K Representative cross - section images showing nuclear localization of H3K27me3 in ES cells after 6h addiction of 500μM IAA ( + IAA 6h ) or equal - volume of alcohol ( - IAA ) . Middle : The zoom - in view of H3K27me3 in one ES cell . The outer dotted line colocalizes with nuclear membrane ; the inner dotted line has the 80 % diameter of the outer dotted line . The average pixel intensity of regions within outer dotted line ( ROI . o ) and regions within inner dotted line ( ROI . i ) were measured separately for each cell . Scale bars , 5μm .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Representative cross-section images showing nuclear localization of H3K27me3 in ES cells after 6h addiction of 500μM IAA (+IAA 6h) or equal-volume of alcohol (-IAA). Middle: The zoom-in view of H3K27me3 in one ES cell. The outer dotted line colocalizes with nuclear membrane; the inner dotted line has the 80% diameter of the outer dotted line. The average pixel intensity of regions within outer dotted line (ROI.o) and regions within inner dotted line (ROI.i) were measured separately for each cell. Scale bars, 5μm.


L Statistics for relative intensity ( ROI . o / ROI . i ) after 6h addiction of 500μM IAA ( + IAA 6h ) ( n = 46 ) or equal - volume of alcohol ( - IAA ) ( n = 38 ) . Each point represents one cell . The P values were calculated using unpaired two - tailed Student ' s t test ; * * * * p < 0 . 0001 . Error bars indicate mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L Statistics for relative intensity (ROI.o/ ROI.i) after 6h addiction of 500μM IAA (+IAA 6h) (n=46) or equal-volume of alcohol (-IAA) (n=38). Each point represents one cell. The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001. Error bars indicate mean ± s.e.m.


A Western blotting showing the distribution of MATR3 proteins in chromatin - non - associated and chromatin - associated extracts before and after DRB ( 75μM for 12h ) or RNase A ( pre - treat with 0 . 05 % Triton x - 100 for 30s , followed by 10μg / ml RNase A for 1h ) treatment in AML12 cells . Representative of two independent replicates with similar results .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blotting showing the distribution of MATR3 proteins in chromatin-non-associated and chromatin-associated extracts before and after DRB (75μM for 12h) or RNase A (pre-treat with 0.05% Triton x-100 for 30s, followed by 10μg/ml RNase A for 1h) treatment in AML12 cells. Representative of two independent replicates with similar results.


B ( Left ) The representative cross - section image showing nuclear distribution of DAPI , MATR3 and H3K27me3 before and after 24h treating of 75μM DRB in AML12 cells . ( Right ) Line charts showing pixel intensity of each channel on the ROIs . r , coefficient of correlation . Scale bars , 5μm .

O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B (Left) The representative cross-section image showing nuclear distribution of DAPI, MATR3 and H3K27me3 before and after 24h treating of 75μM DRB in AML12 cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation. Scale bars, 5μm.


G ( Left ) Representative cross - section images showing relative distribution between AS L1 RNA with MATR3 and with histone modification marks ( H3K27me3 , H3K9me3 and H3K27ac ) in AML12 cells . Probes for RNA FISH were designed towards the consensus sequence of antisense L1 _ Mus1 RNAs . ( Right ) Line charts showing pixel intensity of each channel on the ROIs . Scale bars , 5μm . r , coefficient of correlation .

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

G (Left) Representative cross-section images showing relative distribution between AS L1 RNA with MATR3 and with histone modification marks (H3K27me3, H3K9me3 and H3K27ac) in AML12 cells. Probes for RNA FISH were designed towards the consensus sequence of antisense L1_Mus1 RNAs. (Right) Line charts showing pixel intensity of each channel on the ROIs. Scale bars, 5μm. r, coefficient of correlation.


H Normalized average density of the marks ( top ) and heatmaps ( bottom ) for the two groups of L1 loci . The two groups are antisense L1 RNAs or sense L1 RNAs that interacted with MATR3 . L1 loci with RIP ( MATR3 - IgG ) count number > = 10 of antisense RNA were identified as MATR3 - associated antisense L1 , and the same cutoff for MATR3 - associated sense L1 .

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

H Normalized average density of the marks (top) and heatmaps(bottom) for the two groups of L1 loci. The two groups are antisense L1 RNAs or sense L1 RNAs that interacted with MATR3. L1 loci with RIP (MATR3 -IgG) count number >= 10 of antisense RNA were identified as MATR3-associated antisense L1, and the same cutoff for MATR3-associated sense L1.


A ( Left ) The representative cross - section image showing nuclear distribution of MATR3 and H3K27me3 before and after 12h treating with antisense L1 ASOs in AML12 cells . ( Right ) Line charts showing pixel intensity of each channel on the ROIs . r , coefficient of correlation . Scale bars , 5μm .

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

A (Left) The representative cross-section image showing nuclear distribution of MATR3 and H3K27me3 before and after 12h treating with antisense L1 ASOs in AML12 cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation. Scale bars, 5μm.


C ( Left ) The representative cross - section image showing nuclear distribution of H3K27me3 and AS L1 RNA before and after MATR3 knockdown ( Dox treatment for 3d ) in AML12 cells . ( Right ) The normal distribution curve for the AS L1 pixel intensity . Scale bars , 5μm .

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

C (Left) The representative cross-section image showing nuclear distribution of H3K27me3 and AS L1 RNA before and after MATR3 knockdown (Dox treatment for 3d) in AML12 cells. (Right) The normal distribution curve for the AS L1 pixel intensity. Scale bars, 5μm.


E Representative images showing nuclear colocalization of AS L1 RNAs with wild - type and truncated GFP - MATR3 proteins in AML12 cells . Scale bars , 5μm .

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

E Representative images showing nuclear colocalization of AS L1 RNAs with wild-type and truncated GFP-MATR3 proteins in AML12 cells. Scale bars, 5μm.


G Representative cross - section images showing nuclear localization of H3K27me3 upon Ctrl , MATR3 knockdown and exogenous GFP - MATR3 ( WT / △ ZF1 / △ ZF2 / △ RRM1 / △ RRM2 ) overexpression in AML12 cells . Scale bars , 5μm .

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

G Representative cross-section images showing nuclear localization of H3K27me3 upon Ctrl, MATR3 knockdown and exogenous GFP-MATR3 (WT/△ZF1/△ZF2/△RRM1/△RRM2) overexpression in AML12 cells. Scale bars, 5μm.


A Representative images of the GFP - MATR3 ( WT / △ RRM2 ) FRAP experiments in AML2 cells . Solid arrows indicate the bleached points . Scale bars , 5μm .

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

A Representative images of the GFP-MATR3 (WT/△RRM2) FRAP experiments in AML2 cells. Solid arrows indicate the bleached points. Scale bars, 5μm.


C Representative images of droplet formation assays with different concentrations of GFP - MATR3 proteins . NaCl concentration , 50mM . Scale bars , 5μm . D Representative images of droplet formation assays with different NaCl concentrations . GFP - MATR3 protein concentration , 3 μM . Scale bars , 5μm . E Areas of MATR3 protein droplets formed in different protein concentration ( 3 μM : n = 315 ; 2 μM : n = 196 ; 1 μM : n = 165 ; 0 . 5 μM : n = 50 ) . The P values were calculated using unpaired two - tailed Student ' s t test ; * * * * p < 0 . 0001 . F Areas of MATR3 protein droplets formed in different NaCl concentration ( 50 mM : n = 248 ; 100 mM : n = 196 ; 400 mM : n = 127 ; 800 mM : none ) . The P values were calculated using unpaired two - tailed Student ' s t test ; * * * * p < 0 . 0001 .

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

C Representative images of droplet formation assays with different concentrations of GFP-MATR3 proteins. NaCl concentration, 50mM. Scale bars, 5μm. D Representative images of droplet formation assays with different NaCl concentrations. GFP-MATR3 protein concentration, 3 μM. Scale bars, 5μm. E Areas of MATR3 protein droplets formed in different protein concentration (3 μM: n = 315; 2 μM: n = 196; 1 μM: n = 165; 0.5 μM: n = 50). The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001. F Areas of MATR3 protein droplets formed in different NaCl concentration (50 mM: n = 248; 100 mM: n = 196; 400 mM: n = 127; 800 mM: none). The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001.


H Representative images of droplet formation assays by GFP - MATR3 with different concentration ( 0nM , 5nM , 50nM , 200nM , 500nM ) of AS L1 RNAs . GFP - MATR3 protein concentration , 3 μM . NaCl concentration , 50mM . Scale bars , 5μm .

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

H Representative images of droplet formation assays by GFP-MATR3 with different concentration (0nM, 5nM, 50nM, 200nM, 500nM) of AS L1 RNAs. GFP-MATR3 protein concentration, 3 μM. NaCl concentration, 50mM. Scale bars, 5μm.


E Snapshot of an example region , showing Hi - C , H3K27me3 ChIP - seq and AS L1 in MATR3 RIP - seq in Ctrl and / or shMatr3 samples , using HiCExplorer . The values on the y - axis for Hi - C contact and O / E heatmap are iced normalized read counts at 100kb resolution . The values on the y - axis for ChIP - seq and RIP - seq are average reads per million of mapped reads ( RPM ) .

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

E Snapshot of an example region, showing Hi-C, H3K27me3 ChIP-seq and AS L1 in MATR3 RIP-seq in Ctrl and/or shMatr3 samples, using HiCExplorer. The values on the y-axis for Hi-C contact and O/E heatmap are iced normalized read counts at 100kb resolution. The values on the y-axis for ChIP-seq and RIP-seq are average reads per million of mapped reads (RPM).


K Percentage of TADs located in compartment A or B regions from five TAD groups ( same groups in J ) .

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

K Percentage of TADs located in compartment A or B regions from five TAD groups (same groups in J).


L Boxplot shows gene expression changes for TAD groups from J . The number of genes in each groups are 2050 , 2094 , 2356 , 2071 and 1605 . The box of the boxplot covers the 50 % of the values between 1st quartile and 3rd quartile of each group ; Boxplot whiskers represent the minimal and maximal values of each group ; The horizontal line of box represent the median value of each group ; Red dashed lines indicate the zero .

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

L Boxplot shows gene expression changes for TAD groups from J. The number of genes in each groups are 2050, 2094, 2356, 2071 and 1605. The box of the boxplot covers the 50% of the values between 1st quartile and 3rd quartile of each group; Boxplot whiskers represent the minimal and maximal values of each group; The horizontal line of box represent the median value of each group; Red dashed lines indicate the zero.


A Representative Western blot on chromatin fractionation experiments of Ctrl and shMatr3 cells . Each fraction was loaded with same number of cells . GAPDH , histone H3 , Lamin A / C were used as positive controls for S1 , S2 and S3 , S4 , respectively .

A Representative Western blot on chromatin fractionation experiments of Ctrl and shMatr3 cells. Each fraction was loaded with same number of cells. GAPDH, histone H3, Lamin A/C were used as positive controls for S1, S2 and S3, S4, respectively.


B Genome browser view of the SAMMY - Seq and H3K27me3 ChIP - seq in Ctrl and shMatr3 ( replicate 1 and 2 merged ) cells at a representative region . Gray boxes under each SAMMY - seq track showing the SAMMY - seq domains . The shadows showing the shMatr3 loss SAMMY - seq domains .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Genome browser view of the SAMMY-Seq and H3K27me3 ChIP-seq in Ctrl and shMatr3 (replicate 1 and 2 merged) cells at a representative region. Gray boxes under each SAMMY-seq track showing the SAMMY-seq domains. The shadows showing the shMatr3 loss SAMMY-seq domains.


H Average MATR3 RIP - seq sense and antisense signal at L1 loci around H3K27me3 domains ( groups as in F ) with 200kb upstream and downstream flanking regions from AML12 cells .

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

H Average MATR3 RIP-seq sense and antisense signal at L1 loci around H3K27me3 domains (groups as in F) with 200kb upstream and downstream flanking regions from AML12 cells.


C MATR3 knockdown and GFP - tagged WT / S85C / F115C MATR3 protein replacement in N2A cells . The efficiency of endogenous MATR3 knockdown and exogenous GFP - MATR3 ( WT / S85C / F115C ) over - expression as detected by western blotting . Representative of two independent replicates with similar results .

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

C MATR3 knockdown and GFP-tagged WT/S85C/F115C MATR3 protein replacement in N2A cells. The efficiency of endogenous MATR3 knockdown and exogenous GFP-MATR3 (WT/S85C/F115C) over-expression as detected by western blotting. Representative of two independent replicates with similar results.


D Representative images of the GFP - MATR3 ( WT / S85C / F115C ) FRAP experiments in N2A cells . Solid arrows point to the bleached points . Scale bars , 5μm .

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

D Representative images of the GFP-MATR3 (WT/S85C/F115C) FRAP experiments in N2A cells. Solid arrows point to the bleached points. Scale bars, 5μm.


G Representative cross - section images showing nuclear localization of H3K27me3 upon Ctrl , MATR3 knockdown and exogenous MATR3 ( WT / S85C / F115C ) overexpression in N2A cells . Scale bars , 5μm . H Standard deviation of H3K27me3 pixel intensity upon Ctrl ( n = 93 ) , MATR3 knock down ( n = 100 ) , GFP - MATR3 ( WT ) rescue ( n = 104 ) , GFP - MATR3 ( S85C ) rescue ( n = 82 ) and GFP - MATR3 ( F115C ) rescue ( n = 120 ) . The P values were calculated using unpaired two - tailed Student ' s t test ; ns , not significant , * p < 0 . 05 , * * * * p < 0 . 0001 . Error bars indicate mean ± s . e . m .

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

G Representative cross-section images showing nuclear localization of H3K27me3 upon Ctrl, MATR3 knockdown and exogenous MATR3 (WT/S85C/F115C) overexpression in N2A cells. Scale bars, 5μm. H Standard deviation of H3K27me3 pixel intensity upon Ctrl (n=93), MATR3 knock down (n=100), GFP-MATR3 (WT) rescue (n=104), GFP-MATR3 (S85C) rescue (n=82) and GFP-MATR3 (F115C) rescue (n=120). The P values were calculated using unpaired two-tailed Student's t test; ns, not significant, *p<0.05, ****p<0.0001. Error bars indicate mean ± s.e.m.


a , Wild - type ( WT ) and Park2 - / - BMDMs were infected with mCherry - expressing M . tuberculosis ( Mtb ) for 4 h and immunostained using anti - parkin antibodies .

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

a, Wild-type (WT) and Park2-/- BMDMs were infected with mCherry-expressing M. tuberculosis (Mtb) for 4 h and immunostained using anti-parkin antibodies.


b , Wild - type BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained for polyubiquitin .

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

b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin.


b , Wild - type BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained for polyubiquitin . c , Quantification of ubiquitin - positive M . tuberculosis from b . Results are means ± s . e . m . of three independent experiments ( * * P < 0 . 001 , paired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin. c, Quantification of ubiquitin-positive M. tuberculosis from b. Results are means ± s.e.m. of three independent experiments (**P < 0.001, paired Student's t-test).


d , U937 human macrophages expressing a scrambled shRNA ( Control ) or one of two different shRNAs targeting PARK2 ( shRNA no . 1 , shRNA no . 2 ) were infected with mCherry - expressing M . tuberculosis for 12 h and immunostained for polyubiquitin .

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

d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin.


d , U937 human macrophages expressing a scrambled shRNA ( Control ) or one of two different shRNAs targeting PARK2 ( shRNA no . 1 , shRNA no . 2 ) were infected with mCherry - expressing M . tuberculosis for 12 h and immunostained for polyubiquitin . e , Quantification of ubiquitin positive M . tuberculosis from d , results are means ± s . e . m . of three independent experiments ( * * P 0 . 005 , Student ' s t - test ) . f , The parkin and actin expression in cells from e was determined by western blotting .

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

d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin. e, Quantification of ubiquitin positive M. tuberculosis from d, results are means ± s.e.m. of three independent experiments (**P  0.005, Student's t-test). f, The parkin and actin expression in cells from e was determined by western blotting.


g , Park2 − / − BMDMs were transduced with lentivirus expressing BFP ( − ) , wild - type parkin , or two separate mutant parkin alleles ( T240R , P437L ) . Cells were infected with M . tuberculosis for 4 h and ubiquitin and M . tuberculosis co - localization was quantified and expressed relative to control BMDMs . Results are means ± s . e . m . of three independent experiments ( * * P 0 . 005 , paired Student ' s t - test ) . h , The parkin and actin expression in cells from g was determined by western blotting .

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

g, Park2−/− BMDMs were transduced with lentivirus expressing BFP (−), wild-type parkin, or two separate mutant parkin alleles (T240R, P437L). Cells were infected with M. tuberculosis for 4 h and ubiquitin and M. tuberculosis co-localization was quantified and expressed relative to control BMDMs. Results are means ± s.e.m. of three independent experiments (**P  0.005, paired Student's t-test). h, The parkin and actin expression in cells from g was determined by western blotting.


a , BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained using linkage - specific ubiquitin antibodies . b , Quantification of K63 and K48 ubiquitin - positive M . tuberculosis from a .

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

a, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained using linkage-specific ubiquitin antibodies. b, Quantification of K63 and K48 ubiquitin-positive M. tuberculosis from a.


c , BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h and immunostained for NDP52 , p62 , phospho - TBK1 or NBR1 . d , Quantification of co - localization from c ( n = 3 per group , * * P 0 . 001 ) . All errors , s . e . m .

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

c, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for NDP52, p62, phospho-TBK1 or NBR1. d, Quantification of co-localization from c (n = 3 per group, **P  0.001). All errors, s.e.m.


a , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 4 h and immunostained for LC3 ( LC3B antibody ) or ATG12 . b , Quantification of results from a ( n = 3 per group , * * P 0 . 001 ) . c , Western blot analysis of LC3 ( LC3B antibody ) from cell lysates from a .

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

a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a.


d , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 6 h and immunostained for LAMP1 .

d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1.


d , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 6 h and immunostained for LAMP1 . e , Quantification of results from d expressed relative to control BMDMs ( n = 3 per group , * * P 0 . 001 ) .

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

d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001).


f , BMDMs were infected with M . tuberculosis and colony - forming units ( c . f . u . ) at t = 0 and t = 16 h were determined by plating . Results were normalized to t = 0 ( n = 3 per group , * P < 0 . 02 ) .

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

f, BMDMs were infected with M. tuberculosis and colony-forming units (c.f.u.) at t = 0 and t = 16 h were determined by plating. Results were normalized to t = 0 (n = 3 per group, *P < 0.02).


g , RAW264 . 7 macrophages transduced with lentivirus expressing green fluorescent protein ( GFP ) or parkin were infected with M . tuberculosis and c . f . u . were determined after 16 h . Results were normalized to GFP - expressing cells ( n = 3 per group , * * P < 0 . 0076 ) .

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

g, RAW264.7 macrophages transduced with lentivirus expressing green fluorescent protein (GFP) or parkin were infected with M. tuberculosis and c.f.u. were determined after 16 h. Results were normalized to GFP-expressing cells (n = 3 per group, **P < 0.0076).


h , U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M . tuberculosis for 36 h and c . f . u were determined . Results were normalized to t = 0 ( N = 3 per group , * P < 0 . 02 ) . All errors , s . e . m .

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

h, U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M. tuberculosis for 36 h and c.f.u were determined. Results were normalized to t = 0 (N = 3 per group, *P< 0.02). All errors, s.e.m.


a , WT and Park2 - / - mice were infected with wild - type M . tuberculosis by aerosol and lung bacterial burdens were determined by plating ( means ± s . d . , n = 5 per group , * P < 0 . 02 by Student ' s t - test ) .

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a, WT and Park2-/- mice were infected with wild-type M. tuberculosis by aerosol and lung bacterial burdens were determined by plating (means ± s.d., n = 5 per group, *P < 0.02 by Student's t-test).


b , Enumeration of liver and spleenc . f . u . from mice infected in a , 21 days post - infection ( means ± s . d , n = 5 per group , * P < 0 . 03 by Student ' s t - test ) .

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b, Enumeration of liver and spleenc.f.u. from mice infected in a, 21 days post-infection (means ± s.d, n = 5 per group, *P < 0.03 by Student's t-test).


c , Survival of M . tuberculosis - infected wild - type and Park2 - / - mice ( n = 10 , * * P < 0 . 001 by log - rank test ) .

c, Survival of M. tuberculosis-infected wild-type and Park2-/- mice (n = 10, **P < 0.001 by log-rank test).


d , Immunohistochemistry staining of lung sections from infected mice21 days post - infection , scale bar , 50 µm .

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d, Immunohistochemistry staining of lung sections from infected mice21 days post-infection, scale bar, 50 µm.


e , Immunohistochemistry staining of lung sections from a human patient with active tuberculosis . Scale bars , 500 µm ( × 4 ) , 50 µm ( × 40 ) .

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e, Immunohistochemistry staining of lung sections from a human patient with active tuberculosis. Scale bars, 500 µm (×4), 50 µm (×40).


f , Confocal microscopy images of humanlung sections from e immunostained for M . tuberculosis and parkin ; DNA was visualized using DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) . Scale bars , 25 µm .

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f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm.


g , Mice were infected with wild - type L . monocytogenes by i . p . injection and bacterial burdens in livers and spleens were determined by plating ( means ± s . d . , n = 7 per group , * P < 0 . 04 by Student ' s t - test ) .

g, Mice were infected with wild-type L. monocytogenes by i.p. injection and bacterial burdens in livers and spleens were determined by plating (means ± s.d., n = 7 per group, *P < 0.04 by Student's t-test).


a , Wild - type and two independent parkin - deficient D . melanogaster lines ( parkc00062 , parkf01950 ) were infected with L . monocytogenes by anterior abdomen injection . Atg8 processing was monitored in whole - fly protein lysates by western blotting .

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a, Wild-type and two independent parkin-deficient D. melanogaster lines (parkc00062, parkf01950) were infected with L. monocytogenes by anterior abdomen injection. Atg8 processing was monitored in whole-fly protein lysates by western blotting.


b , L . monocytogenes c . f . u . from a were determined by plating ( means ± s . d . , n = 3 - 5 , * * P < 0 . 001 by Student ' s t - test ) .

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b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3-5, **P < 0.001 by Student's t-test).


c , Survival of D . melanogaster was determined ( n = 33 , * * P < 0 . 001 by log - rank test ) .

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c, Survival of D. melanogaster was determined (n = 33, **P < 0.001 by log-rank test).


d , e , Flies were infected with S . enterica serovar Typhimurium and bacterial c . f . u . ( d ) ( means ± s . d . , n = 3 - 5 per group , * * P 0 . 009 by Student ' s t - test ) and fly survival ( e ) were determined ( n = 59 , * * P 0 . 001 by log - rank test ) .

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d, e, Flies were infected with S. enterica serovar Typhimurium and bacterial c.f.u. (d) (means ± s.d., n = 3-5 per group, **P  0.009 by Student's t-test) and fly survival (e) were determined (n = 59, **P  0.001 by log-rank test).


f , Survival of flies infected with M . marinum ( n = 37 , * * P 0 . 0045 by log - rank test ) .

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f, Survival of flies infected with M. marinum (n = 37, **P  0.0045 by log-rank test).


( B ) The EC50 values ( 95 % confidence interval ) for VHH - B9 , VHH - 10C4 and VHH - 4A2 are 99 . 2 nM ( 83 . 8 - 117 . 5 nM ) , 112 . 5 nM ( 101 . 1 - 125 . 2 nM ) and 788 . 7 nM ( 682 . 7 - 911 . 2 nM ) , respectively . Values are mean ± S . D . , n = 3 , technical replicates .

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(B) The EC50 values (95% confidence interval) for VHH-B9, VHH-10C4 and VHH-4A2 are 99.2 nM (83.8-117.5 nM), 112.5 nM (101.1-125.2 nM) and 788.7 nM (682.7-911.2 nM), respectively. Values are mean ± S.D., n=3, technical replicates.


( C , D ) Primary cultured neurons were transduced by Semliki Forest virus ( SFV ) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs . PBS and anti - GFP VHH were used as controls . ( C ) Western blot analysis of conditioned media for sAPP ⍺ and sAPPβ , as well as cell extracts for full length APP , CTF ⍺ and CTFβ . ( D ) ELISA measurements of Aβ1 - 40 and Aβ1 - 42 in conditioned media . Values are mean ± S . E . M . , n = 3 cultures for each analysis . One - way ANOVA , * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 0001 .

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(C, D) Primary cultured neurons were transduced by Semliki Forest virus (SFV) expressing wild type human APP695 and treated with 3 µM of the indicated VHHs. PBS and anti-GFP VHH were used as controls. (C) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (D) ELISA measurements of Aβ1-40 and Aβ1-42 in conditioned media. Values are mean ± S.E.M., n=3 cultures for each analysis. One-way ANOVA, *p<0.5, **p<0.01, *** p<0.0001.


( E , F ) Dose - dependent inhibition of BACE1 in primary cultured neurons by VHH - B9 . Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS ( control : CT ) or decreasing concentrations of VHH - B9 , ranging from 10 µM to 0 . 7 nM . ( E ) Western blot analysis of conditioned media for sAPP ⍺ and sAPPβ , as well as cell extracts for full length APP , CTF ⍺ and CTFβ . ( F ) Conditioned media was analyzed by ELISA to assess levels of Aβ1 - 40 . Values are mean ± S . D . , n = 3 cultures for each analysis . The EC50 value ( 95 % confidence interval ) was estimated as 85 . 4 nM ( 52 . 5 - 125 . 6 nM ) .

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(E, F) Dose-dependent inhibition of BACE1 in primary cultured neurons by VHH-B9. Cultured neurons were transduced by SFV expressing wild type human APP695 and treated with PBS (control: CT) or decreasing concentrations of VHH-B9, ranging from 10 µM to 0.7 nM. (E) Western blot analysis of conditioned media for sAPP⍺ and sAPPβ, as well as cell extracts for full length APP, CTF⍺ and CTFβ. (F) Conditioned media was analyzed by ELISA to assess levels of Aβ1-40. Values are mean ± S.D., n=3 cultures for each analysis. The EC50 value (95 % confidence interval) was estimated as 85.4 nM (52.5-125.6 nM).


( A ) Co - staining with anti - N - Terminal Aβ 82E1 ( green ) and Thioflavin - S ( magenta ) reveals Aβ plaques ( arrow heads ) in regions of interest . Representative images are shown . Scale bar : 50 μm . ( B , C ) The number of Aβ plaques in regions of interest was significantly reduced by treatment AAV - VHH - B9 ( B ) , as was total plaque area ( C ) . (

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(A) Co-staining with anti-N-Terminal Aβ 82E1 (green) and Thioflavin-S (magenta) reveals Aβ plaques (arrow heads) in regions of interest. Representative images are shown. Scale bar: 50 μm. (B, C) The number of Aβ plaques in regions of interest was significantly reduced by treatment AAV-VHH-B9 (B), as was total plaque area (C). (


( A ) ( left ) Representative images of anti - GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL - G - F mice receiving either AAV - VHH - B9 or AAV - GFP ; ( right ) quantification of the GFAP + cell number in the indicated brain regions across experimental cohorts .

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(A) (left) Representative images of anti-GFAP immunostaining in Prefrontal Cortex and Amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the GFAP+ cell number in the indicated brain regions across experimental cohorts.


( B ) ( left ) Representative images of anti - IBA1 immunostaining in dentate gyrus and amygdala from AppNL - G - F mice receiving either AAV - VHH - B9 or AAV - GFP ; ( right ) quantification of the IBA1 + cell number in the indicated brain regions across experimental cohorts .

(B) (left) Representative images of anti-IBA1 immunostaining in dentate gyrus and amygdala from AppNL-G-F mice receiving either AAV-VHH-B9 or AAV-GFP; (right) quantification of the IBA1+ cell number in the indicated brain regions across experimental cohorts.


( A ) Mitochondrial extracts from 143B cells , COX1 ∆ and COX2 ∆ mutant cybrids and their respective isogenic controls ( CON - 1 and CON - 2 ) were analyzed by SDS - PAGE and immunoblotting with the indicated antibodies . The graphs ( right ) show densitometric quantifications of the signals normalized to CII subunit SDHA . The mean of three independent controls ( CON ) was set to 100 and all measurements were referenced to that value . The values represent mean ± SD ( n = 3 - 8 ) . Mann - Whitney U test was applied for statistical analyses . Variations between controls and mutants : α , p < 0 . 05 ; β , p < 0 . 01 ; γ , p < 0 . 001 . Specific variations between COX1 ∆ and COX2 ∆ mutants : ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 .

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(A) Mitochondrial extracts from 143B cells, COX1∆ and COX2∆ mutant cybrids and their respective isogenic controls (CON-1 and CON-2) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The graphs (right) show densitometric quantifications of the signals normalized to CII subunit SDHA. The mean of three independent controls (CON) was set to 100 and all measurements were referenced to that value. The values represent mean ± SD (n=3-8). Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; β, p<0.01; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05; ∗∗p<0.01.


( B ) Digitonized mitochondria ( digitonin - to - protein ratio of 2 : 1 ) were analyzed by BN - PAGE , followed by CI in gel activity ( IGA ) assays and Coomassie staining . The SC I + III2 + IV1 band ( b1 ) was excised from the control ( 143B ) lane and the SC I + III2 bands ( b2 to b4 ) were excised from the COX1 ∆ and COX2 ∆ lanes . Their protein compositions were subsequently analyzed by nano - LC / ESI - MS ( see also Figure EV2 ) . The relative position of the molecular weight marker is indicated on the left .

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(B) Digitonized mitochondria (digitonin-to-protein ratio of 2:1) were analyzed by BN-PAGE, followed by CI in gel activity (IGA) assays and Coomassie staining. The SC I+III2+IV1 band (b1) was excised from the control (143B) lane and the SC I+III2 bands (b2 to b4) were excised from the COX1∆ and COX2∆ lanes. Their protein compositions were subsequently analyzed by nano-LC/ESI-MS (see also Figure EV2). The relative position of the molecular weight marker is indicated on the left.


( C ) BN - PAGE was followed by CI - IGA assay and immunoblotting with the indicated antibodies ( in brackets ) . The identity of the MRC complexes and SCs is : I + III2 + IV1 , SC containing CI , CIII2 , and CIV ; I + III2 , SC containing CI and CIII2 ; I + III2plus , SC containing CI , CIII2 and a COX1 - submodule ; III2 + IV , SC containing CIII2 and CIV ; I , complex I ; III2 , CIII dimer ; IV2 , CIV dimer ; subCOX1 , COX1 - containing subcomplexes .

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(C) BN-PAGE was followed by CI-IGA assay and immunoblotting with the indicated antibodies (in brackets). The identity of the MRC complexes and SCs is: I+III2+IV1, SC containing CI, CIII2, and CIV; I+III2, SC containing CI and CIII2; I+III2plus, SC containing CI, CIII2 and a COX1-submodule; III2+IV, SC containing CIII2 and CIV; I, complex I; III2, CIII dimer; IV2, CIV dimer; subCOX1, COX1-containing subcomplexes.


( A ) Control 143B cells ( left panels ) , COX1 ∆ cybrids ( middle panels ) , and COX2 ∆ cybrids ( right panels ) . The right lane highlights structural subunits of CI ( yellow ) , CIII ( light green ) , and CIV ( orange brown ) , and assembly factors ( orange ) . Intensity - based absolute quantification ( iBAQ ) values were normalized to the sum of all control values . Top lanes indicate gel slice numbers and their relative native masses .

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(A) Control 143B cells (left panels), COX1∆ cybrids (middle panels), and COX2∆ cybrids (right panels). The right lane highlights structural subunits of CI (yellow), CIII (light green), and CIV (orange brown), and assembly factors (orange). Intensity-based absolute quantification (iBAQ) values were normalized to the sum of all control values. Top lanes indicate gel slice numbers and their relative native masses.


( B ) Intensity profiles showing abundance and relative distribution of structural subunits from CI ( upper graphs ) , CIII ( middle graphs ) , and CIV ( bottom graphs ) . The identities of MRC complexes and SCs are as in Figure 1 .

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(B) Intensity profiles showing abundance and relative distribution of structural subunits from CI (upper graphs), CIII (middle graphs), and CIV (bottom graphs). The identities of MRC complexes and SCs are as in Figure 1.


( A ) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A , HIGD2A , CMC1 and COA3 in control ( 143B ) , COX1 ∆ and COX2 ∆ cybrids . The relative abundance and distribution of detected proteins is shown in the lower graphs . The localization of SC I + III2plus in COX2Δ cybrids is highlighted with a red line . The approximate molecular weights of CIV - containing structures are indicated on the top ( in kDa ) .

(A) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A, HIGD2A, CMC1 and COA3 in control (143B), COX1∆ and COX2∆ cybrids. The relative abundance and distribution of detected proteins is shown in the lower graphs. The localization of SC I+III2plus in COX2Δ cybrids is highlighted with a red line. The approximate molecular weights of CIV-containing structures are indicated on the top (in kDa).


( B ) 2D - BN / SDS - PAGE analysis from control ( 143B ) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies . I + III2 + IV1 , SC containing CI , CIII , and CIV ; I + III2plus , SC containing CI , CIII and a COX1 - submodule ; III2 + IV , SC containing CIII and CIV ; IV2 , CIV dimer ; IV , CIV monomer . The presence of a COX1 - CMC1 - COA3 - containing subcomplex is indicated .

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(B) 2D-BN/SDS-PAGE analysis from control (143B) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies. I+III2+IV1, SC containing CI, CIII, and CIV; I+III2plus, SC containing CI, CIII and a COX1-submodule; III2+IV, SC containing CIII and CIV; IV2, CIV dimer; IV, CIV monomer. The presence of a COX1-CMC1-COA3-containing subcomplex is indicated.


Mitochondrial extracts from control 143B cells ( left ) , COX1 ∆ cybrids ( right ) , and COX2 ∆ cybrids ( middle ) were immunoprecipitated using antibodies against : ( A ) CIV subunits COX1 and COX5B . ( B ) CI subunit NDUFB7 . Samples were subsequently analyzed by SDS - PAGE and immunoblotting with the indicated antibodies . In , input . IP , immuno - precipitate . FT , flow - through . Ig , unrelated immunoglobulin used as a negative control .

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Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (A) CIV subunits COX1 and COX5B. (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.


Mitochondrial extracts from control 143B cells ( left ) , COX1 ∆ cybrids ( right ) , and COX2 ∆ cybrids ( middle ) were immunoprecipitated using antibodies against : ( B ) CI subunit NDUFB7 . Samples were subsequently analyzed by SDS - PAGE and immunoblotting with the indicated antibodies . In , input . IP , immuno - precipitate . FT , flow - through . Ig , unrelated immunoglobulin used as a negative control .

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Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.


Mitochondrial extracts from control 143B cells ( left ) , COX1 ∆ cybrids ( right ) , and COX2 ∆ cybrids ( middle ) were immunoprecipitated using antibodies against : ( C ) CIII subunit CORE2 . Samples were subsequently analyzed by SDS - PAGE and immunoblotting with the indicated antibodies . In , input . IP , immuno - precipitate . FT , flow - through . Ig , unrelated immunoglobulin used as a negative control .

Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (C) CIII subunit CORE2. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.


( A ) Digitonin - solubilized mitochondria from doxycycline - treated 143B cells , COX1 ∆ and COX2 ∆ cybrids were analyzed by BN - PAGE followed by CI - IGA assays ( upper panels ) or immunoblotting ( medium and lower panels ) with antibodies against CI subunits NDUFA9 and NDUFS1 . The identities of MRC complexes and SCs are as in Figure 1 .

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(A) Digitonin-solubilized mitochondria from doxycycline-treated 143B cells, COX1∆ and COX2∆ cybrids were analyzed by BN-PAGE followed by CI-IGA assays (upper panels) or immunoblotting (medium and lower panels) with antibodies against CI subunits NDUFA9 and NDUFS1. The identities of MRC complexes and SCs are as in Figure 1.


( B ) Mean incorporation rates of NDUFA9 and NDUFS1 into SC I + III2 + IV1 from 143B cells and into SCs I + III2 from the CIV - KO cybrids . Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels . Time - point values are expressed as percentages of untreated cells ( SS ) and indicated as mean ± SD .

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(B) Mean incorporation rates of NDUFA9 and NDUFS1 into SC I+III2+IV1 from 143B cells and into SCs I+III2 from the CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.


( C ) Samples were subsequently analyzed by 2D - BN / SDS - PAGE and immunoblotting with the indicated antibodies . The identities of MRC complexes and SCs are as in Figure 1 .

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(C) Samples were subsequently analyzed by 2D-BN/SDS-PAGE and immunoblotting with the indicated antibodies. The identities of MRC complexes and SCs are as in Figure 1.


( D ) Mean incorporation rates of CIII subunits CORE2 and RISP into SC I + III2 + IV1 from control cells and into SCs I + III2 from CIV - KO cybrids . Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels . Time - point values are expressed as percentages of untreated cells ( SS ) and indicated as mean ± SD .

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(D) Mean incorporation rates of CIII subunits CORE2 and RISP into SC I+III2+IV1 from control cells and into SCs I+III2 from CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.


( A ) Digitonized mitochondria from doxycycline - treated 143B cells and COX2 ∆ cybrids were analyzed by 2D - BN / SDS - PAGE and western blot with the indicated antibodies .

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(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies.


( B ) Mean incorporation rates of COX subunits into SC I + III2plus from COX2 ∆ cybrids . Signals from three independent experiments were quantified and normalized by CII . Time - point values are expressed as percentages of the untreated cells ( SS ) and indicated as mean ± SD . Control ( 143B ) values for subunits COX4 , COX5A and COX6C were obtained from ( Moreno - Lastres et al . , 2012 ) .

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(B) Mean incorporation rates of COX subunits into SC I+III2plus from COX2∆ cybrids. Signals from three independent experiments were quantified and normalized by CII. Time-point values are expressed as percentages of the untreated cells (SS) and indicated as mean ± SD. Control (143B) values for subunits COX4, COX5A and COX6C were obtained from (Moreno-Lastres et al., 2012).


C - D SDS - PAGE ( C ) and BN - PAGE ( D ) analyses from 143B cells and COX2 ∆ cybrids transiently transfected either with a COX5B - Myc - DDK construct or with the empty vector , followed by immunoblotting with antibodies against COX5B and CII subunit SDHA ( loading control ) . The identities of MRC complexes and SCs are as in Figure 1 . The red arrow points to the accumulation of over expressed COX5B bound to SC I + III2 in control cells .

C-D SDS-PAGE (C) and BN-PAGE (D) analyses from 143B cells and COX2∆ cybrids transiently transfected either with a COX5B-Myc-DDK construct or with the empty vector, followed by immunoblotting with antibodies against COX5B and CII subunit SDHA (loading control). The identities of MRC complexes and SCs are as in Figure 1. The red arrow points to the accumulation of over expressed COX5B bound to SC I+III2 in control cells.


Mitochondrial translation products were pulse - labelled in control 143B cells ( left panel ) , COX1 ∆ ( middle panel ) and COX2 ∆ ( right panel ) cybrids with [ 35S ] - methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation . For chase , cells were washed and incubated with fresh culture medium for the indicated time points ( T0 - T24 hours ) . Radiolabeled mitochondrial proteins ( indicated on the right ) were separated by 2D - BN / SDS - PAGE and visualized by autoradiography . A enlarged section of the SCs region at T24 ( delimited by a square ) shows the co - migration of COX1 with SC I + III2plus ( black arrow ) in the COX2 ∆ cybrids relative to the position of canonical SC I + III2 ( gray arrow ) . The identities of MRC complexes and SCs ( bottom ) are as in Figure 1 . Molecular weight markers are indicated on the left .

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

Mitochondrial translation products were pulse-labelled in control 143B cells (left panel), COX1∆ (middle panel) and COX2∆ (right panel) cybrids with [35S]-methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation. For chase, cells were washed and incubated with fresh culture medium for the indicated time points (T0-T24 hours). Radiolabeled mitochondrial proteins (indicated on the right) were separated by 2D-BN/SDS-PAGE and visualized by autoradiography. A enlarged section of the SCs region at T24 (delimited by a square) shows the co-migration of COX1 with SC I+III2plus (black arrow) in the COX2∆ cybrids relative to the position of canonical SC I+III2 (gray arrow). The identities of MRC complexes and SCs (bottom) are as in Figure 1. Molecular weight markers are indicated on the left.


( A ) Respiratory chain enzyme activities of control ( blue ) , COX1 ∆ ( red ) and COX2 ∆ ( green ) cybrids . Enzyme activities were measured in three experimental replicates . Data are expressed as the percentages of control cells , and indicated as means ± SD . Mann - Whitney U test was applied for statistical analyses . Variations between controls and mutants : α , p < 0 . 05 ; γ , p < 0 . 001 . Specific variations between COX1 ∆ and COX2 ∆ mutants : ∗ p < 0 . 05 .

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

(A) Respiratory chain enzyme activities of control (blue), COX1∆ (red) and COX2∆ (green) cybrids. Enzyme activities were measured in three experimental replicates. Data are expressed as the percentages of control cells, and indicated as means ± SD. Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05.


D Ratio of toxicity and viability of HCT116 , U87 and BxPC3 cells grown in spheroids ( 3D ) and treated as indicated for 24h ( BxPC3 ) or 48h ( HCT116 , U87 ) ( NEN 1 . 2 µM ; Domperidone ( Domp ) , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM ) . Data information : Data are presented as mean ( SD ) ( N = 3 ) and were analyzed by two - way ANOVA with Tukey post - hoc test Significance is indicated for multiple comparisons as indicated . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Ratio of toxicity and viability of HCT116, U87 and BxPC3 cells grown in spheroids (3D) and treated as indicated for 24h (BxPC3) or 48h (HCT116, U87) (NEN 1.2 µM; Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM). Data information: Data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test Significance is indicated for multiple comparisons as indicated. * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Immunoblot analysis of CHOP , ATF4 , phosphorylated ( p - ) and t - ) eIF2alpha ( eIF2a ) of HCT116 cells treated as indicated for 16h ( NEN ( N ) 1 . 2 µM ; Domperidone ( Domp ) , Amitriptyline ( Ami ) , each 30 µM ) .

O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

B Immunoblot analysis of CHOP, ATF4, phosphorylated (p-) and t-) eIF2alpha (eIF2a) of HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM; Domperidone (Domp), Amitriptyline (Ami), each 30 µM).


F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h ( NEN 1 . 2 µM , Domperidone ( Domp ) , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM , ISRIB 1 µM ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O

F Ratio of toxicity and viability in HCT116 spheroids treated as indicated for 48h (NEN 1.2 µM, Domperidone (Domp), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM, ISRIB 1 µM).


A Relative mRNA expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ( ( NEN ( N ) 1 . 2 µM ; Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 30 µM ; Clomipramine ( Clomi ) 20 µM ) , determined by qPCR . Data information : In ( A ) ( first ( TFE3 ) and third ( CD68 ) graph ( N = 3 ) ; second ( TFE3 ) and fourth ( CD68 ) graph ( N = 5 , besides NEN + Clomi ( N = 4 ) and NEN + Desi ( N = 4 ) ) data are presented as mean ( SD ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . In the second and the fourth graph , significance is indicated for the comparison of combinatorial treatments ( NEN + TCAs ) to controls ( Ctrl ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Relative mRNA expression levels of indicated CLEAR network genes in HCT116 cells treated as indicated ((NEN (N) 1.2 µM; Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 30 µM; Clomipramine (Clomi) 20 µM), determined by qPCR. Data information: In (A) (first (TFE3) and third (CD68) graph (N=3); second (TFE3) and fourth (CD68) graph (N=5, besides NEN+Clomi (N=4) and NEN+Desi (N=4)) data are presented as mean (SD) and were analyzed by a one-way ANOVA with Tukey post-hoc test. In the second and the fourth graph, significance is indicated for the comparison of combinatorial treatments (NEN+TCAs) to controls (Ctrl). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Immunofluorescence ( IF ) of HCT116 cells treated with DMSO ( Control ( Ctrl ) ) , NEN , Domperidone ( Domp ) or NEN + Domp as indicated ( NEN , 1 . 2 µM ; Domp , 30 µM ) for 16 h and stained with anti - Lamp1 ( top panel ) , Alexa - 555 LC3 ( middle panel ) co - stained with Alexa - 647 p62 ( bottom panel ) . Nuclei , Blue ( dapi ) . Scale bar 20 µm . Mean ( SEM ) fluorescence of Lamp1 ( Ctrl , NEN , Domp ( N = 20 cells ) ; NEN + Domp ( N = 24 cells ) ) , LC3 - II ( Ctrl , NEN + Domp ( N = 16 cells ) ) ; NEN , Domp ( N = 16 cells ) ) or P62 ( Ctrl ( N = 38 cells ) ; NEN ( N = 27 cells ) ; Domp ( N = 28 cells ) ; NEN + Domp ( N = 21 cells ) ) was quantified and analyzed by one - way ANOVA with Tukey post - hoc test ( for LC3 - II ) or Kruskal - Wallis with Dunn ´ s post - hoc test ( for Lamp1 and p62 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Immunofluorescence (IF) of HCT116 cells treated with DMSO (Control (Ctrl)), NEN, Domperidone (Domp) or NEN+Domp as indicated (NEN, 1.2 µM; Domp, 30 µM) for 16 h and stained with anti-Lamp1 (top panel), Alexa-555 LC3 (middle panel) co-stained with Alexa-647 p62 (bottom panel). Nuclei, Blue (dapi). Scale bar 20 µm. Mean (SEM) fluorescence of Lamp1 (Ctrl, NEN, Domp (N=20 cells); NEN+Domp (N=24 cells)), LC3-II (Ctrl, NEN+Domp (N=16 cells)); NEN, Domp (N=16 cells)) or P62 (Ctrl (N=38 cells); NEN (N=27 cells); Domp (N=28 cells); NEN+Domp (N=21 cells)) was quantified and analyzed by one-way ANOVA with Tukey post-hoc test (for LC3-II) or Kruskal-Wallis with Dunn´s post-hoc test (for Lamp1 and p62). * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control ( siCtrl ) or combined TFE3 - and MITF - targeting ( siTFE3 / siMITF ) siRNAs and treated as indicated ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , ISRIB 1 µM ) , determined by qPCR . Data information : data ( N = 3 ) are presented as mean ( SD ) and were analyzed by two - way ANOVA with Tukey post - hoc

O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Relative UPP1 mRNA expression levels in HCT116 cells either transfected with control (siCtrl) or combined TFE3- and MITF- targeting (siTFE3/siMITF) siRNAs and treated as indicated (NEN (N) 1.2 µM, Domperidone (D) 30 µM, ISRIB 1 µM), determined by qPCR. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h ( NEN ( low ) 0 . 6 µM or NEN ( High ) 1 . 2 µM , Domperidone ( Domp ) 30 µM ) Cells were transfected either with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 48h prior to treatment .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment.


H Ratio of toxicity and viability of HCT116 spheroids ( 3D ) treated as indicated ( NEN 1 . 2 µM , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM ) . Cells were transfected with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 24h prior to spheroid formation . Data information : data ( N = 3 ) are presented as mean ( SD ) and were analyzed by two - way ANOVA with Tukey post - hoc

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM , Clomipramine ( Clomi ) 20 µM ) . Data information : , data are presented as mean ( SD ) ( N = 3 ) and were analyzed by Kruskal - Wallis with Dunn ´ s post - hoc test ( A , left graph ) , one - way ANOVA with Tukey post - hoc test ( A , right graph ) In ( A ) , significance is indicated for the comparison of the different treatments to controls . * * p < 0 , 01 ; * * * * p < 0 , 0001 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM, Clomipramine (Clomi) 20 µM). Data information: , data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001.


D Caspase activity in HCT116 cells treated as indicated for 24h ( NEN 0 . 6 µM , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 20 µM , Clomipramine ( Clomi ) 10 µM , A77126 ( A77 ) 50 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by two - way ANOVA with Tukey post - hoc

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 20 µM, Clomipramine (Clomi) 10 µM, A77126 (A77) 50 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test


A Immunofluorescence ( IF ) images of HCT116 cells treated as indicated for 12h ( Imipramine and Domperidone , each 30 µM ) and stained for cholesterol content using filipin III . B Immunofluorescence ( IF ) images of HCT116 cells treated with Domperidone and co - stained for cholesterol and Lamp1 .

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

A Immunofluorescence (IF) images of HCT116 cells treated as indicated for 12h (Imipramine and Domperidone, each 30 µM) and stained for cholesterol content using filipin III. B Immunofluorescence (IF) images of HCT116 cells treated with Domperidone and co-stained for cholesterol and Lamp1.


E Relative mRNA expression levels of the specified genes in HCT116 cells , treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , Cyclodextrin ( CyD ) 0 . 75 % ) . F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h ( NEN 1 . 2 µM , Domperidone ( Domp ) 30 µM , Cyclodextrin ( CD ) 0 . 75 % ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Relative mRNA expression levels of the specified genes in HCT116 cells, treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (D) 30 µM, Cyclodextrin (CyD) 0.75%). F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h (NEN 1.2 µM, Domperidone (Domp) 30 µM, Cyclodextrin (CD) 0.75%). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A Ratio of toxicity and viability determined in pancreatic cancer - derived organoids from two patients ( ( left ) PDO - 42 and ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for the comparison between single and combined drug treatments as indicated ( NEN 1 . 2 µM or 2 . 5 µM , Amitriptyline ( Ami , A ) , Imipramine ( Imi , I ) , Desipramine ( Desi , D ) , each 30 µM , Clomipramine ( Clomi , C ) 20 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between single and combined drug treatments as indicated (NEN 1.2 µM or 2.5 µM, Amitriptyline (Ami, A), Imipramine (Imi, I), Desipramine (Desi, D), each 30 µM, Clomipramine (Clomi, C) 20 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Ratio of toxicity and viability determined in pancreatic cancer - derived organoids from two patients ( ( left ) PDO - 42 and ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for the comparison between drug treatments as indicated to the control condition ( NEN ( N ) 1 . 2 µM or 2 . 5 µM , Amitriptyline ( A ) , Imipramine ( I ) 20 µM , Paclitaxel ( Pacli ) , 2 , 20 or 200nM ) . As references for sensitization effects to standard chemotherapy , the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue . Bars for controls ( Ctrl ) , Paclitaxel ( Pacli ) , NEN ( N ) alone and combined NEN + Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between drug treatments as indicated to the control condition (NEN (N) 1.2 µM or 2.5 µM, Amitriptyline (A), Imipramine (I) 20 µM, Paclitaxel (Pacli), 2, 20 or 200nM). As references for sensitization effects to standard chemotherapy, the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue. Bars for controls (Ctrl), Paclitaxel (Pacli), NEN (N) alone and combined NEN+Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated. Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


CSF PGRN is increased in mutation carriers ( MC ) compared to non - carriers ( NC ) .

CSF PGRN is increased in mutation carriers (MC) compared to non-carriers (NC).


CSF PGRN is not associated with age in either NC or MC .

CSF PGRN is not associated with age in either NC or MC.


CSF PGRN is increased in males compare to females .

CSF PGRN is increased in males compare to females.


CSF PGRN levels do not differ among MC participants carrying a PSEN1 , PSEN2 or APP mutation .

CSF PGRN levels do not differ among MC participants carrying a PSEN1, PSEN2 or APP mutation.


CSF PGRN as a function of EYO in mutation carriers ( MC , red ) and non - carriers ( NC , blue ) . The solid lines indicate the regression line for each of the groups and the 95 % confidence interval ( CI ) calculated by a linear model adjusting by gender . The interaction term of mutation status and EYO is significant ( P = 0 . 041 ) , also when including PGRN outliers and participants with EYO > + 20 ( P = 0 . 030 ) . Individual data points are not displayed to prevent disclosure of mutation status .

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

CSF PGRN as a function of EYO in mutation carriers (MC, red) and non-carriers (NC, blue). The solid lines indicate the regression line for each of the groups and the 95% confidence interval (CI) calculated by a linear model adjusting by gender. The interaction term of mutation status and EYO is significant (P = 0.041), also when including PGRN outliers and participants with EYO > +20 (P = 0.030). Individual data points are not displayed to prevent disclosure of mutation status.


The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO , in the context of other biomarker and cognitive changes . The curves were generated by the linear model that best fit each marker ( see statistics section and Appendix Table S2 ) . CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset ( shadowed area ) after brain amyloidosis and brain injury ( as measured by CSF T - tau ) have started , and shortly before CSF sTREM2 starts to increase . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; CSF , cerebrospinal fluid ; MC , mutation carriers ; MMSE , Mini - Mental State Examination ; NC , non - carriers ; T - tau , total tau .

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

The graph depicts the standardised differences of CSF PGRN between MCs and NCs as a function of EYO, in the context of other biomarker and cognitive changes. The curves were generated by the linear model that best fit each marker (see statistics section and Appendix Table S2). CSF PGRN is significantly increased in MC compared to NC 10 years before the expected symptom onset (shadowed area) after brain amyloidosis and brain injury (as measured by CSF T-tau) have started, and shortly before CSF sTREM2 starts to increase. Abbreviations: Aβ1-42: amyloid-β 42; CSF, cerebrospinal fluid; MC, mutation carriers; MMSE, Mini-Mental State Examination; NC, non-carriers; T-tau, total tau.


Scatter plot representing the levels of CSF PGRN in healthy controls ( depicted in blue ) and the different stages of the Alzheimer ' s continuum ( depicted in red ) . The analysis and graphs were performed excluding CSF PGRN outliers ( 1 ' healthy control ' , 1 ' Preclinical AD A + / TN - ' , 4 ' AD CDR = 0 . 5 ' and 1 ' AD CDR = 1 ' ) . Including them yielded a similar result and CSF PGRN was still significantly higher in the ' AD CDR = 1 ' group compared to the ' healthy controls ' ( P = 0 . 001 ) and ' Preclinical AD A + / TN - ' ( P = 0 . 0001 ) groups . Abbreviations : A : amyloid - β biomarker status ; AD : Alzheimer ' s disease ; CDR : clinical dementia rating ; CSF , cerebrospinal fluid ; N : neurodegeneration biomarker status ; T : tau pathology biomarker status .

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

Scatter plot representing the levels of CSF PGRN in healthy controls (depicted in blue) and the different stages of the Alzheimer's continuum (depicted in red). The analysis and graphs were performed excluding CSF PGRN outliers (1 'healthy control', 1 'Preclinical AD A+/TN-', 4 'AD CDR = 0.5' and 1 'AD CDR = 1'). Including them yielded a similar result and CSF PGRN was still significantly higher in the 'AD CDR = 1' group compared to the 'healthy controls' (P = 0.001) and 'Preclinical AD A+/TN-' (P = 0.0001) groups. Abbreviations: A: amyloid-β biomarker status; AD: Alzheimer's disease; CDR: clinical dementia rating; CSF, cerebrospinal fluid; N: neurodegeneration biomarker status; T: tau pathology biomarker status.


Scatter plots representing the association of CSF PGRN with different cognitive tests . Only the subjects of the Alzheimer ' s continuum group ( n = 474 ) were included . In all tests studied , higher levels of CSF PGRN were associated with worse cognitive performance ( namely , lower scores in ADNI - Mem , ADNI - EF and MMSE and higher scores in ADAS - Cog11 , ADAS - Cog13 and CDR - SB ) . The analysis and the graphs are excluding PGRN outliers ; including them rendered similar results ( Appendix Table S6 ) . Abbreviations : ADAS - Cog , Alzheimer ' s disease Assessment Scale - cognitive subscale ; ADNI - Mem : ADNI memory composite score ; ADNI - EF : ADNI executive function composite score ; CDR - SB : clinical dementia rating Sum of Boxes ; MMSE , Mini - Mental State Examination .

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

Scatter plots representing the association of CSF PGRN with different cognitive tests. Only the subjects of the Alzheimer's continuum group (n = 474) were included. In all tests studied, higher levels of CSF PGRN were associated with worse cognitive performance (namely, lower scores in ADNI-Mem, ADNI-EF and MMSE and higher scores in ADAS-Cog11, ADAS-Cog13 and CDR-SB). The analysis and the graphs are excluding PGRN outliers; including them rendered similar results (Appendix Table S6). Abbreviations: ADAS-Cog, Alzheimer's disease Assessment Scale - cognitive subscale; ADNI-Mem: ADNI memory composite score; ADNI-EF: ADNI executive function composite score; CDR-SB: clinical dementia rating Sum of Boxes; MMSE, Mini-Mental State Examination.


Scatter plots representing the association of CSF PGRN with temporo - parietal FDG - PET uptake within the subjects of the Alzheimer ' s continuum group ( n = 474 ) . Abbreviations : FDG - PET : Fludeoxyglucose positron emission

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

Scatter plots representing the association of CSF PGRN with temporo-parietal FDG-PET uptake within the subjects of the Alzheimer's continuum group (n = 474). Abbreviations: FDG-PET: Fludeoxyglucose positron emission tomography


Scatter plots representing the association of CSF PGRN with total hippocampal volume ( B ) within the subjects of the Alzheimer ' s continuum group ( n = 474 ) .

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

Scatter plots representing the association of CSF PGRN with total hippocampal volume (B) within the subjects of the Alzheimer's continuum group (n = 474).


Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers ( T - tau , P - tau181P and Aβ1 − 42 ) in non - carriers ( NC , blue ; A , C , E and G ) and in mutation carriers ( MC , red ; B , D , F and H ) . Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95 % confidence interval ( CI ) for each of the groups . The standardized regression coefficients ( β ) and the P - values are shown and were computed using a linear model adjusting for age , gender and APOE ε4 . The sample contained some outliers ( defined as 3 SDs below or above the group mean ) of the CSF core markers of AD . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 .

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

Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in non-carriers (NC, blue; A, C, E and G) and in mutation carriers (MC, red; B, D, F and H). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181.


Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers ( T - tau , P - tau181P and Aβ1 − 42 ) in healthy controls ( blue ; A , D , G and J ) , Alzheimer ' s continuum ( red ; B , E , H and K ) and ' suspected non - Alzheimer ' s pathophysiology ( SNAP ) ' groups ( green ; C , F , I and L ) . Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95 % confidence interval ( CI ) for each of the groups . The standardized regression coefficients ( β ) and the P - values are shown and were computed using a linear model adjusting for age , gender and APOE ε4 . The sample contained some outliers ( defined as 3 SDs below or above the group mean ) of the CSF core markers of AD , the analysis including these outliers yielded similar results ( Appendix Table S8 ) . The Aβ1 - 42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg / ml . We also tested the associations with Aβ1 - 42 values truncated at the upper technical limit and the result was similar . Abbreviations : Aβ1 - 42 : amyloid - β 42 ; T - tau : total tau ; P - tau : tau phosphorylated at Threonine 181 ; SNAP : suspected non - Alzheimer ' s pathophysiology .

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

Scatter plots representing the associations of CSF PGRN with CSF sTREM2 and each of the AD CSF core biomarkers (T-tau, P-tau181P and Aβ1−42) in healthy controls (blue; A, D, G and J), Alzheimer's continuum (red; B, E, H and K) and 'suspected non-Alzheimer's pathophysiology (SNAP)' groups (green; C, F, I and L). Each point depicts the value of CSF PGRN and the corresponding biomarker of a subject and the solid lines indicate the regression line and the 95% confidence interval (CI) for each of the groups. The standardized regression coefficients (β) and the P-values are shown and were computed using a linear model adjusting for age, gender and APOE ε4. The sample contained some outliers (defined as 3 SDs below or above the group mean) of the CSF core markers of AD, the analysis including these outliers yielded similar results (Appendix Table S8). The Aβ1-42 values used for the associations test are those based on an extrapolation curve since the upper technical limit is 1700pg/ml. We also tested the associations with Aβ1-42 values truncated at the upper technical limit and the result was similar. Abbreviations: Aβ1-42: amyloid-β 42; T-tau: total tau; P-tau: tau phosphorylated at Threonine 181; SNAP: suspected non-Alzheimer's pathophysiology.


Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing , indicated by increased % callus mineralization , at day 28 after operation in a dose - dependent manner . Data are presented as mean ± SEM . Treatment with 1 ng TNF versus PBS control , * * * * P < 0 . 0001 by one - way ANOVA with Dunnett ' s multiple comparisons test .

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

Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus mineralization, at day 28 after operation in a dose-dependent manner. Data are presented as mean ± SEM. Treatment with 1 ng TNF versus PBS control, ****P < 0.0001 by one-way ANOVA with Dunnett's multiple comparisons test.


Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing , indicated by % callus mineralization . Data are presented as mean ± SEM . 1 ng TNF versus PBS control treatment on days 0 and 1 , * * * P = 0 . 0009 by one - way ANOVA with Dunnett ' s multiple comparisons test .

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

Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing, indicated by % callus mineralization. Data are presented as mean ± SEM. 1 ng TNF versus PBS control treatment on days 0 and 1, ***P = 0.0009 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with systemic anti - TNF or local rmIL - 10 led to impaired fracture repair at day 28 , indicated by % callus mineralization . Data are presented as mean ± SEM . Treatment with PBS control versus anti - TNF , * P = 0 . 037 , PBS control versus rmIL - 10 , * P = 0 . 037 , by unpaired two - sided t - tests .

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Treatment with systemic anti-TNF or local rmIL-10 led to impaired fracture repair at day 28, indicated by % callus mineralization. Data are presented as mean ± SEM. Treatment with PBS control versus anti-TNF, *P = 0.037, PBS control versus rmIL-10, *P = 0.037, by unpaired two-sided t-tests.


Representative micro - CT images at the fracture site showing ( from left to right ) : lateral view of tibia , cross - section , and cross and longitudinal sections with color overlay and 3D reconstruction . In the color overlays , the shade of blue corresponds to percentage mineralization : light blue denotes soft immature callus , whereas dark blue denotes hard mineralized callus . Scale bar , 2 mm .

Representative micro-CT images at the fracture site showing (from left to right): lateral view of tibia, cross-section, and cross and longitudinal sections with color overlay and 3D reconstruction. In the color overlays, the shade of blue corresponds to percentage mineralization: light blue denotes soft immature callus, whereas dark blue denotes hard mineralized callus. Scale bar, 2 mm.


Representative ISH image ( light field ) showing TNF expression at the murine fracture site at 24 h after fracture . Scale bar , 250 μm . Red box indicates region of interest .

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Representative ISH image (light field) showing TNF expression at the murine fracture site at 24 h after fracture. Scale bar, 250 μm. Red box indicates region of interest.


High - power micrographs of region of interest from ( E ) : at 24 h after fracture , mTNF expression ( white signal on dark field , above ) co - localized with polymorphonuclear cells found on the adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm . Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti - neutrophil elastase .

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High-power micrographs of region of interest from (E): at 24 h after fracture, mTNF expression (white signal on dark field, above) co-localized with polymorphonuclear cells found on the adjacent H&amp;E section (below). Scale bar, 25 μm. Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti-neutrophil elastase.


High - power micrographs : at 7 days , TNF expression ( white signal on dark field , above ) co - localized with F4 / 80 - positive cells ( stained dark brown ) extravasating from a blood vessel on the adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm .

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High-power micrographs: at 7 days, TNF expression (white signal on dark field, above) co-localized with F4/80-positive cells (stained dark brown) extravasating from a blood vessel on the adjacent H&amp;E section (below). Scale bar, 25 μm.


Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model . Compared to control and number of blood neutrophils at 3 h * * * P = 0 . 0006 and at 6 h * P = 0 . 042 , by one - way ANOVA with Dunnett correction .

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

Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model. Compared to control and number of blood neutrophils at 3 h ***P = 0.0006 and at 6 h *P = 0.042, by one-way ANOVA with Dunnett correction.


Depletion of neutrophils using anti - Ly6G . Intravenous anti - Ly6G treatment led to depletion of neutrophils in the systemic circulation ( left ) as well the local peri - fracture soft tissues ( right ) . Left : counts of neutrophils in the blood harvested by cardiac puncture at 3 h post - injury . Data are presented as mean ± SEM ( * * P = 0 . 0074 using unpaired one - tailed t - test ) . Right : counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti - Ly6G antibody versus IgG control at day 1 and day 7 post - fracture . At day 1 , number of neutrophils in control group as detected by Ly6G and NE in control versus treatment : * * * * P = 0 . 0001 for both Ly6G and NE , by unpaired two - tailed t - test . At day 7 , number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups : * * * * P = 0 . 0001 for Ly6G and * P = 0 . 020 for NE , by unpaired two - tailed t - test . Number of neutrophils at day 1 versus day 7 in control groups , * P = 0 . 037 for Ly6G and * * * P = 0 . 0006 for NE , by unpaired two - tailed t - test .

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

Depletion of neutrophils using anti-Ly6G. Intravenous anti-Ly6G treatment led to depletion of neutrophils in the systemic circulation (left) as well the local peri-fracture soft tissues (right). Left: counts of neutrophils in the blood harvested by cardiac puncture at 3 h post-injury. Data are presented as mean ± SEM (**P = 0.0074 using unpaired one-tailed t-test). Right: counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti-Ly6G antibody versus IgG control at day 1 and day 7 post-fracture. At day 1, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment: ****P = 0.0001 for both Ly6G and NE, by unpaired two-tailed t-test. At day 7, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups: ****P = 0.0001 for Ly6G and *P = 0.020 for NE, by unpaired two-tailed t-test. Number of neutrophils at day 1 versus day 7 in control groups, *P = 0.037 for Ly6G and ***P = 0.0006 for NE, by unpaired two-tailed t-test.


Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti - Ly6G and anti - neutrophil elastase primary antibodies at day 1 post - fracture following treatment with anti - Ly6G antibody or IgG control . Scale bar , 100 μm .

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

Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti-Ly6G and anti-neutrophil elastase primary antibodies at day 1 post-fracture following treatment with anti-Ly6G antibody or IgG control. Scale bar, 100 μm.


Depletion of neutrophils using anti - Ly6G led to impaired fracture healing . Representative section stained with Masson ' s trichrome at day 14 comparing fracture healing in mouse treated with isotype control ( top ) versus anti - Ly6G ( bottom ) . Black and white images in the right column are identical to the color images in the left column ; they have been labeled to clearly demonstrate the anatomical structures . Control section shows advanced mineralized callus , while treatment section shows a large immature unmineralized callus . Muscle fibers : red ; collagen , bone , and mineralized callus : green . Scale bar , 1 mm .

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

Depletion of neutrophils using anti-Ly6G led to impaired fracture healing. Representative section stained with Masson's trichrome at day 14 comparing fracture healing in mouse treated with isotype control (top) versus anti-Ly6G (bottom). Black and white images in the right column are identical to the color images in the left column; they have been labeled to clearly demonstrate the anatomical structures. Control section shows advanced mineralized callus, while treatment section shows a large immature unmineralized callus. Muscle fibers: red; collagen, bone, and mineralized callus: green. Scale bar, 1 mm.