 h ) as indicated . Cells were fixed , immunostained with HA antibody and counter - stained with DAPI . Data information : scale bars , 10 μm . U2OS cells stably expressing ZAKβ - GFP were treated with sorbitol ( 500 mM , 1 h ) , pre - extracted , fixed and immunostained with Lamin A / C antibody . Lower right : Intensity distribution graph showing fluorescence intensities along the magenta line . As in ( B ) except that Hela cells were transfected with ZAKbeta - GFP and immunostained with 53BP1 antibody . Data information : scale bars , 10 μm . Cells from ( B ) were imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm . As in ( D ) , except that cells were co - transfected with mCherry - FAK . Inserts show higher magnification of the yellow and red regions , white arrow indicates the direction of stress fibers . Data information : scale bars , 10 μm . U2OS cells were transfected with constructs from ( F ) and imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm . U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were treated with sorbitol ( 500 mM , 5 min ) . Lysates were analyzed by immunoblotting with the indicated antibodies .

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Figure 2U2OS cells stably expressing Strep-HA-ZAKβ or -ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody and counter-stained with DAPI. Data information: scale bars, 10 μm.U2OS cells stably expressing ZAKβ-GFP were treated with sorbitol (500 mM, 1 h), pre-extracted, fixed and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line. As in (B) except that Hela cells were transfected with ZAKbeta-GFP and immunostained with 53BP1 antibody. Data information: scale bars, 10 μm.Cells from (B) were imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.As in (D), except that cells were co-transfected with mCherry-FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers. Data information: scale bars, 10 μm.U2OS cells were transfected with constructs from (F) and imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.


Figure 3U2OS and ∆ ZAK cells were embedded in agarose and subjected to cyclic compression ( 0 . 5 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min . Lysates were analyzed as in ( B ) . U2OS and ∆ ZAK cells were pretreated with ZAK inhibitor ( ZAKi ) for 30 min and subjected to equibiaxial ( Equi ) or uniaxial ( Uni ) cyclic stretch ( 0 . 5 h ) as indicated . Lysates were analyzed as in ( B ) . U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch ( 5 min ) as indicated . Lysates were analyzed as in ( B ) . Expression levels of MAP3 kinase genes in human skeletal muscle ( GTEx Portal ) . TPM , Transcripts Per Million . C2C12 cells were incubated in diff . medium for 3 or 6 days . Lysates were analyzed for ZAK isoform expression by immunoblotting . C2C12 myoblasts and myotubes ( 6 d differentiation ) were pre - treated with ZAK inhibitor ( ZAKi , 0 . 5 h ) and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies .

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Figure 3U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


Figure 4Skeletal muscle ( tibialis anterior , TA ) and liver from WT and ZAK - / - mice were lysed and analyzed for ZAK isoform expression by immunoblotting . Ponceau staining of the membrane indicates equal loading . Mouse embryonic fibroblasts ( MEF ) isolated from WT and ZAK - / - mice were treated with 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . * , unspecific band . 16 - 18 - week - old WT and ZAK - / - male mice ( n = 3 biological replicates ) were subjected to the protocol in ( D ) . TA lysates were analyzed by immunoblotting with the indicated antibodies . ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from ( D ) . Values indicate absolute phospho - peptide abundances and error bars represent the standard deviation ( n = 3 biological replicates ) . TA muscle in an 8 - week old WT male mouse was electroporated with a ZAKβ - tdTomato construct . After 7 days the muscle was harvested , sectioned longitudinally and immunostained with an antibody against the Z - disc marker α - actinin1 . The right panel shows a higher magnification of the region highlighted in yellow . Data information : scale bars , 20 μm . C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP - ZAKβ constructs . Cells were fixed and immunostained with an antibody against α - actinin1 . Data information : scale bars , 20 μm . 16 - 18 - week - old WT and ZAK - / - male mice were subjected to the experimental protocol in ( I ) . TA muscle lysates were analyzed by immunoblotting with the indicated antibodies .

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Figure 4Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow. Data information: scale bars, 20 μm.C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1. Data information: scale bars, 20 μm.16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.


Figure 5H & E staining of soleus muscle cross sections from 8 - and 22 - week - old WT and ZAK - / - male mice . Arrows indicate the presence of centralized nuclei . Scale bars , 50 μm . Quantification of ( A ) . Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and * * * , p < 0 . 001 in t - test with Bonferroni - Dunn correction for multiple comparison . Muscles from ( A ) were immunostained for type I ( red ) and type IIa ( green ) fibers using myosin isoform - specific antibodies . Scale bars , 500 μm . Quantification of ( C ) . Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and ns , not significant in t - test with Bonferroni - Dunn correction for multiple comparison .

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Figure 5H&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm. Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm. Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.


Figure 4 Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect - size heat maps of six drugs ( see titles of panels ) targeting EGFR . It is evident that the different drugs showed different profiles but also that high MAP2K1 expression ( blue / red gradient across cell lines ) was consistently associated with drug resistance ( dark blue / yellow gradient across cell lines ; AUC : area under the curve ; see main text and Appendix for details ) . See also Figure EV5 .

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Figure 4 Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect-size heat maps of six drugs (see titles of panels) targeting EGFR. It is evident that the different drugs showed different profiles but also that high MAP2K1 expression (blue/red gradient across cell lines) was consistently associated with drug resistance (dark blue/yellow gradient across cell lines; AUC: area under the curve; see main text and Appendix for details). See also Figure EV5.


Figure 5 ( A ) Effect - size heat maps of two drugs ( one from two different drug sensitivity screens ) targeting MEK1 / 2 show consistent association of high MERTK expression with drug resistance . The colour scheme is the same as in Figure 4 . ( C ) Dose - response curves of two drugs for which high MERTK ( left panels ) or ACVR2A ( right panels ) expression was predicted to confer drug resistance . For each drug , three cell lines predicted to be sensitive ( dark blue ) and three cell lines predicted to be resistant ( yellow ) were assayed for viability . The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype ( data represents the average of three technical replicates ; see Appendix for details ) . ( D ) Boxplots summarizing the data shown in panel ( C ) using the area under the curve ( AUC ) as a measure for drug sensitivity . High AUC values indicate drug resistance . Again , it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly ( p ≤ 0 . 05 , one - sided Mann - Whitney test ) more resistant than cell lines predicted to be more sensitive . ( E ) Western blot of and cells , visualising successful knockout by CRISPR / Cas9 . ( F ) Dose - response curve of RDEA119 ( MEK1 / 2 inhibitor ) in and cells . The knockout is more sensitive to MEK1 / 2 inhibition than the wild type . See also Figure EV6 .

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Figure 5(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4.(C) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details).(D) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.(E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9.(F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.


Figure 6 ( A ) Western Blot visualising MERTK expression in two negative ( RKO , CC07 ) and two positive control cell lines ( CaCo - 2 ; HT55 ) . ( B ) IHC staining of MERTK expression in RKO ( negative control ) and CaCo - 2 ( positive control ) cells . ( C ) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists . ( D ) Kaplan - Meier plots showing worse overall , disease - free and recurrence - free survival of patients with high cytoplasmic / membranous expression of MERTK .

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Figure 6(A) Western Blot visualising MERTK expression in two negative (RKO, CC07) and two positive control cell lines (CaCo-2; HT55).(B) IHC staining of MERTK expression in RKO (negative control) and CaCo-2 (positive control) cells.(C) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists.(D) Kaplan-Meier plots showing worse overall, disease-free and recurrence-free survival of patients with high cytoplasmic/membranous expression of MERTK.


Figure 1 ( A ) Neutrophil bacterial killing were assessed at day 6 ( section iii ) . Controls were without treatment . . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve . ( A ) Neutrophil morphologic differentiation were assessed at day 6 ( section ii ) . Controls were without treatment . White arrows indicate neutrophilnuclear segmentation . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve . ( A ) Neutrophil ROS production were assessed at day 6 ( iv ) . Controls were without treatment . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve . ( A ) Proliferation analysis of hematopoietic CD34 + precursors for up to 6 days ( section i ) . ( B ) qRT - PCR assessed RA - target gene expression after culturing CD34 + cells for 1 , 2 , and 6 days . ( C ) Flow cytometry analysis of CD66 - CD18 co - expression after culturing CD34 + cells for 6 days , using anti - human CD66 - PE and CD18 - FITC antibodies ( section i ) . Isotypes were used for controls ( section i , top panel ) . CD66 - CD18 co - expression was quantified in section ii . ( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear cells were evaluated by ROS production ( section ii ) . ( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bacterial killing was assessed in the presence or absence of PBneutrophils ( section i ) . ( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear cells were evaluated by bacterial killing assays ( section iii ) . ( E ) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies . Controls were with or without IgG . Normal IgG , φ ; Ab , antibodies ; Comb Ab , combined anti - CD18 and - CD11b antibodies . ( F ) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days . Bacterial killing ( sections i ) and neutralization of bacterial killing in the presence of IgG or anti - CD18 antibody were assessed at day 3 ( section ii ) .

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Figure 1(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i).(B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days.(C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii.(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).(D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i).(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.(F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).


Figure 2 ( A ) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia ( AML ) patient . ROS production ( section ii ) were analyzed after cells were treated for 4 days . ( A ) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia ( AML ) patient . Proliferation ( section i ) were analyzed after cells were treated for 4 days . ( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . Proliferation was assessed at day 3 in parallel ( section ii ) . ( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . Transcription of RA - target genes was analyzed with qRT - PCR at day 2 , 3 , and 6 ( sections i ) . ( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . ROS production were assessed at day 3 in parallel ( section iii ) .

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Figure 2(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days.(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).


Figure 3 ( A ) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF ( 25 ng / ml ) for up to 9 days . Dynamic changes in proliferation ( left sections ) and live cells on day 9 ( right sections ) were illustrated for each of specimens . Controls were without treatment . ( B ) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . Dynamic changes in proliferation ( left sections ) and live cells on day 12 ( right sections ) were illustrated for each of specimens . ( C ) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF . Proliferation ( left sections ) and bacterial killing ( right sections ) were analyzed on day 12 for each of samples . ( B ) Fresh BM mononuclear cells collected from AML patients were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . ROS production was assessed at day 12 ( section ii ) . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Morphologic differentiation was assessed at day 12 ( section iv ) . White arrows indicate neutrophil nuclear segmentation . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Proliferation was assessed for up to 12 days ( section i ) . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Bacterial killing was assessed at day 12 ( sections iii ) . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . RA - target gene expression was assessed at day 12 ( section v ) .

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Figure 3(A) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).


Figure 4 ( A ) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide ( CPA ) consists of both neutrophil decrease and neutrophil recovery stages . ANC , absolute neutrophil count . ( D ) Vetscan counting PBleukocytes . ( E ) Neutrophils induced by low dose treatment in PB ( section i ) and BM ( section ii ) . ( F ) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post - infection and in spleen ( sections i - iii ) , using blood agar analysis of total extracellular viable bacteria . ( H ) Vetscan counting PBleukocytes . ( I ) Neutrophils induced by medium dose treatment in PB ( section i ) and BM ( section ii ) . ( J ) Similar to panel F , bacterial killing by PBneutrophils was evaluated 3 and 16 hr post - infection and in heart .

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Figure 4(A) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide (CPA) consists of both neutrophil decrease and neutrophil recovery stages. ANC, absolute neutrophil count.(D) Vetscan counting PBleukocytes.(E) Neutrophils induced by low dose treatment in PB (section i) and BM (section ii).(F) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post-infection and in spleen (sections i-iii), using blood agar analysis of total extracellular viable bacteria.(H) Vetscan counting PBleukocytes.(I) Neutrophils induced by medium dose treatment in PB (section i) and BM (section ii).(J) Similar to panel F, bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection and in heart.


Figure 5 ( B ) Vetscan counting PBleukocytes . ( C ) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post - infection ( sections i , ii ) , using blood agar analysis of extracellular viable bacteria . ( D E ) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and / or GCSF in BM ( panel D ) and PB ( panel E ) . White arrows indicate neutrophilnuclear segmentation .

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Figure 5(B) Vetscan counting PBleukocytes.(C) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection (sections i, ii), using blood agar analysis of extracellular viable bacteria.(D E) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and/or GCSF in BM (panel D) and PB (panel E). White arrows indicate neutrophilnuclear segmentation.


Figure 6 ( B ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 3 independent experiments with similar results . ( C ) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9 . White arrows indicate phagocytosed S . aureus in neutrophils . ( E ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 2 independent experiments with similar results . ( G ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) .

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Figure 6(B) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 3 independent experiments with similar results.(C) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9. White arrows indicate phagocytosed S. aureus in neutrophils.(E) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 2 independent experiments with similar results.(G) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii).


Figure 7 ( A B ) Neutrophil recovery and morphologic differentiation were assessed in BM ( panel A ) and PB ( panel B ) of CCIN mice surviving on day 9 . White arrows indicate neutrophil nuclear segmentation . ( C ) PBneutrophil production and nuclear segmentation were assessed in control and Am80 - GCSF mice surviving on day 13 ( section i ) . ( C ) PBneutrophil production and nuclear segmentation were assessed in control and Am80 - GCSF mice surviving on day 13 ( section ii ) . ( C ) The increased spleen weight on day 9 in Am80 - GCSF mice dropped to the levels similar to those in control mice on day 13 ( sections iii , iv ) . ( C ) The increased spleen size on day 9 in Am80 - GCSF mice dropped to the levels similar to those in control mice on day 13 ( section iii ) . ( D ) Significant loss of body weight occurred in GCSFmice on day 9 . ( E ) Ultrastructure analysis of neutrophils collected from mice surviving on day 11 . Neutrophil from GCSFmice showed less granules ( section i , images 1 vs . 2 or 3 ) , more intracytoplasmic vacuoles ( sections i , ii ) , and inner nuclear membrane dilation ( sections i , iii ) . Pg : primary granules ; Sg : secondary granules .

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Figure 7(A B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation.(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i).(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii).(C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv).(C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii).(D) Significant loss of body weight occurred in GCSFmice on day 9.(E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSFmice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules.


Figure 2Sqr mRNA levels ( A , B , C ) , in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . SQR protein levels ( D , E , F ) in cerebrum ( A , D ) , kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . Note that SQRwestern - blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum . In kidneys and muscle the western - blots were performed in tissue homogenates . SQR activity ( G , H ) in kidneys ( B , E , G ) and muscle ( C , F , H ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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Figure 2Sqr mRNA levels (A, B, C), in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.SQR protein levels (D, E, F) in cerebrum (A, D), kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice. Note that SQRwestern-blots were performed in isolated cerebralmitochondria due to the low levels of this protein in cerebrum. In kidneys and muscle the western-blots were performed in tissue homogenates.SQR activity (G, H) in kidneys (B, E, G) and muscle (C, F, H) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Figure 3Levels of CoQ10 in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency ( A ) . Levels of SQR protein in fibroblasts of controls ( C ) and patients ( P1 - 4 ) with primary CoQ10 deficiency cultured without ( vehicle ) and with 5μM of CoQ10 ( + CoQ10 ) during 1 day or 7 days ( B ) . Total CoQ levels ( CoQ9 + CoQ10 ) in kidneys ( C ) and muscle ( D ) of Coq9 + / + , Coq9R239X and Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice . Levels of SQR protein in kidneys ( E ) and muscle ( F ) of Coq9 + / + , Coq9R239X and Coq9R239X + ubiquinol - 10 ( Q10H2 ) mice .

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Figure 3Levels of CoQ10 in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency (A).Levels of SQR protein in fibroblasts of controls (C) and patients (P1-4) with primary CoQ10 deficiency cultured without (vehicle) and with 5μM of CoQ10 (+ CoQ10) during 1 day or 7 days (B).Total CoQ levels (CoQ9 + CoQ10) in kidneys (C) and muscle (D) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.Levels of SQR protein in kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9R239X + ubiquinol-10 (Q10H2) mice.


Figure 4TST protein levels in cerebrum ( A ) , kidneys ( B ) and muscle ( C ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . TST activity in cerebrum ( D ) , kidneys ( E ) and muscle ( F ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . ETHE1 ( SDO ) protein levels in cerebrum ( G ) , kidneys ( H ) and muscle ( I ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . SUOX protein levels in cerebrum ( J ) , kidneys ( K ) and muscle ( L ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice .

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Figure 4TST protein levels in cerebrum (A), kidneys (B) and muscle (C) of Coq9+/+, Coq9R239X and Coq9Q95X mice.TST activity in cerebrum (D), kidneys (E) and muscle (F) of Coq9+/+, Coq9R239X and Coq9Q95X mice.ETHE1 (SDO) protein levels in cerebrum (G), kidneys (H) and muscle (I) of Coq9+/+, Coq9R239X and Coq9Q95X mice.SUOX protein levels in cerebrum (J), kidneys (K) and muscle (L) of Coq9+/+, Coq9R239X and Coq9Q95X mice.


Figure 5Quantification of sulfide levels in cerebrum and kidneys of Coq9 + / + , Coq9R239X and Coq9Q95X mice ( A ) . Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9 + / + and Coq9R239X mice ( B ) .

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Figure 5Quantification of sulfide levels in cerebrum and kidneys of Coq9+/+, Coq9R239X and Coq9Q95X mice (A).Qualitative measurement of hydrogen sulfide in cerebrum and kidneys of Coq9+/+ and Coq9R239X mice (B).


Figure 6Total GSH in cytosol and mitochondria of cerebrum of Coq9 + / + and Coq9R239X mice ( A ) . Cytosolic GPx and GRd activities in cerebrum of Coq9 + / + and Coq9R239X mice ( B ) . Levels of GPx4 ( C ) and GRd ( D ) protein in cerebral homogenate of Coq9 + / + and Coq9R239X mice . Levels of L - Glutamate ( L - Glu ) , N - Acetyl - Glutamate ( NacGlu ) , L - Tryptophan ( L - Trp ) , 5HIAA , N - Acetyl - Tryptophan ( NALT ) , L - Tyrosine ( L - Tyr ) in cerebrum of Coq9 + / + and Coq9R239X mice ( E ) .

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Figure 6Total GSH in cytosol and mitochondria of cerebrum of Coq9+/+ and Coq9R239X mice(A).Cytosolic GPx and GRd activities in cerebrum of Coq9+/+ and Coq9R239X mice (B).Levels of GPx4 (C) and GRd (D) protein in cerebral homogenate of Coq9+/+ and Coq9R239X mice.Levels of L-Glutamate (L-Glu), N-Acetyl-Glutamate (NacGlu), L-Tryptophan (L-Trp), 5HIAA, N-Acetyl-Tryptophan (NALT), L-Tyrosine (L-Tyr) in cerebrum of Coq9+/+ and Coq9R239X mice (E).


Figure 7SQR ( A ) and TST ( B ) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137 . TST protein level in kidneys ( C ) and cerebrum ( D ) of Coq9 + / + mice supplemented with the H2S donor GYY4137 . Levels of neurotransmitters in cerebrum of Coq9 + / + mice supplemented with the H2S donor GYY4137 ( E ) .

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Figure 7SQR (A) and TST (B) protein levels in human skin fibroblasts supplemented with the H2S donor GYY4137.TST protein level in kidneys (C) and cerebrum (D) of Coq9+/+ mice supplemented with the H2S donor GYY4137.Levels of neurotransmitters in cerebrum of Coq9+/+ mice supplemented with the H2S donor GYY4137 (E).


Figure 8COX activity in cerebrum , kidneys and muscle ( A ) of Coq9 + / + , Coq9R239X and Coq9Q95X mice . Images COX histochemistry ( B ) in gastrocnemious of Coq9 + / + , Coq9R239X and Coq9Q95X mice ; scale bars : 100 μm .

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Figure 8COX activity in cerebrum, kidneys and muscle (A) of Coq9+/+, Coq9R239X and Coq9Q95X mice.Images COX histochemistry (B) in gastrocnemious of Coq9+/+, Coq9R239X and Coq9Q95X mice; scale bars: 100 μm.


Figure 9Systolic , diastolic and mean blood pressure in Coq9 + / + and Coq9R239X mice ( A ) . Heart rate in Coq9 + / + and Coq9R239X mice ( B ) . * * P < 0 . 01 ; Coq9R239X mice versus Coq9 + / + mice ( t test ; n = 5 for each group ) .

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Figure 9Systolic, diastolic and mean blood pressure in Coq9+/+ and Coq9R239X mice (A).Heart rate in Coq9+/+ and Coq9R239X mice (B). **P < 0.01; Coq9R239X mice versus Coq9+/+ mice (t test; n = 5 for each group).


Figure 1A . Enrichment of lamin B1 in the SGZ of DG . B . Immunohistochemical analyses of lamin B1 - high cells . The lamin B1 - high cells express Sox2 or PSA - NCAM , but not Prox1 . Arrowheads indicate Prox1 negative lamin B1 - high cells in the SGZ . C . Immunohistochemical analyses of lamin B1 in pNestin - GFP mice . An open arrowhead indicates RGL - ANSC and a closed arrowhead indicates a non - RGL ( NR ) - NPC . E . Immunocytochemistry of lamin B1 in NPCs and Tuj1 + differentiating neurons 4 days after the induction of differentiation . F . Relative expression of Lmnb1 and Tubb3 revealed by qRT - PCR from NPCs and differentiating neurons at the indicated days . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 , one sample t - test ( n = 4 ) . G . Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types . F ( 3 , 82 ) = 173 . 1 , P = 1 . 2 e - 34 , n = 12 - 30 cells per cell type , one - way ANOVA followed by Tukey - Kramer test ( * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O B-PANEL_START 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 B-PANEL_START 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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 B-PANEL_START 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

Figure 1A. Enrichment of lamin B1 in the SGZ of DG.B. Immunohistochemical analyses of lamin B1-high cells. The lamin B1-high cells express Sox2 or PSA-NCAM, but not Prox1. Arrowheads indicate Prox1 negative lamin B1-high cells in the SGZ.C. Immunohistochemical analyses of lamin B1 in pNestin-GFP mice. An open arrowhead indicates RGL-ANSC and a closed arrowhead indicates a non-RGL(NR)-NPC.E. Immunocytochemistry of lamin B1 in NPCs and Tuj1+ differentiating neurons 4 days after the induction of differentiation.F. Relative expression of Lmnb1 and Tubb3 revealed by qRT-PCR from NPCs and differentiating neurons at the indicated days. ***P < 0.001, **P < 0.01, *P < 0.05, one sample t-test (n = 4).G. Quantification of signal intensity of lamin B1 immunoreactivity in different neural cell types. F (3,82) = 173.1, P = 1.2 e-34, n = 12-30 cells per cell type, one-way ANOVA followed by Tukey-Kramer test (**P < 0.01).


Figure 2A . Age - dependent reduction of lamin B1 in the SGZ . As early as 5 . 5 months of age , the levels of lamin B1 in the SGZ were markedly reduced . Insets show higher magnification images in the SGZ . B - D . Quantification of lamin B1 levels using immunofluorescent signals . Lamin B1 levels in Sox2 - positive , PSA - NCAM - negative - ANSPCs were selectively downregulated at 5 . 5 months of age ( ANPCs , P = 1 . 02129e - 19 ; Neuroblasts , P = 0 . 09 , DGCs , P = 6 . 4011e - 15 , ANOVA ) . * * P < 0 . 001 , ANOVA followed by Tukey - Kramer test . ( 3 animals , 30 cells per conditions ) E . Age - dependent reduction of lamin B1 in RGL - ANSCs ( upper ) and NR - ANPC ( lower ) from 2 to 5 . 5 months of age . Arrowheads indicate RGL - ANSCs or NR - ANPCs . F . Quantification of lamin B1 levels in RGL - ANSCs and ANPCs . * P < 0 . 05 , * * * P < 0 . 001 , ( 4 animals , 18 - 32 cells per conditions , Mann - Whitney test ) .

O B-PANEL_START 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 B-PANEL_START 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 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-PANEL_START 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 O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Age-dependent reduction of lamin B1 in the SGZ. As early as 5.5 months of age, the levels of lamin B1 in the SGZ were markedly reduced. Insets show higher magnification images in the SGZ.B-D. Quantification of lamin B1 levels using immunofluorescent signals. Lamin B1 levels in Sox2-positive, PSA-NCAM-negative-ANSPCs were selectively downregulated at 5.5 months of age (ANPCs, P = 1.02129e-19; Neuroblasts, P = 0.09, DGCs, P = 6.4011e-15, ANOVA). ** P < 0.001, ANOVA followed by Tukey-Kramer test. (3 animals, 30 cells per conditions)E. Age-dependent reduction of lamin B1 in RGL-ANSCs (upper) and NR-ANPC (lower) from 2 to 5.5 months of age. Arrowheads indicate RGL-ANSCs or NR-ANPCs. F. Quantification of lamin B1 levels in RGL -ANSCs and ANPCs. * P < 0.05, *** P < 0.001, (4 animals, 18-32 cells per conditions, Mann-Whitney test).


Figure 3Open field ( OF ) test . ( B ) Color - coded distribution of the exploratory path in the open field test . X - Y axes of squares correspond to the OF arena . Old WT mice as well as Lmnb1 - cKO mice spend more time in the periphery of the OF arena . Open field ( OF ) test . ( C ) Total distance traveled during the OF test . No significant difference among groups . F ( 2 , 25 ) = 2 . 23 , P = 0 . 13 , ANOVA . ( D ) Velocity of travel during the OF test . F ( 2 , 25 ) = 1 . 60 , P = 0 . 22 , ANOVA . N . S . ( not significant ) . ( E ) Fraction of time spent in the periphery . F ( 2 , 25 ) = 10 . 71 , P = 0 . 00044 , ANOVA followed by Tukey Kramer , * * P < 0 . 01 . ( F ) Fraction of traveled distance in the periphery , F ( 2 , 25 ) = 3 . 84 , P = 0 . 035 , ANOVA followed by Tukey Kramer , * P < 0 . 05 ( n : WT = 10 , WT - old = 7 , Lmnb1 - cKO = 11 for the OF test ) . H - J . Novelty suppressed feeding . ( H ) Total feeding time during the NSF test , P = 0 . 0068 , Kruskal - Wallis test followed by Dunn ' s test . * * P < 0 . 01 . ( n : WT = 6 , WT - old = 7 , Lmnb1 - cKO = 6 for novelty suppressed feeding ) . ( I ) Food consumption during the NSF test , P = 0 . 014 , Kruskal - Wallis test followed by Dunn ' s test . * P < 0 . 05 . ( J ) Total number of feeding instances during NSF test . N . S . ( not significant ) .

O B-PANEL_START 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 B-PANEL_START 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 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 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 B-PANEL_START 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 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

Figure 3Open field (OF) test. (B) Color-coded distribution of the exploratory path in the open field test. X-Y axes of squares correspond to the OF arena. Old WT mice as well as Lmnb1-cKO mice spend more time in the periphery of the OF arena.Open field (OF) test. (C) Total distance traveled during the OF test. No significant difference among groups. F(2, 25) = 2.23, P = 0.13, ANOVA. (D) Velocity of travel during the OF test. F(2, 25) = 1.60, P = 0.22, ANOVA. N.S. (not significant). (E) Fraction of time spent in the periphery. F(2, 25) = 10.71, P = 0.00044, ANOVA followed by Tukey Kramer, ** P < 0.01. (F) Fraction of traveled distance in the periphery, F(2, 25) = 3.84, P = 0.035, ANOVA followed by Tukey Kramer, * P < 0.05 (n: WT = 10, WT-old =7, Lmnb1-cKO = 11 for the OF test).H-J. Novelty suppressed feeding. (H) Total feeding time during the NSF test, P = 0.0068, Kruskal-Wallis test followed by Dunn's test. ** P < 0.01. (n: WT = 6, WT-old =7, Lmnb1-cKO = 6 for novelty suppressed feeding). (I) Food consumption during the NSF test, P = 0.014, Kruskal-Wallis test followed by Dunn's test. * P < 0.05. (J) Total number of feeding instances during NSF test. N.S. (not significant).


Figure 4B , C . Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment . Note that the numbers of DCX - positive cells are increased . Boxes indicate magnified regions in C . D , E , F . Quantification three weeks after TAM treatment . Among EYFP - positive cells , the density of RGL - ANSCs is not different ( P = 0 . 65 , t - test ) ( D ) . The density of ANPCs is significantly decreased ( P = 0 . 0037 , t - test ) ( E ) whereas the density of DCX - positive cells is markedly increased ( P = 0 . 014 , t - test ) ( F ) . ( n = 4 for Control , n = 3 for cKO ) . Open arrowheads indicate respective cells . G , H , I . Quantification two months after the induction of KO ( n = 3 for Control , n = 4 for cKO ) . The density of RGL - ANSCs is not significantly different ( P = 0 . 29 , t - test ) ( G ) . The density of ANPCs is significantly decreased . ( P = 0 . 0092 , t - test ) ( H ) . The density of neuroblasts is not significantly different ( P = 0 . 76 , t - test ) ( I ) . J , K , L . Quantification of proliferating cells three weeks after TAM treatment . The density of Ki67 + RGL - ANSCs is significantly decreased ( P = 0 . 027 , Mann Whitney test ) ( J ) , and that of Ki67 + ANPCs is also significantly decreased . ( P = 0 . 034 , t - test ) ( K ) . The density of Ki67 + neuroblasts is significantly increased ( P = 0 . 0011 , t - test ) ( N ) ( n = 4 for Control , n = 4 for cKO ) ( L ) . M , N , O . Quantification of proliferating cells two months after TAM treatment . The density of Ki67 + RGL - ANSCs is significantly decreased ( P = 0 . 0086 , t - test ) ( M ) , and densities of Ki67 + ANPCs and Ki67 + neuroblasts are also significantly decreased . ( P = 0 . 023 , P = 0 . 025 , t - test ) ( n = 3 for Control , n = 3 for cKO ) ( N , O ) . P . Declined return to RGL - ANSCs . Left , experimental scheme . Analysis was performed 3 weeks after BrdU injections . Middle , a representative image of BrdU + GFAP + RGL - ANSCs . Right , Quantification of BrdU + GFAP + RGL - ANSCs ( P = 0 . 019 , n = 4 for Control , n = 4 for cKO ) .

O B-PANEL_START 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 B-PANEL_START 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 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 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 O O O O O O O B-PANEL_START 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 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 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 B-PANEL_START 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

Figure 4B, C. Representative images of Control and cKO with EYFP and DCX staining three weeks after TAM treatment. Note that the numbers of DCX-positive cells are increased. Boxes indicate magnified regions in C.D, E, F. Quantification three weeks after TAM treatment. Among EYFP-positive cells, the density of RGL-ANSCs is not different (P = 0.65, t-test) (D). The density of ANPCs is significantly decreased (P = 0.0037, t-test) (E) whereas the density of DCX-positive cells is markedly increased (P = 0.014, t-test) (F). (n = 4 for Control, n =3 for cKO). Open arrowheads indicate respective cells. G, H, I. Quantification two months after the induction of KO (n = 3 for Control, n = 4 for cKO). The density of RGL-ANSCs is not significantly different (P = 0.29, t-test) (G). The density of ANPCs is significantly decreased. (P = 0.0092, t-test) (H). The density of neuroblasts is not significantly different (P = 0.76, t-test) (I). J, K, L. Quantification of proliferating cells three weeks after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.027, Mann Whitney test) (J), and that of Ki67+ANPCs is also significantly decreased. (P = 0.034, t-test) (K). The density of Ki67+ neuroblasts is significantly increased (P = 0.0011, t-test) (N) (n = 4 for Control, n = 4 for cKO) (L). M, N, O. Quantification of proliferating cells two months after TAM treatment. The density of Ki67+RGL-ANSCs is significantly decreased (P = 0.0086, t-test) (M), and densities of Ki67+ANPCs and Ki67+neuroblasts are also significantly decreased. (P = 0.023, P = 0.025, t-test) (n = 3 for Control, n = 3 for cKO) (N, O). P. Declined return to RGL-ANSCs. Left, experimental scheme. Analysis was performed 3 weeks after BrdU injections. Middle, a representative image of BrdU+GFAP+ RGL-ANSCs. Right, Quantification of BrdU+GFAP+RGL-ANSCs (P = 0.019, n = 4 for Control, n = 4 for cKO).


Figure 5Representative confocal images of the SGZ 6 . 5 months after the treatment with TAM . C . Representative confocal images of the SGZ 6 . 5 months after the treatment with TAM . Quantification of cell numbers 6 . 5 months after the induction of KO ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M , ) . ( D ) The density of RGL - ANSCs tends to be reduced in cKO . F ( 2 , 8 ) = 3 . 98 , P = 0 . 063 , One - way ANOVA . ( E ) The density of ANPCs was reduced in cKO - 8M and Cont - 15M mice . F ( 2 , 8 ) = 11 . 49 , P = 0 . 0044 , One - way ANOVA followed by Tukey ' s HSD test ( * * P < 0 . 01 ) . Quantification of cell numbers 6 . 5 months after the induction of KO ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M , ) . ( F ) The density of DCX - positive cells was reduced in WT - old and cKO mice . p = 0 . 0091 , Kruskal - Wallis test followed by Dunn ' s test ( * P < 0 . 05 ) . ( G ) The density of Ki67 + RGL - ANSCs was reduced in cKO - 8M mice ( * P = 0 . 029 , Kruskal - Wallis test followed by Dunn ' s test ) . ( H ) The density of Ki67 + ANPC was not significantly different among three groups ( P = 0 . 49 , Kruskal - Wallis test ) . ( I ) The density of NeuN + EYFP + neurons was reduced in cKO - 8M mice . F ( 2 , 8 ) = 34 . 85 , P = 0 . 00011 , One - way ANOVA followed by Tukey ' s HSD test ( * * P < 0 . 001 ) . ( n = 4 for Cont - 8M , n = 3 for cKO - 8M , n = 4 for Cont - 15M ) .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START 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 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 B-PANEL_START 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 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 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 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

Figure 5Representative confocal images of the SGZ 6.5 months after the treatment with TAM.C. Representative confocal images of the SGZ 6.5 months after the treatment with TAM.Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (D) The density of RGL-ANSCs tends to be reduced in cKO. F (2, 8) = 3.98, P = 0.063, One-way ANOVA. (E) The density of ANPCs was reduced in cKO-8M and Cont-15M mice. F (2, 8) = 11.49, P = 0.0044, One-way ANOVA followed by Tukey's HSD test (** P < 0.01).Quantification of cell numbers 6.5 months after the induction of KO (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M,). (F) The density of DCX-positive cells was reduced in WT-old and cKO mice. p = 0.0091, Kruskal-Wallis test followed by Dunn's test (* P < 0.05). (G) The density of Ki67+RGL-ANSCs was reduced in cKO-8M mice (* P = 0.029, Kruskal-Wallis test followed by Dunn's test). (H) The density of Ki67+ANPC was not significantly different among three groups (P = 0.49, Kruskal-Wallis test). (I) The density of NeuN+EYFP+ neurons was reduced in cKO-8M mice. F (2, 8) = 34.85, P = 0.00011, One-way ANOVA followed by Tukey's HSD test (** P < 0.001). (n = 4 for Cont-8M, n = 3 for cKO-8M, n = 4 for Cont-15M).


Figure 6MA plot of differentially expressed genes between control and Lmnb1 - KO NPCs . E . Fraction of direction of gene regulation by LaminB1 . Genes were allocated dependent on where LaminB1 interacts with genes . Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout .

O B-PANEL_START O O O O O O O O O O O O O B-PANEL_START 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

Figure 6MA plot of differentially expressed genes between control and Lmnb1-KO NPCs.E. Fraction of direction of gene regulation by LaminB1. Genes were allocated dependent on where LaminB1 interacts with genes. Red bars indicated upregulated genes and blue bars indicate downregulated genes after lamin B1 knockout.


Figure 7Confirmation of Lamin B1 overexpression by qRT - PCR ( * P = 0 . 020 , n = 3 , one - sample t - test ) C . Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs . Scale bar = 10 µm . D , Relative induced expression of differentiation markers upon the withdrawal of FGF2 . 0 % indicates the same expression levels with control . Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation ( Tuj1 , * P = 0 . 023 ; Prox1 , * * * P = 0 . 00012 ; NeuroD1 , * * P = 0 . 0046 ; Ascl1 , * * * P = 0 . 00064 ; S100β , * * * P = 0 . 000097 ; n = 3 , one - sample t - test ) . F , Confocal images after exogenous expression of lamin B1 in EGFP + cells with RV ; LaminB1 - IRES - GFP ( laminB1 - OE . ) Scale bar = 20 µm . G , H , Exogenous expression of laminB1 repressed neuronal differentiation in vivo . An arrowhead indicate DCX + GFP + cell in control ( G , left ) and open arrowheads indicate Sox2 + GFP + cells in lamin B1 - OE cells . The fraction of EGFP + DCX + cells was significantly reduced by lamin B1 overexpression ( * * * P < 0 . 0002 , n = 3 ) , whereas the fractions of EGFP + DCX + Sox2 + cells and the EGFP + Sox2 + were increased ( EGFP + DCX + Sox2 + , * * P = 0 . 0024 ; EGFP + Sox2 + , P = 0 . 058 ; others , * P = 0 . 02 ) . Scale bar = 20 µm .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START 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 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 B-PANEL_START 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 B-PANEL_START 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 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 O O

Figure 7Confirmation of Lamin B1 overexpression by qRT-PCR (*P = 0.020, n = 3, one-sample t-test)C. Confirmation of Lamin B1 overexpression by immunocytochemistry in NPCs. Scale bar = 10 µm.D, Relative induced expression of differentiation markers upon the withdrawal of FGF2. 0 % indicates the same expression levels with control. Exogenous expression of lamin B1 inhibits the induction of genes related to neural differentiation (Tuj1, *P = 0.023; Prox1, ***P = 0.00012; NeuroD1, ** P = 0.0046; Ascl1, ***P = 0.00064; S100β, ***P = 0.000097; n = 3, one-sample t-test).F, Confocal images after exogenous expression of lamin B1 in EGFP+ cells with RV;LaminB1-IRES-GFP (laminB1-OE.) Scale bar = 20 µm.G, H, Exogenous expression of laminB1 repressed neuronal differentiation in vivo. An arrowhead indicate DCX+GFP+ cell in control (G, left) and open arrowheads indicate Sox2+GFP+ cells in lamin B1-OE cells. The fraction of EGFP+ DCX+ cells was significantly reduced by lamin B1 overexpression (***P < 0.0002, n = 3), whereas the fractions of EGFP+DCX+Sox2+ cells and the EGFP+Sox2+ were increased (EGFP+DCX+Sox2+, **P = 0.0024; EGFP+ Sox2+, P = 0.058; others, *P = 0.02). Scale bar = 20 µm.