Figure 5A ) Manhattan plot of genome - wide association for TAG ( 48 : 2 ) . Red line shows Bonferroni - corrected threshold of significance calculated based on FaST - LMM pvalues . B ) Locuszoom plots showing the focused genomic region ( x - axis ) plotted against the - log10 ( p - value ) of association for liver mRNA expression of Map2k6 . P - values for GWAS associations were calculated using FaST - LMM ( C ) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study . Blue represents negative correlations . E ) Western blots of liver homogenate using anti - Map2k6 and anti - β - actin . F ) Comparison of total hepatic TAG between Map2k6 - overexpressing mice ( black bar ) and control mice ( empty bar ) . G ) Differences in total phospholipid ( PC ) , total cholesterol ( TC ) and unesterified cholesterol ( UC ) between Map2k6 - overexpressing mice ( black bars ) and control mice ( empty bars ) . H ) Body fat % of both experimental cohorts prior to ( week 0 ) or 8 weeks on a HF / HS diet . I , J ) Plasma concentration of glucose ( I ) and insulin ( J ) at the end of the 8 weeks study .

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 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 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 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 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

Figure 5A) Manhattan plot of genome-wide association for TAG(48:2). Red line shows Bonferroni-corrected threshold of significance calculated based on FaST-LMM pvalues.B) Locuszoom plots showing the focused genomic region (x-axis) plotted against the -log10 (p-value) of association for liver mRNA expression of Map2k6. P-values for GWAS associations were calculated using FaST-LMM(C) Correlations between hepatic expression of Map2k6 and all TAGs identified in the study. Blue represents negative correlations.E) Western blots of liver homogenate using anti-Map2k6 and anti-β-actin.F) Comparison of total hepatic TAG between Map2k6-overexpressing mice (black bar) and control mice (empty bar).G) Differences in total phospholipid (PC), total cholesterol (TC) and unesterified cholesterol (UC) between Map2k6-overexpressing mice (black bars) and control mice (empty bars).H) Body fat % of both experimental cohorts prior to (week 0) or 8 weeks on a HF/HS diet.I,J) Plasma concentration of glucose (I) and insulin (J) at the end of the 8 weeks study.


Figure 6A ) Manhattan plot of genome - wide association for expression of Ifi203 in liver . Red line shows Bonferroni - corrected threshold and blue shows an FDR = 0 . 01 pvalue of significance calculated based on FaST - LMM pvalues . B - D ) Allelic variation plots showing the peak SNP for Ifi203 expression ( rs31614030 ) at the CC or TT allele ( x - axis ) plotted against expression of Ifi203 ( B ) , levels of hepatic PC ( 38 : 3 ) ( C ) , and plasma insulin levels ( D ) . Red line shows Bonferroni - corrected threshold and blue shows an FDR = 0 . 01 pvalue of significance calculated based on FaST - LMM pvalues . E - G ) Correlation between the parameters listed above showing significant relationships between hepatic Ifi203 and PC ( 38 : 3 ) ( E ) , hepatic Ifi203 and plasma insulin levels ( F ) or PC ( 38 : 3 ) and plasma insulin ( G ) . Pvalues were calculated based on significance of regression ( students test ) and adjusted for multiple comparisons ( FDR = 0 . 05 ) Mice receiving the control virus ( open bars ) or shIfi203 ( black bars ) were analyzed for liver expression of Ifi203 ( I ) , total PC levels in liver ( J ) P - values calculated using a students t - test between groups . Data represent means ± SEM ( n = 4 - 5 per group ) . Mice receiving the control virus ( open bars ) or shIfi203 ( black bars ) were analyzed for liver expression of Pemt ( K ) , total liver TAG content ( L ) , or plasma insulin levels ( M ) . P - values calculated using a students t - test between groups . Data represent means ± SEM ( n = 4 - 5 per group ) .

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 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 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 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 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

Figure 6A) Manhattan plot of genome-wide association for expression of Ifi203 in liver. Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.B-D) Allelic variation plots showing the peak SNP for Ifi203 expression (rs31614030) at the CC or TT allele (x-axis) plotted against expression of Ifi203 (B), levels of hepatic PC(38:3) (C), and plasma insulin levels (D). Red line shows Bonferroni-corrected threshold and blue shows an FDR =0.01 pvalue of significance calculated based on FaST-LMM pvalues.E-G) Correlation between the parameters listed above showing significant relationships between hepatic Ifi203 and PC(38:3) (E), hepatic Ifi203 and plasma insulin levels (F) or PC (38:3) and plasma insulin (G). Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Ifi203 (I), total PC levels in liver (J) P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).Mice receiving the control virus (open bars) or shIfi203 (black bars) were analyzed for liver expression of Pemt (K), total liver TAG content (L), or plasma insulin levels (M). P-values calculated using a students t-test between groups. Data represent means ± SEM (n =4-5 per group).


Figure 1 ( A ) Proof - of - principle experiment . Time - lapse images show overexpression of Myc - tagged CPAP - WT ( i ) or CPAPΔT ( ii ) has no effect on two centrosome - containing MCF10A cells ( - Dox , two centrosomes ) . The cells were imaged from interphase to Cytokinesis . Bar graph at right quantifies mitotic duration of cells overexpressing CPAP - WT and CPAPΔT . We define mitotic duration as time consumed from the onset of cell rounding until cytokinesis . Number of cells ( n ) analyzed in each condition is indicated at the top of each bar . Number of independent experiments ( N ) , ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test . CPAP - WT ( iii ) or CPAPΔT ( iv ) expressing extra centrosome - containing MCF10A cells ( + Dox , extra centrosomes ) . Note compared to CPAP - WT expressing centrosomes ( iii ) CPAPΔT expressing centrosomes nucleate enhanced levels of microtubule asters already at prophase - like stage ( iv ) . White arrows indicate centrosomal dots . Red arrows indicate centrosomes nucleating microtubule asters causing multipolar spindles . Scale bar , 2µm . Bar graph at right quantifies mitotic duration and percentage of cells exhibiting prolonged mitosis in cells expressing CPAP - WT and CPAPΔT . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * * P < 0 . 0001 ( B - D ) Fixed cell images showing the expression of CPAP - WT ( top panel ) and CPAPΔT ( bottom panel ) in extra centrosome containing MCF10A ( + Dox , extra centrosomes ) , MDA - MB - 231 and H1975T790M cells . Note , in CPAP - WT expressing cells , clustered extra centrosomes remain inactive with no or reduced microtubule nucleation . On the other hand , CPAPΔT expression enhances microtubule nucleation in extra centrosomes ( arrows ) and preventing them from clustering at G2 - like interphase and mitosis . Cells were stained with CPAP ( GFP ) , microtubules ( α - tubulin , red ) , Cep152 ( magenta ) . Scale bar , 2µm ( E ) Bar graph quantifies percentages of mitotic cells exhibiting multipolar spindles upon CPAP - WT or CPAPΔT expression . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * P < 0 . 001 ( F ) Subcutaneous xenograft tumor volume measurements of MDA - MB - 231 cells expressing CPAP - WT ( red ) as control or CPAPΔT ( blue ) in nude mice . ( i ) Bar graph at right shows total tumor volume reduction at day 22 . ( ii ) Each condition used 4 mice each containing 4 xenograft tumors . Error bars , mean ± SEM with n = 16 for CPAP - WT and n = 16 for CPAPΔT . Unpaired t - test * * * P < 0 . 0001 .

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 O O O O 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 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 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

Figure 1(A) Proof-of-principle experiment. Time-lapse images show overexpression of Myc-tagged CPAP-WT (i) or CPAPΔT (ii) has no effect on two centrosome-containing MCF10A cells (-Dox, two centrosomes). The cells were imaged from interphase to Cytokinesis. Bar graph at right quantifies mitotic duration of cells overexpressing CPAP-WT and CPAPΔT. We define mitotic duration as time consumed from the onset of cell rounding until cytokinesis. Number of cells (n) analyzed in each condition is indicated at the top of each bar. Number of independent experiments (N), (N)=3. Error bars, mean ± SEM. Unpaired t-test. CPAP-WT (iii) or CPAPΔT (iv) expressing extra centrosome-containing MCF10A cells (+Dox, extra centrosomes). Note compared to CPAP-WT expressing centrosomes (iii) CPAPΔT expressing centrosomes nucleate enhanced levels of microtubule asters already at prophase-like stage (iv). White arrows indicate centrosomal dots. Red arrows indicate centrosomes nucleating microtubule asters causing multipolar spindles. Scale bar, 2µm. Bar graph at right quantifies mitotic duration and percentage of cells exhibiting prolonged mitosis in cells expressing CPAP-WT and CPAPΔT. (N)=3. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001(B-D) Fixed cell images showing the expression of CPAP-WT (top panel) and CPAPΔT (bottom panel) in extra centrosome containing MCF10A (+Dox, extra centrosomes), MDA-MB-231 and H1975T790M cells. Note, in CPAP-WT expressing cells, clustered extra centrosomes remain inactive with no or reduced microtubule nucleation. On the other hand, CPAPΔT expression enhances microtubule nucleation in extra centrosomes (arrows) and preventing them from clustering at G2-like interphase and mitosis. Cells were stained with CPAP (GFP), microtubules (α-tubulin, red), Cep152 (magenta). Scale bar, 2µm(E) Bar graph quantifies percentages of mitotic cells exhibiting multipolar spindles upon CPAP-WT or CPAPΔT expression. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001(F) Subcutaneous xenograft tumor volume measurements of MDA-MB-231 cells expressing CPAP-WT (red) as control or CPAPΔT (blue) in nude mice. (i) Bar graph at right shows total tumor volume reduction at day 22. (ii) Each condition used 4 mice each containing 4 xenograft tumors. Error bars, mean ± SEM with n = 16 for CPAP-WT and n = 16 for CPAPΔT. Unpaired t-test ***P < 0.0001.


Figure 2 ( A ) 1H NMR spectra of free CCB02 ( black ) and bound to tubulin in 50 : 1 ratio ( orange ) . Broadening of CCB02 proton peaks indicate its binding ability to tubulin ( B ) NOESY spectrum recorded for a solution comprising tubulin ( 6 . 5µM ) , CPAP peptide ( 400µM ) and CCB02 . 1 ( 200µM ) with 70ms mixing time . The signals colored in green and ( highlighted by boxes ) correspond to inter - ligand trNOE signals arising due to protein - mediated spin - diffusion ( INPHARMA NOEs ) , while the black peaks represent intra - ligand trNOE signals .

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 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

Figure 2(A) 1H NMR spectra of free CCB02 (black) and bound to tubulin in 50:1 ratio (orange). Broadening of CCB02 proton peaks indicate its binding ability to tubulin(B) NOESY spectrum recorded for a solution comprising tubulin (6.5µM), CPAP peptide (400µM) and CCB02.1 (200µM) with 70ms mixing time. The signals colored in green and (highlighted by boxes) correspond to inter-ligand trNOE signals arising due to protein-mediated spin-diffusion (INPHARMA NOEs), while the black peaks represent intra-ligand trNOE signals.


Figure 3 ( A ) 14 day CCB02 treatment of MDA - MB - 231 , HCC827 - GR , Calu - 6 and MCF10A ( + Dox , extra centrosomes ) mostly eliminated cells with extra centrosomes . At the end of the treatment , cells were fixed and stained for centrosomes . Bar graph shows the percentage of cells displaying two - and more than two centrosomes . ' Vehicle ' at the bar graph indicates percentages of cells with extra centrosome Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02 - treatment ( open bars ) . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 70 ) . p values were obtained using unpaired t test * * P < 0 . 001 , * * * P < 0 . 0001 ( B ) CCB02 treatment prevents centrosome clustering in HCC827 - GR cells . Activated centrosomes ( arrows ) fail to cluster leading to multipolar mitosis . Cells were stained with Cep152 ( green ) , CPAP ( magenta ) and microtubules ( α - tubulin , red ) . Scale bar , 2µm , Insets , 0 . 5µm . Time - lapse images show the effect of CCB02 - mediated centrosome activation in cell cycle progression of MCF10A ( + Dox , extra centrosomes ) ( C cells . White arrows indicate centrosome dots . Red arrows ( in CCB02 - treated cells ) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells . Note , centrosome activation , declustering and prolonged multipolar mitosis occur only with CCB02 treatment . Cartoons of a cell with extra centrosomes below to the panel ( C ) represent various events occurring upon vehicle or CCB02 treatment . Scale bar , 2µm . Bar graphs at right to panel ( C quantifies mitotic duration ( ii ) , quantifies microtubule intensity prior to mitosis ( iii ) and depicts relative events occurring in cells treated with vehicle or CCB02 ( iv ) At least 100 cells were analyzed in each condition . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * P < 0 . 001 , * * * P < 0 . 0001Time - lapse images show the effect of CCB02 - mediated centrosome activation in cell cycle progression o HCC827 - GR ( D ) cells . White arrows indicate centrosome dots . Red arrows ( in CCB02 - treated cells ) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells . Note , centrosome activation , declustering and prolonged multipolar mitosis occur only with CCB02 treatment Bar graphs at right to pane ( D ) quantifies mitotic duration ( ii ) , quantifies microtubule intensity prior to mitosis ( iii ) and depicts relative events occurring in cells treated with vehicle or CCB02 ( iv ) . At least 100 cells were analyzed in each condition . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * P < 0 . 001 , * * * P < 0 .

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 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 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 O O O O O O O O O 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 3(A) 14 day CCB02 treatment of MDA-MB-231, HCC827-GR, Calu-6 and MCF10A (+Dox, extra centrosomes) mostly eliminated cells with extra centrosomes. At the end of the treatment, cells were fixed and stained for centrosomes. Bar graph shows the percentage of cells displaying two- and more than two centrosomes. 'Vehicle' at the bar graph indicates percentages of cells with extra centrosome Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02-treatment (open bars). Data represent mean ± SEM of three independent experiments. (N)=3, (n=70). p values were obtained using unpaired t test **P < 0.001, ***P < 0.0001(B) CCB02 treatment prevents centrosome clustering in HCC827-GR cells. Activated centrosomes (arrows) fail to cluster leading to multipolar mitosis. Cells were stained with Cep152 (green), CPAP (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm, Insets, 0.5µm.Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression of MCF10A (+Dox, extra centrosomes) (C cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment. Cartoons of a cell with extra centrosomes below to the panel (C) represent various events occurring upon vehicle or CCB02 treatment. Scale bar, 2µm. Bar graphs at right to panel (C quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv) At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression o HCC827-GR (D) cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment Bar graphs at right to pane (D) quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv). At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001


Figure 4 ( i ) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes ( + Dox , extra centrosomes ) ( A Cells were treated with CPAP siRNA and control ( scramble ) siRNA for 48 hours . ( ii ) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAPExperimental strategy to test if the effect of CCB02 is CPAP dependent i MDA - MB - 23 cell ( B ) . Cells were treated with CPAP siRNA and control ( scramble ) siRNA for 48 hours . ( ii ) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP ( C ) Fractions of cells containing centrosomes after siRNA treatment . 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments . Bar graph shows the percentage of cells with extra centrosomes in MCF10A ( + Dox , extra centrosomes ) in the presence of control siRNA or CPAP siRNA . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 300 cells per condition ) . p values were obtained using Ordinary One - way ANOVA test * P < 0 . 01 , * * P < 0 . 001 ( D ) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A ( i ) MDA - MB - 231 cells ( ii ) CPAP depletion does not prevent centrosomes from clustering ( second rows of i and ii ) . Note , in CPAP depleted cultures , centrosomes are marked with Cep152 ( green ) where CPAP ( magenta ) is detected faintly . In CPAP depleted cells , CCB02 does not prevent extra centrosomes from clustering ( fourth rows of i and ii ) . Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA ( third rows of i and ii ) . Cells are stained for Cep152 ( green ) , CPAP ( magenta ) , microtubules ( α - tubulin , red ) and DNA ( DAPI , blue ) . Scale bar , 2µm ( E ) Bar graph shows the percentage of cells with multipolar mitosis . Higher percentage of multipolar mitotic cells is observed in control siRNA - treated cultures . Bar graph on right shows the percentage of cells with multipolar mitosis . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 680 cells ) . p values were obtained using unpaired t test . * * * P < 0 . 0001 ( F ) Bar graph shows the percentage of living ( TUNEL - negative ) and dyeing ( TUNEL - positive ) cells under various conditions tested . Higher percentage of TUNEL - positive cells is observed in CCB02 - treated cultures . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 500 cells per condition ) . p values were obtained using Ordinary One - way ANOVA test * P < 0 . 01 .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(i) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes (+Dox, extra centrosomes) (A Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAPExperimental strategy to test if the effect of CCB02 is CPAP dependent i MDA-MB-23 cell (B). Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP(C) Fractions of cells containing centrosomes after siRNA treatment. 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments. Bar graph shows the percentage of cells with extra centrosomes in MCF10A (+Dox, extra centrosomes) in the presence of control siRNA or CPAP siRNA. Data represent mean ± SEM of three independent experiments. (N)=3, (n=300 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01, **P < 0.001(D) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A (i) MDA-MB-231 cells (ii) CPAP depletion does not prevent centrosomes from clustering (second rows of i and ii). Note, in CPAP depleted cultures, centrosomes are marked with Cep152 (green) where CPAP (magenta) is detected faintly. In CPAP depleted cells, CCB02 does not prevent extra centrosomes from clustering (fourth rows of i and ii). Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA (third rows of i and ii). Cells are stained for Cep152 (green), CPAP (magenta), microtubules (α-tubulin, red) and DNA (DAPI, blue). Scale bar, 2µm(E) Bar graph shows the percentage of cells with multipolar mitosis. Higher percentage of multipolar mitotic cells is observed in control siRNA-treated cultures. Bar graph on right shows the percentage of cells with multipolar mitosis. Data represent mean ± SEM of three independent experiments. (N)=3, (n=680 cells). p values were obtained using unpaired t test. ***P < 0.0001(F) Bar graph shows the percentage of living (TUNEL-negative) and dyeing (TUNEL-positive) cells under various conditions tested. Higher percentage of TUNEL-positive cells is observed in CCB02-treated cultures. Data represent mean ± SEM of three independent experiments. (N)=3, (n=500 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01.


Figure 5 ( A ) Interphase centrosomes of CCB02 - treated two - centrosome containing MCF10A cells display an enhanced level of PCM proteins . CPAP ( green ) intensity remains same . Cells are stained for CPAP ( green ) , PCM proteins ( magenta ) Cep152 ( i ) , PCNT ( ii ) and CDK5RAP2 ( iii ) and microtubules ( α - tubulin , red ) . Scale bars , 2µm for left panels and 0 . 2µm for right panels . Heat map images show intensity saturation ( blue ) for CPAP , Cep152 , PCNT and CDK5RAP2 upon CCB02 treatment . Line graphs at right to each panels indicate the heat map intensity of single centrosomes of cells ( given in boxes at heat map panels ) treated with vehicle ( blue line ) or CCB02 ( red line ) . Images were recorded with low laser excitation and high zoom factor ( B ) Bar graph quantifies relative intensity of CPAP - interacting proteins in interphase centrosomes compared to vehicle treatment . At least 150 centrosomes ( for vehicle ) ; 130 centrosomes ( for CCB02 ) were analyzed in each condition . Error bars , mean ± SEM . ( N ) = 3 . Ordinary Two - way ANOVA test * * * P < 0 . 0001 ( C ) Immunopurification of CPAP complexes from CCB02 - treated cell extracts . ( i ) CCB02 specifically prevents CPAP - Tubulin interaction . This allows CPAP to bind an enhanced amount of Cep152 and γ - tubulin . Control IP experiment is shown below . ( ii ) Plot showing PCM protein intensities co - immunoprecipitated with CPAP at various concentrations of CCB02 treatment . CPAP amount does not change with CCB02 treatment . Data represent mean ± SEM . ( N ) = 3 .

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 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 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

Figure 5(A) Interphase centrosomes of CCB02-treated two-centrosome containing MCF10A cells display an enhanced level of PCM proteins. CPAP (green) intensity remains same. Cells are stained for CPAP (green), PCM proteins (magenta) Cep152 (i), PCNT (ii) and CDK5RAP2 (iii) and microtubules (α-tubulin, red). Scale bars, 2µm for left panels and 0.2µm for right panels. Heat map images show intensity saturation (blue) for CPAP, Cep152, PCNT and CDK5RAP2 upon CCB02 treatment. Line graphs at right to each panels indicate the heat map intensity of single centrosomes of cells (given in boxes at heat map panels) treated with vehicle (blue line) or CCB02 (red line). Images were recorded with low laser excitation and high zoom factor(B) Bar graph quantifies relative intensity of CPAP-interacting proteins in interphase centrosomes compared to vehicle treatment. At least 150 centrosomes (for vehicle); 130 centrosomes (for CCB02) were analyzed in each condition. Error bars, mean ± SEM. (N)=3. Ordinary Two-way ANOVA test ***P < 0.0001(C) Immunopurification of CPAP complexes from CCB02-treated cell extracts. (i) CCB02 specifically prevents CPAP-Tubulin interaction. This allows CPAP to bind an enhanced amount of Cep152 and γ-tubulin. Control IP experiment is shown below. (ii) Plot showing PCM protein intensities co-immunoprecipitated with CPAP at various concentrations of CCB02 treatment. CPAP amount does not change with CCB02 treatment. Data represent mean ± SEM. (N)=3.


Figure 6 ( B - D ) MT - regrowth assays at 0 , 1 . 5 and 3 . 0 min using MCF10A ( - Dox , two centrosomes ) , MCF10A ( + Dox , extra centrosomes ) and MDA - MB - 231 cells . MT nucleation panel is shown in grey scale ( inset images are inverted ) and γ - tubulin is shown in red . Note that in contrast to vehicle treatment , CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1 . 5 min after induction of regrowth with simultaneous increase in γ - tubulin recruitment . All these cells were stained with γ - tubulin ( red ) , microtubules ( α - tubulin , grey ) and DNA ( DAPI blue ) . Scale bar , 2µm and insets , 0 . 5 µm . Bar graphs show MT and γ - tubulin intensities at 3 min after induction of MT regrowth . ( N ) = 3 . At least 80 centrosomes were considered to calculate intensities from each cell line . Error bars , mean ± SEM . Unpaired t - test * * P < 0 . 001 , * * * P < 0 . 0001 .

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

Figure 6(B-D) MT-regrowth assays at 0, 1.5 and 3.0 min using MCF10A (-Dox, two centrosomes), MCF10A (+Dox, extra centrosomes) and MDA-MB-231 cells. MT nucleation panel is shown in grey scale (inset images are inverted) and γ-tubulin is shown in red. Note that in contrast to vehicle treatment, CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1.5 min after induction of regrowth with simultaneous increase in γ-tubulin recruitment. All these cells were stained with γ-tubulin (red), microtubules (α-tubulin, grey) and DNA (DAPI blue). Scale bar, 2µm and insets, 0.5 µm. Bar graphs show MT and γ-tubulin intensities at 3 min after induction of MT regrowth. (N)=3. At least 80 centrosomes were considered to calculate intensities from each cell line. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001.


Figure 7 ( A ) Bar graph quantifies relative intensity of PCM proteins at interphase centrosomes of cells treated with vehicle , CCB02 , and known tubulin binders . Two centrosomes containing MCF10A ( - Dox , two centrosomes ) cells were used . Elevated PCM proteins intensities were detected only with CCB02 compared to vehicle treated cells . However , a slight increase in PCNT was observed with Docetaxel treatment . Number of centrosomes tested , n = 100 ( vehicle ) ; n = 100 ( for CCB02 , Taxol , Bacatalin III , Docetaxel and Vinblastine ) . ( N ) = 3 . Error bars , mean ± SEM . Ordinary Two - way ANOVA test * * P < 0 . 001 , * * * P < 0 . 0001 ( B ) CCB02 but not conventional tubulin binders ( Taxol , Bacatalin III , Docetaxel and Vinblastine ; concentrations used 30nM ) causes activation of extra centrosomes and prevents them from clustering . MCF10A cells with extra centrosomes ( + Dox , extra centrosomes ) were used . In contrast to CCB02 - treatment , interphase centrosomes of tubulin binders - treated cells consistently display clustered centrosomes . Note , disrupted mitotic spindles were observed in tubulin binders - treated cells due to their general toxicity to microtubules . Cells were stained with CPAP ( green ) , Cep152 ( magenta ) and microtubules ( α - tubulin , red ) . Scale bar , 2µm . Three independent experiments ( N ) = 3 with each at least 100 cells were examined per treatment ( C ) Bar graph quantifies percentages of cells exhibiting multipolar spindles in interphase and mitosis of vehicle , CCB02 and conventional tubulin binders - treated cells . At least 150 cells were analyzed for each independent experiment ( N ) = 3 . Error bars , mean ± SEM . Ordinary Two - way ANOVA test * * P < 0 . 001 ( D ) Kymographs ( i ) and averaged microtubule polymerization velocities mediated by vehicle and CCB02 . Graph below represents the microtubule growth velocities mediated by vehicle ( red ) and CCB02 ( blue ) during microtubule end - tracking assay ( ii ) . CCB02 does not alter the microtubule growth although there is a slight increase in growth at 5µM . At least 75 filaments were analyzed for each condition . The error bars represent mean ± SEM of three independent experiments ( N ) = 3 ( E ) Snap - shot images show the effect of CCB02 on microtubule dynamics in two - centrosome containing MCF10A cells . The microtubule was stained with SiR - tubulin and + end tip of microtubule was marked with EB3 - EGFP . A 5µM CCB02 did not alter localization and dynamics of EB3 at the microtubules indicating that the CCB02 does not affect microtubule dynamic At least , 50 cells were analyzed for each condition . Scale bar , 2µm ( F ) Box plots represent the speed ( µm / sec ) ( i ) , lifetime ( sec ) ( ii ) , and length ( µm ) ( iii ) of growing microtubule tracks in MCF10A cells treated with 1 , 2 and 5µM of CCB02 . At least , 60 filaments were analyzed from each cell from a total of 30 - 40 cells . ( N ) = 3 . Error bars in boxplots mean ± SD . Ordinary One - way ANOVA test was performed . Boxes in all plots show the growing microtubule parameters such as speed ( µm / sec ) , lifetime ( sec ) and length ( µm / sec ) . Horizontal lines in boxes show the median value . Whiskers at both extremes of boxes indicate minimum and maximum values .

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 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 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 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 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 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

Figure 7(A) Bar graph quantifies relative intensity of PCM proteins at interphase centrosomes of cells treated with vehicle, CCB02, and known tubulin binders. Two centrosomes containing MCF10A (-Dox, two centrosomes) cells were used. Elevated PCM proteins intensities were detected only with CCB02 compared to vehicle treated cells. However, a slight increase in PCNT was observed with Docetaxel treatment. Number of centrosomes tested, n=100 (vehicle); n=100 (for CCB02, Taxol, Bacatalin III, Docetaxel and Vinblastine). (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001, ***P < 0.0001(B) CCB02 but not conventional tubulin binders (Taxol, Bacatalin III, Docetaxel and Vinblastine; concentrations used 30nM) causes activation of extra centrosomes and prevents them from clustering. MCF10A cells with extra centrosomes (+Dox, extra centrosomes) were used. In contrast to CCB02-treatment, interphase centrosomes of tubulin binders-treated cells consistently display clustered centrosomes. Note, disrupted mitotic spindles were observed in tubulin binders-treated cells due to their general toxicity to microtubules. Cells were stained with CPAP (green), Cep152 (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm. Three independent experiments (N)=3 with each at least 100 cells were examined per treatment(C) Bar graph quantifies percentages of cells exhibiting multipolar spindles in interphase and mitosis of vehicle, CCB02 and conventional tubulin binders-treated cells. At least 150 cells were analyzed for each independent experiment (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001(D) Kymographs (i) and averaged microtubule polymerization velocities mediated by vehicle and CCB02. Graph below represents the microtubule growth velocities mediated by vehicle (red) and CCB02 (blue) during microtubule end-tracking assay (ii). CCB02 does not alter the microtubule growth although there is a slight increase in growth at 5µM. At least 75 filaments were analyzed for each condition. The error bars represent mean ± SEM of three independent experiments (N)=3(E) Snap-shot images show the effect of CCB02 on microtubule dynamics in two-centrosome containing MCF10A cells. The microtubule was stained with SiR-tubulin and +end tip of microtubule was marked with EB3-EGFP. A 5µM CCB02 did not alter localization and dynamics of EB3 at the microtubules indicating that the CCB02 does not affect microtubule dynamic At least, 50 cells were analyzed for each condition. Scale bar, 2µm(F) Box plots represent the speed (µm/sec) (i), lifetime (sec) (ii), and length (µm) (iii) of growing microtubule tracks in MCF10A cells treated with 1, 2 and 5µM of CCB02. At least, 60 filaments were analyzed from each cell from a total of 30-40 cells. (N)=3. Error bars in boxplots mean ± SD. Ordinary One-way ANOVA test was performed. Boxes in all plots show the growing microtubule parameters such as speed (µm/sec), lifetime (sec) and length (µm/sec). Horizontal lines in boxes show the median value. Whiskers at both extremes of boxes indicate minimum and maximum values.


Figure 8 ( A ) Real time live imaging of NSCLC ( H1975T790M ) 3D spheroids with vehicle , 5µM erlotinib or 5µM CCB02 . At the end of live imaging , spheroids were fixed and stained for F - actin ( green ) . Arrow indicates invasive ( vehicle - treated ) and non - invasive ( CCB02 - treated ) structures in spheroids . Scale bar , 100µm ( B ) Time series plot shows the spheroid growth rate measurements . CCB02 treatment prevents the spheroid growth rate by inhibiting the formation of invasive protrusions At least 10 spheroids were measured for each condition . Error bars , mean ± SEM . Unpaired t - test * * * P < 0 . 0001 ( D ) Antitumor activity of CCB02 in vivo . Left : Subcutaneous xenograft tumor volume measurements of H1975T790M ( 30mg / kg of weight , daily ) in nude mice treated with vehicle or CCB02 . Right : Bar graph shows total tumor volume at day 1 and day 24 of vehicle or CCB02 treated xenograft . Error bars , mean ± SD with n = 8 for control vehicle and n = 8 for CCB02 treatment . Unpaired t - test * P < 0 . 01 .

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 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

Figure 8(A) Real time live imaging of NSCLC (H1975T790M) 3D spheroids with vehicle, 5µM erlotinib or 5µM CCB02. At the end of live imaging, spheroids were fixed and stained for F-actin (green). Arrow indicates invasive (vehicle-treated) and non-invasive (CCB02-treated) structures in spheroids. Scale bar, 100µm(B) Time series plot shows the spheroid growth rate measurements. CCB02 treatment prevents the spheroid growth rate by inhibiting the formation of invasive protrusions At least 10 spheroids were measured for each condition. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001(D) Antitumor activity of CCB02 in vivo. Left: Subcutaneous xenograft tumor volume measurements of H1975T790M (30mg/kg of weight, daily) in nude mice treated with vehicle or CCB02. Right: Bar graph shows total tumor volume at day 1 and day 24 of vehicle or CCB02 treated xenograft. Error bars, mean ± SD with n = 8 for control vehicle and n = 8 for CCB02 treatment. Unpaired t-test *P < 0.01.


Figure 1A Lung function data of COPD ( II - IV ) and no COPD ( ex - smoker controls ) donors used in this study . The lung function between the two groups is significantly different . In ( A ) , data points represent each donor values and horizontal bars the group median . An unpaired non - parametric t test ( Mann - Whitney test , GraphPad Prism software , version 8 . 0 . 1 ) was employed to compare the lung function ( FEV1 and FEV1 / FVC values ) between control ( no COPD , n = 3 ) and COPD II - IV donors ( n = 5 ) , * p - value < 0 . 05 . Exact p - values are : FEV1 , p - value = 0 . 0357 ; FEV1 / FVC , p - value = 0 . 0357 . FEV1 , forced expiratory volume in 1 s ; FVC , forced vital capacity ; T - WGBS data of primary fibroblasts from no COPD and COPD ( II - IV ) patients was analyzed DMRs E Detailed view of a representative hypo - ( top ) and hypermethylated ( bottom ) DMR ( grey box ) . CpG methylation levels of each individual donor ( dots ) and the group average ( lines ) methylation profile of three no COPD ( blue ) and five COPD ( II - IV ) ( dark green ) donors are displayed . RefSeq annotated genes and CpG islands are indicated .

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Figure 1A Lung function data of COPD (II-IV) and no COPD (ex-smoker controls) donors used in this study. The lung function between the two groups is significantly different. In (A), data points represent each donor values and horizontal bars the group median. An unpaired non-parametric t test (Mann-Whitney test, GraphPad Prism software, version 8.0.1) was employed to compare the lung function (FEV1 and FEV1/FVC values) between control (no COPD, n=3) and COPD II-IV donors (n=5), *p-value<0.05. Exact p-values are: FEV1, p-value= 0.0357; FEV1/FVC, p-value= 0.0357. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity;T-WGBS data of primary fibroblasts from no COPD and COPD (II-IV) patients was analyzed DMRs E Detailed view of a representative hypo- (top) and hypermethylated (bottom) DMR (grey box). CpG methylation levels of each individual donor (dots) and the group average (lines) methylation profile of three no COPD (blue) and five COPD (II-IV) (dark green) donors are displayed. RefSeq annotated genes and CpG islands are indicated.


Figure 2D Genome browser view of an example DMR at a putative enhancer region . Group median CpG methylation is shown for no COPD ( blue ) , COPD ( I ) ( light green ) and COPD ( II - IV ) ( dark green ) . At the bottom the level of enhancer marks is depicted as fold - change over control : H3K4me1 ( ENCODE accession : ENCFF102BGI ) and H3K27ac ( ENCODE accession : ENCFF386FDQ ) . H Enrichment of methylation sensitive transcription factor motifs at hypo - ( right ) and hypermethylated ( left ) DMRs . Methylation sensitive motifs were derived from the study Transcription factors , whose binding affinity was impaired upon methylation of their corresponding DNA motif are shown in red ( MethylMinus ) and transcription factors , whose binding affinity was increased , in blue ( MethylPlus ) . TTS , transcription termination site . In H , Motif enrichment was calculated using HOMER , which uses ZOOPS scoring ( zero or one occurrence per sequence ) coupled with the hypergeometric enrichment calculations .

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Figure 2D Genome browser view of an example DMR at a putative enhancer region. Group median CpG methylation is shown for no COPD (blue), COPD (I) (light green) and COPD (II-IV) (dark green). At the bottom the level of enhancer marks is depicted as fold-change over control: H3K4me1 (ENCODE accession: ENCFF102BGI) and H3K27ac (ENCODE accession: ENCFF386FDQ).H Enrichment of methylation sensitive transcription factor motifs at hypo- (right) and hypermethylated (left) DMRs. Methylation sensitive motifs were derived from the study Transcription factors, whose binding affinity was impaired upon methylation of their corresponding DNA motif are shown in red (MethylMinus) and transcription factors, whose binding affinity was increased, in blue (MethylPlus). TTS, transcription termination site. In H, Motif enrichment was calculated using HOMER, which uses ZOOPS scoring (zero or one occurrence per sequence) coupled with the hypergeometric enrichment calculations.


Figure 3D Selected examples of DEG across disease stages from clusters 2 - 5 defined in ( C ) . In ( D ) each donor is represented by an individual point ( no COPD n = 3 , COPD I n = 3 , COPD II - IV n = 5 ) , the group mean is shown as black dot and the error bar represents the standard deviation . In ( D ) FDRs and log2 ( fold - changes ) ( in A ) were calculated using DESeq2 , which uses a negative binominal GLM ( generalized linear model ) and Wald statistics . * : FDR < 0 . 05 ; * * : FDR < 0 . 01 ; * * * : FDR < 0 . 001 . The specific statistically significant FDR values are the following : RAD18 in COPD II - IV = 0 . 000434 ; ADAMTSL1 in COPD II - IV = 0 . 02771 ; STEPA3 in COPD I = 0 . 04219 and in COPD II - IV = 0 . 001272 ; BMP4 in COPD II - IV = 0 . 0000535 .

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

Figure 3D Selected examples of DEG across disease stages from clusters 2-5 defined in (C). In (D) each donor is represented by an individual point (no COPD n=3, COPD I n=3, COPD II-IV n=5), the group mean is shown as black dot and the error bar represents the standard deviation. In (D) FDRs and log2(fold-changes) (in A) were calculated using DESeq2, which uses a negative binominal GLM (generalized linear model) and Wald statistics. *: FDR <0.05; **: FDR <0.01; ***: FDR <0.001. The specific statistically significant FDR values are the following: RAD18 in COPD II-IV= 0.000434; ADAMTSL1 in COPD II-IV=0.02771; STEPA3 in COPD I= 0.04219 and in COPD II-IV= 0.001272; BMP4 in COPD II-IV= 0.0000535.


Figure 4F Scatter plots showing examples of correlations between gene expression and methylation of promoter associated DMRs . Each dot represents an individual donor . Dots are color coded according to disease state . Gene expression is illustrated as normalized counts . Methylation is illustrated as average beta value of the corresponding DMR . In ( F ) linear regression analysis was performed ( black line ) and the 95 % confidence interval is indicated ( grey area ) . Correlation coefficients and p - values were calculated by the Spearman correlation method .

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

Figure 4F Scatter plots showing examples of correlations between gene expression and methylation of promoter associated DMRs. Each dot represents an individual donor. Dots are color coded according to disease state. Gene expression is illustrated as normalized counts. Methylation is illustrated as average beta value of the corresponding DMR. In (F) linear regression analysis was performed (black line) and the 95% confidence interval is indicated (grey area). Correlation coefficients and p-values were calculated by the Spearman correlation method.


Figure 5B Examples of primary pictures obtained in the screens showing the performance of the siRNA controls as well as positive hits upon KD . Scale bars : 0 . 05 mm . KD , knockdown ; FGF , fibroblast growth factor ; TGFβ , transforming growth factor beta ; αSMA , alpha smooth - muscle actin ; col1 , collagen 1 ; NTC , non - targeting siRNA control ; C Heatmap showing the effect of the KD of each candidate gene on the three measured readouts ( αSMA , col1 and proliferation ) in primary normal ( NHLF ) or COPD ( DHLF ) human lung fibroblasts , red : readout higher than NTC , blue : readout lower than NTC . | SSMD values | ≥ 2 are shown in lighter shade and | SSMD values | ≥ 3 in stronger shade . ; αSMA , alpha smooth - muscle actin ; col1 , collagen 1 SSMD , strictly standardized mean difference .

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 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

Figure 5B Examples of primary pictures obtained in the screens showing the performance of the siRNA controls as well as positive hits upon KD. Scale bars: 0.05 mm. KD, knockdown; FGF, fibroblast growth factor; TGFβ, transforming growth factor beta; αSMA, alpha smooth-muscle actin; col1, collagen 1; NTC, non-targeting siRNA control;C Heatmap showing the effect of the KD of each candidate gene on the three measured readouts (αSMA, col1 and proliferation) in primary normal (NHLF) or COPD (DHLF) human lung fibroblasts, red: readout higher than NTC, blue: readout lower than NTC. |SSMD values| ≥ 2 are shown in lighter shade and |SSMD values| ≥ 3 in stronger shade. ; αSMA, alpha smooth-muscle actin; col1, collagen 1 SSMD, strictly standardized mean difference.


Figure 1A , B X - gal staining showed NB - 3 / LacZ expression in the sham - operated ( A ) and injured ( B ) spinal cords from adult NB - 3 + / − mice . C , D Western blot analysis ( C ) and quantification ( D ) of NB - 3 expression in the lesion area post - injury . NB - 3 expression levels were normalized by b - tubulin . Data are represented as mean ± SEM . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - test . Data were analyzed from 10 independent experiments each including 2 mice per group . E , F Single - labeling immunostaining showed NB - 3 expression in the sham - operated ( E ) and injured ( F ) spinal cords from adult NB - 3 + / + mice . G - K Co - immunostaining of NB - 3 ( green ) , PDGFR - β ( red ) , and GFAP ( blue ) on serial sagittal sections from the same NB - 3 + / + spinal cord 14 days post - injury . ( G1 - K1 ) High - magnification images of boxed areas in ( G - K ) , respectively . L Co - immunostaining of NB - 3 ( green ) , PDGFR - β ( red ) , and GFAP ( blue ) on sagittal sections from the NB - 3 − / − spinal cord 14 days post - injury . ( L1 ) High - magnification image of the boxed area in ( L ) . The arrows indicate the lesion sites . M - Q Co - immunostaining of NB - 3 ( green ) , Fibronectin ( red ) , and GFAP ( blue ) on serial sagittal sections from the same NB - 3 + / + spinal cord 14 days post - injury . ( M1 - Q1 ) High - magnification images of boxed areas in ( M - Q ) , respectively . R Co - immunostaining of NB - 3 ( green ) , Fibronectin ( red ) , and GFAP ( blue ) on sagittal sections from the NB - 3 − / − spinal cord 14 days post - injury . ( R1 ) High - magnification image of the boxed area in ( R ) . The arrows in ( G - R ) indicate the lesion sites .

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 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 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 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

Figure 1A, B X-gal staining showed NB-3/LacZ expression in the sham-operated (A) and injured (B) spinal cords from adult NB-3+/−mice.C, D Western blot analysis (C) and quantification (D) of NB-3 expression in the lesion area post-injury. NB-3 expression levels were normalized by b-tubulin. Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 10 independent experiments each including 2 mice per group.E, F Single-labeling immunostaining showed NB-3 expression in the sham-operated (E) and injured (F) spinal cords from adult NB-3+/+mice.G-K Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (G1-K1) High-magnification images of boxed areas in (G-K), respectively.L Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (L1) High-magnification image of the boxed area in (L). The arrows indicate the lesion sites.M-Q Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (M1-Q1) High-magnification images of boxed areas in (M-Q), respectively.R Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (R1) High-magnification image of the boxed area in (R). The arrows in (G-R) indicate the lesion sites.


Figure 2B NB - 3 expression in corticospinal neurons 14 and 28 days post - injury ( dpi ) . FG ( blue ) was applied to retrogradely label corticospinal neurons . C Quantification of the fluorescence intensity of NB - 3 in ( B ) . * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified . D , E Western blot analysis ( D ) and quantification ( E ) of NB - 3 expression in the sensorimotor cortex after SCI . NB - 3 expression levels were normalized by b - tubulin . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - test . Data were analyzed from 3 independent experiments each including 3 mice per group . F - K Induced NB - 3 expression at the distal terminations of post - traumatic corticospinal axons and in reactive astrocytes at the lesion border 14 ( H and I ) and 28 days post - injury ( dpi ) ( J and K ) . BDA ( red ) was applied to anterogradely label corticospinal axons . GFAP ( blue ) was used to label reactive astrocytes . ( G ) High - magnification image of the boxed area in ( F ) . ( I and K ) High - magnification and x - y - z reconstruction images of boxed areas in ( H and J ) , respectively , show co - localization of NB - 3 and GFAP in astrocytes at the lesion border and co - localization of NB - 3 in BDA - labeled axons and GFAP - positive astrocytes at the contact surface of corticospinal axons and astrocytes ( arrowheads ) . The arrows in ( H and J ) indicate the lesion sites . The asterisks in ( H and J ) indicate the terminations of the BDA - labeled projecting axons .

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 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

Figure 2B NB-3 expression in corticospinal neurons 14 and 28 days post-injury (dpi). FG (blue) was applied to retrogradely label corticospinal neurons.C Quantification of the fluorescence intensity of NB-3 in (B). **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.D, E Western blot analysis (D) and quantification (E) of NB-3 expression in the sensorimotor cortex after SCI. NB-3 expression levels were normalized by b-tubulin. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.F-K Induced NB-3 expression at the distal terminations of post-traumatic corticospinal axons and in reactive astrocytes at the lesion border 14 (H and I) and 28 days post-injury (dpi) (J and K). BDA (red) was applied to anterogradely label corticospinal axons. GFAP (blue) was used to label reactive astrocytes. (G) High-magnification image of the boxed area in (F). (I and K) High-magnification and x-y-z reconstruction images of boxed areas in (H and J), respectively, show co-localization of NB-3 and GFAP in astrocytes at the lesion border and co-localization of NB-3 in BDA-labeled axons and GFAP-positive astrocytes at the contact surface of corticospinal axons and astrocytes (arrowheads). The arrows in (H and J) indicate the lesion sites. The asterisks in (H and J) indicate the terminations of the BDA-labeled projecting axons.


Figure 3B - K Representative images of serial sagittal sections showing the main BDA - labeled corticospinal axons in NB - 3 + / + ( B - D ) and NB - 3 − / − ( E - G ) mice 14 weeks after complete spinal transection . ( H - J ) High - magnification images of the areas in ( E - G ) ( single asterisks ) , respectively . ( K ) High - magnification image of the area in ( E ) ( double asterisk ) . The arrows indicate the lesion sites ; the white dashed lines indicate the regenerative corticospinal axons extending into the distal spinal cord ; the single asterisks in ( E - G ) indicate the lesion epicenters ; the double asterisk in ( E ) indicates the axons extending from the caudal lesion border . Scale bars , 400 µm ( B - G ) and 100 µm ( H - K ) . L Quantification of the intensity index of BDA - labeled axons at certain distances from the lesion border in ( B - G ) . Data are represented as mean ± SEM . * p < 0 . 05 , and * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 25 mice per group .

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

Figure 3B-K Representative images of serial sagittal sections showing the main BDA-labeled corticospinal axons in NB-3+/+ (B-D) and NB-3−/− (E-G) mice 14 weeks after complete spinal transection. (H-J) High-magnification images of the areas in (E-G) (single asterisks), respectively. (K) High-magnification image of the area in (E) (double asterisk). The arrows indicate the lesion sites; the white dashed lines indicate the regenerative corticospinal axons extending into the distal spinal cord; the single asterisks in (E-G) indicate the lesion epicenters; the double asterisk in (E) indicates the axons extending from the caudal lesion border. Scale bars, 400 µm (B-G) and 100 µm (H-K).L Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (B-G). Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 25 mice per group.


Figure 4A Time course of the change in BMS scores in NB - 3 + / + and NB - 3 − / − mice as measured with the BMS rating scale after spinal cord complete transection . * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s post - test . n = 25 mice per group . C Electrophysiology transmission across the spinal lesion site . In each group of NB - 3 + / + and NB - 3 − / − mice , the electrophysiological responses in the caudal spinal cord were recorded in the condition of sham - operated or 12 weeks post - injury . The latencies and amplitudes of responses were analyzed and quantified in each group . n . s . , not significant , * p < 0 . 05 , and * * p < 0 . 01 ; two - way ANOVA followed by Bonferroni ' s post - test . n = 10 mice per group . E Representative images of coronal sections showing synapse formation between the glutamatergic corticospinal terminals and spinal neurons in NB - 3 + / + and NB - 3 − / − mice . Corticospinal axons , neurons , and glutamatergic terminals of corticospinal axons were labeled with BDA ( red ) , Tuj1 ( blue ) and vGlut1 / 2 ( green ) , respectively . Each group showed the data from the rostral and caudal spinal cord in the condition of sham - operated or 12 weeks post - injury . Scale bar , 20 μm . F Quantification of the number of glutamatergic corticospinal synapses per neuron in each group . n . s . , not significant , * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 15 mice per group .

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 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 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

Figure 4A Time course of the change in BMS scores in NB-3+/+ and NB-3−/−mice as measured with the BMS rating scale after spinal cord complete transection. *p < 0.05; two-way ANOVA followed by Bonferroni's post-test. n= 25 mice per group.C Electrophysiology transmission across the spinal lesion site. In each group of NB-3+/+ and NB-3−/−mice, the electrophysiological responses in the caudal spinal cord were recorded in the condition of sham-operated or 12 weeks post-injury. The latencies and amplitudes of responses were analyzed and quantified in each group. n.s., not significant, *p < 0.05, and **p < 0.01; two-way ANOVA followed by Bonferroni's post-test. n= 10 mice per group.E Representative images of coronal sections showing synapse formation between the glutamatergic corticospinal terminals and spinal neurons in NB-3+/+ and NB-3−/−mice. Corticospinal axons, neurons, and glutamatergic terminals of corticospinal axons were labeled with BDA (red), Tuj1 (blue) and vGlut1/2 (green), respectively. Each group showed the data from the rostral and caudal spinal cord in the condition of sham-operated or 12 weeks post-injury. Scale bar, 20 μm.F Quantification of the number of glutamatergic corticospinal synapses per neuron in each group. n.s., not significant, **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 15 mice per group.


Figure 5A , B Western blot analysis ( A ) and quantification ( B ) of NB - 3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control ( LV - nc - GFP ) and that encoding NB - 3 shRNA ( LV - NB - 3 shRNA - GFP ) . NB - 3 expression levels were normalized by b - tubulin . * * * p < 0 . 001 ; one sample t - test . Data were analyzed from 5 independent experiments . C - E Sagittal sections showing the BDA - labeled corticospinal axons in injured spinal cords following cortical injection of LV - nc - GFP ( C ) and LV - NB - 3 shRNA - GFP ( D ) . High - magnification image ( E ) of the area in ( D ) ( asterisk ) . F , J Quantification of the intensity index of BDA - labeled axons at certain distances from the lesion border in ( C , D , G , and H ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 19 mice per group in ( F ) , and n = 17 mice per group in ( J ) . G - I Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( G ) and LV - NB - 3 shRNA - GFP ( H ) into the lesion sites . High - magnification image ( I ) of the area in ( H ) ( asterisk ) . The arrows in ( C - E and G - H ) indicate the lesion sites ; the asterisks in ( D and H ) indicate the lesion epicenters ; the white dashed lines in ( D and H ) indicate the regenerative corticospinal axons extending into the distal spinal cord . F , J Quantification of the intensity index of BDA - labeled axons at certain distances from the lesion border in ( C , D , G , and H ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 19 mice per group in ( F ) , and n = 17 mice per group in ( J ) .

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 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

Figure 5A, B Western blot analysis (A) and quantification (B) of NB-3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control (LV-nc-GFP) and that encoding NB-3 shRNA (LV-NB-3 shRNA-GFP). NB-3 expression levels were normalized by b-tubulin. ***p < 0.001; one sample t-test. Data were analyzed from 5 independent experiments.C-E Sagittal sections showing the BDA-labeled corticospinal axons in injured spinal cords following cortical injection of LV-nc-GFP (C) and LV-NB-3 shRNA-GFP (D). High-magnification image (E) of the area in (D) (asterisk).F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 19 mice per group in (F), and n= 17 mice per group in (J).G-I Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (G) and LV-NB-3 shRNA-GFP (H) into the lesion sites. High-magnification image (I) of the area in (H) (asterisk). The arrows in (C-E and G-H) indicate the lesion sites; the asterisks in (D and H) indicate the lesion epicenters; the white dashed lines in (D and H) indicate the regenerative corticospinal axons extending into the distal spinal cord.F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 19 mice per group in (F), and n= 17 mice per group in (J).


Figure 6A , B NB - 3 - Fc - coated bead aggregation assay ( A ) . BSA - coated beads as a negative control ( B ) . C Co - immunoprecipitation of NB - 3 - Myc with NB - 3 - HA from co - transfected COS1 cells . D , E Cis interactions of NB - 3 - Myc with NB - 3 - HA in COS1 cells . ( E ) High - magnification image of the boxed area in ( D ) . Cells were co - stained for Myc ( red ) , HA ( green ) , and DAPI ( blue ) . The arrowheads indicate cell surface co - localization of NB - 3 - Myc and NB - 3 - HA . F , G Trans interactions between NB - 3 expressed in COS1 cells and treated NB - 3 - Fc . Cells were co - stained for Myc ( red ) , Fc or NB - 3 - Fc ( green ) , and DAPI ( blue ) . The arrows indicate co - localization of NB - 3 - Myc and NB - 3 - Fc at the cell membrane ( G ) . H Cell membrane co - localization of NB - 3 - Myc and NB - 3 - HA separately expressed in 293T and COS1 cells . Cells were co - stained for Myc ( red ) , HA ( green ) , and DAPI ( blue ) . I , J NB - 3 expression in cultured pyramidal neurons and astrocytes . Pyramidal neurons were co - stained for NB - 3 , Ctip2 and Tuj1 ( I ) . Astrocytes were co - stained for NB - 3 , GFAP , and DAPI ( J ) . K Western blot analysis and quantification of NB - 3 in cultured pyramidal neurons and astrocytes . NB - 3 expression levels were normalized by b - tubulin and expressed relative to that of neurons . * * * p < 0 . 001 ; one sample t - test . Data were analyzed from 3 independent experiments . L - Q NB - 3 + / + ( L and M ) and NB - 3 − / − ( O and P ) pyramidal neurons were co - cultured with astrocytes that did not ( L and O ) or did ( M and P ) express NB - 3 , and immunostained for Tuj1 , NB - 3 , and GFAP ( L , M , O and P ) . NB - 3 + / + pyramidal neurons ( N ) and NB - 3 − / − pyramidal neurons that overexpressed NB - 3ΔECD - Myc ( Q ) were co - cultured with astrocytes that overexpressed NB - 3ΔECD - Myc , and immunostained for Tuj1 , Myc , and GFAP ( N and Q ) . R The length of neurites on astrocytes relative to total neurite length . n . s . , not significant , and * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified .

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 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 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 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 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 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

Figure 6A, B NB-3-Fc-coated bead aggregation assay (A). BSA-coated beads as a negative control (B).C Co-immunoprecipitation of NB-3-Myc with NB-3-HA from co-transfected COS1 cells.D, E Cis interactions of NB-3-Myc with NB-3-HA in COS1 cells. (E) High-magnification image of the boxed area in (D). Cells were co-stained for Myc (red), HA (green), and DAPI (blue). The arrowheads indicate cell surface co-localization of NB-3-Myc and NB-3-HA.F, G Trans interactions between NB-3 expressed in COS1 cells and treated NB-3-Fc. Cells were co-stained for Myc (red), Fc or NB-3-Fc (green), and DAPI (blue). The arrows indicate co-localization of NB-3-Myc and NB-3-Fc at the cell membrane (G).H Cell membrane co-localization of NB-3-Myc and NB-3-HA separately expressed in 293T and COS1 cells. Cells were co-stained for Myc (red), HA (green), and DAPI (blue).I, J NB-3 expression in cultured pyramidal neurons and astrocytes. Pyramidal neurons were co-stained for NB-3, Ctip2 and Tuj1 (I). Astrocytes were co-stained for NB-3, GFAP, and DAPI (J).K Western blot analysis and quantification of NB-3 in cultured pyramidal neurons and astrocytes. NB-3 expression levels were normalized by b-tubulin and expressed relative to that of neurons. ***p < 0.001; one sample t-test. Data were analyzed from 3 independent experiments.L-Q NB-3+/+ (L and M) and NB-3−/− (O and P) pyramidal neurons were co-cultured with astrocytes that did not (L and O) or did (M and P) express NB-3, and immunostained for Tuj1, NB-3, and GFAP (L, M, O and P). NB-3+/+ pyramidal neurons (N) and NB-3−/−pyramidal neurons that overexpressed NB-3ΔECD-Myc (Q) were co-cultured with astrocytes that overexpressed NB-3ΔECD-Myc, and immunostained for Tuj1, Myc, and GFAP (N and Q).R The length of neurites on astrocytes relative to total neurite length. n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified.


Figure 7A Layer V cortex explants from E18 . 5 NB - 3 + / + and NB - 3 − / − mice were cultured in medium supplemented with either Fc or NB - 3 - Fc ( 50 μg / ml ) . B Quantification of neurite outgrowth in ( A ) . n . s . , not significant , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group . C Pyramidal neurons of NB - 3 + / + and NB - 3 − / − mice were cultured in medium supplemented with either Fc or NB - 3 - Fc ( 50 μg / ml ) . The neurons were co - stained for Ctip2 , NB - 3 , and Rhodamine phalloidin . ( C1 - C4 ) High - magnification images of the growth cones ( white arrowheads ) of the neurons in ( C ) , respectively . D Quantification of neurite length in ( C ) , and the percentage of collapsed growth cones in ( C ) . n . s . , not significant , * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group .

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 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

Figure 7A Layer V cortex explants from E18.5 NB-3+/+ and NB-3−/−mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml).B Quantification of neurite outgrowth in (A). n.s., not significant, **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group.C Pyramidal neurons of NB-3+/+ and NB-3−/− mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml). The neurons were co-stained for Ctip2, NB-3, and Rhodamine phalloidin. (C1-C4) High-magnification images of the growth cones (white arrowheads) of the neurons in (C), respectively.D Quantification of neurite length in (C), and the percentage of collapsed growth cones in (C). n.s., not significant, *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group.


Figure 8A Images of coronal sections of layer Vsensorimotor cortex from NB - 3 + / + and NB - 3 − / − mice after SCI . Corticospinal neurons were retrogradely labeled with Fluorogold ( FG ) , and coronal sections were co - stained with p - mTOR ( A ) ( arrowheads ) . B Quantification of fluorescence intensity of p - mTOR in FG - labeled corticospinal neurons in ( A ) . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified . C Images of coronal sections of layer Vsensorimotor cortex from NB - 3 + / + and NB - 3 − / − mice after SCI . Corticospinal neurons were retrogradely labeled with Fluorogold ( FG ) , and coronal sections were co - stained with p - S6 ( S240 / 244 ) ( C ) ( arrowheads ) . D Quantification of fluorescence intensity of p - S6 ( S240 / 244 ) in FG - labeled corticospinal neurons in ( C ) . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified . E - H Western blot analysis and quantification of the expression of p - Akt / Akt ( E and F ) and p - S6 ( S240 / 244 ) ( G and H ) in sensorimotor cortex from NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from 3 independent experiments each including 3 mice per group . I , K , M , O Expression of Akt ( I ) , p - Akt ( K ) , p - mTOR ( M ) , and p - S6 ( S240 / 244 ) ( O ) in NB - 3 + / + pyramidal neuron - astrocyte co - cultures . The astrocytes were transfected either with NB - 3 - Myc or a control vector . The dashed lines outline the astrocytes . The asterisks indicate the NB - 3 - expressing astrocytes . J , L , N , P Quantification of fluorescence intensity of Akt ( J ) , p - Akt ( L ) , p - mTOR ( N ) , and p - S6 ( S240 / 244 ) ( P ) , respectively , in pyramidal neurons of the co - cultures in ( I , K , M , and O ) . n . s . , not significant , and * p < 0 . 05 ; one sample t - test . The intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 8A Images of coronal sections of layer Vsensorimotor cortex from NB-3+/+ and NB-3−/−mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-mTOR (A) (arrowheads).B Quantification of fluorescence intensity of p-mTOR in FG-labeled corticospinal neurons in (A). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.C Images of coronal sections of layer Vsensorimotor cortex from NB-3+/+ and NB-3−/−mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-S6 (S240/244) (C) (arrowheads).D Quantification of fluorescence intensity of p-S6 (S240/244) in FG-labeled corticospinal neurons in (C). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.E-H Western blot analysis and quantification of the expression of p-Akt/Akt (E and F) and p-S6 (S240/244) (G and H) in sensorimotor cortex from NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.I, K, M, O Expression of Akt (I), p-Akt (K), p-mTOR (M), and p-S6 (S240/244) (O) in NB-3+/+ pyramidal neuron-astrocyte co-cultures. The astrocytes were transfected either with NB-3-Myc or a control vector. The dashed lines outline the astrocytes. The asterisks indicate the NB-3-expressing astrocytes.J, L, N, P Quantification of fluorescence intensity of Akt (J), p-Akt (L), p-mTOR (N), and p-S6 (S240/244) (P), respectively, in pyramidal neurons of the co-cultures in (I, K, M, and O). n.s., not significant, and *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified.


Figure 9A Co - immunostaining of CHL1 ( blue ) , NB - 3 ( green ) , and BDA ( red ) in injured spinal cords of NB - 3 + / + ( A1 ) and NB - 3 − / − ( A5 ) mice . ( A2 - A4 and A6 - A8 ) High - magnification images of boxed areas in ( A1 and A5 ) , respectively . Images in ( A2 and A6 ) are presented as single - channel fluorescence images in ( A3 - A4 and A7 - A8 ) , respectively . B Quantification of fluorescence intensity of CHL1 at the spinal lesion sites of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . n = 11 mice per group . C , D Western blot analysis ( C ) and quantification ( D ) of CHL1 expression in spinal lesion areas of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from 3 independent experiments each including 2 mice per group . E Co - immunoprecipitation of NB - 3 and CHL1 from spinal cord lesion areas of NB - 3 + / + and NB - 3 − / − mice . F Co - immunostaining of CHL1 and NB - 3 in FG - labeled corticospinal neurons ( arrowheads ) of NB - 3 + / + and NB - 3 − / − mice 14 days post - injury ( dpi ) . G Quantification of the fluorescence intensity of CHL1 in ( F ) . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified . H , I Western blot analysis ( H ) and quantification ( I ) of CHL1 expression in sensorimotor cortices of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from 3 independent experiments each including 3 mice per group . J CHL1 expression in the somas and axons of NB - 3 + / + and NB - 3 − / − pyramidal neurons when co - culturing with NB - 3 - expressing astrocytes ( dashed lines and asterisks ) or astrocytes that didn ' t express NB - 3 ( dashed lines ) . Co - localization of CHL1 and NB - 3 in astrocytes overexpressed NB - 3 ( asterisks ) . Co - localization of CHL1 and NB - 3 in the somas and axons of co - cultured NB - 3 + / + pyramidal neurons ( arrowheads ) . Left inset , high - magnification view of the soma ( white arrowhead ) . Right inset , high - magnification view of the axon tip ( green arrowhead ) . K Quantification of fluorescence intensity of CHL1 in ( J ) . * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified . L - N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( L ) and LV - CHL1 shRNA - GFP ( M ) into both the sensorimotor cortex and lesion sites . High - magnification image ( N ) of the area in ( M ) ( asterisk ) . The arrows indicate the lesion sites ; the asterisk indicates the lesion epicenter . O Quantification of intensity index of BDA - labeled axons at certain distances from the lesion border in ( L and M ) . * p < 0 . 05 and * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 9 mice in ( L ) , and n = 10 mice in ( M ) .

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 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 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 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 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 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

Figure 9A Co-immunostaining of CHL1 (blue), NB-3 (green), and BDA (red) in injured spinal cords of NB-3+/+ (A1) and NB-3−/− (A5) mice. (A2-A4 and A6-A8) High-magnification images of boxed areas in (A1 and A5), respectively. Images in (A2 and A6) are presented as single-channel fluorescence images in (A3-A4 and A7-A8), respectively.B Quantification of fluorescence intensity of CHL1 at the spinal lesion sites of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. n = 11 mice per group.C, D Western blot analysis (C) and quantification (D) of CHL1 expression in spinal lesion areas of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 2 mice per group.E Co-immunoprecipitation of NB-3 and CHL1 from spinal cord lesion areas of NB-3+/+ and NB-3−/−mice.F Co-immunostaining of CHL1 and NB-3 in FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi).G Quantification of the fluorescence intensity of CHL1 in (F). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.H, I Western blot analysis (H) and quantification (I) of CHL1 expression in sensorimotor cortices of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.J CHL1 expression in the somas and axons of NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3-expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Co-localization of CHL1 and NB-3 in astrocytes overexpressed NB-3 (asterisks). Co-localization of CHL1 and NB-3 in the somas and axons of co-cultured NB-3+/+ pyramidal neurons (arrowheads). Left inset, high-magnification view of the soma (white arrowhead). Right inset, high-magnification view of the axon tip (green arrowhead).K Quantification of fluorescence intensity of CHL1 in (J). *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified.L-N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (L) and LV-CHL1 shRNA-GFP (M) into both the sensorimotor cortex and lesion sites. High-magnification image (N) of the area in (M) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.O Quantification of intensity index of BDA-labeled axons at certain distances from the lesion border in (L and M). *p < 0.05 and **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 9 mice in (L), and n= 10 mice in (M).