( A and B ) Top , TIRF images of fixed EL4 - MyD88 - GFP cells incubated with IL - 1 functionalized SLBs for 30 minutes , and stained with antibodies against pp65 ( A ) or pIKK ( B ) . Scale bar , 5 µm . Region of interest ( red box , merge image ) shows an example of MyD88 - GFP puncta that colocalizes with pp65 ( A ) or pIKK ( B ) puncta . Bottom , 2D histograms of the distribution of MyD88 puncta intensity and associated pp65 ( A ) or pIKK ( B ) staining intensity . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient , R , of linear fit labeled on 2D histograms ) . Blue shaded regions on scatter plot high MyD88 puncta classified as clustered Myddosomes . Bottom right , zoomed images of the region of interest ( red box overlaid merge image , top ) show MyD88 - GFP channel and associated pp65 ( A ) and pIKK ( B ) channel ( pp65 / pIKK images are displayed with Fire LUT ) . Red data points on the 2D histogram are from indicated puncta in the MyD88 - GFP image ( numbered red arrows ) . Scale bar , 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with antibodies against pp65 (A) or pIKK (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that colocalizes with pp65 (A) or pIKK (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated pp65 (A) or pIKK (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded regions on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated pp65 (A) and pIKK (B) channel (pp65/pIKK images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.


( E and F ) 2D histogram of MyD88 - GFP puncta intensity and associated pp65 ( E ) or pIKK ( F ) staining intensity on 2 . 5 µm and 1 µm grids . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient , R , of linear fit labeled on 2D histograms ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) 2D histogram of MyD88-GFP puncta intensity and associated pp65 (E) or pIKK (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms).


( G and H ) Quantification of mean pp65 ( G ) or pIKK ( H ) staining intensity for puncta classifieds as single or clustered Myddosomes , and MyD88 puncta formed on 2 . 5 and 1 µm grids . The normalized mean intensity for clusters , single Myddosomes MyD88 puncta on 2 . 5 and 1 µm grids are the following : for pp65 0 . 318 ± 0 . 044 , 0 . 163 ± 0 . 009 , 0 . 059 ± 0 . 005 and 0 . 057 ± 0 . 008 ; for pIKK 0 . 393 ± 0 . 051 , 0 . 130 ± 0 . 004 , 0 . 037 ± 0 . 006 and 0 . 035 ± 0 . 007 ( a . u . , mean ± SEM , mean value states in the order they appear on plot , left to right ) . Violin plots show the distribution of all segmented MyD88 puncta . Data points superimposed on the violin plots are the averages from independent experiments . p values are * < 0 . 05 , * * * < 0 . 001 , * * * * < 0 . 0001 . Bars represent mean ± SEM ( n = 3 - 4 biological replicates for pp65 , with 10273 , 14009 and 2657 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ; n = 4 - 5 biological replicates for pIKK , with 2375 , 35496 and 59593 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ) . Statistical significance is determined using unpaired two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) Quantification of mean pp65 (G) or pIKK (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clusters, single Myddosomes MyD88 puncta on 2.5 and 1 µm grids are the following: for pp65 0.318 ± 0.044, 0.163 ± 0.009, 0.059 ± 0.005 and 0.057 ± 0.008; for pIKK 0.393 ± 0.051, 0.130 ± 0.004, 0.037 ± 0.006 and 0.035 ± 0.007 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are *<0.05, ***<0.001, ****<0.0001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for pp65, with 10273, 14009 and 2657 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 4 - 5 biological replicates for pIKK, with 2375, 35496 and 59593 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.


( A and B ) Top , TIRF images of fixed EL4 - MyD88 - GFP cells incubated with IL - 1 functionalized SLBs for 30 minutes , and stained with anti - K63 - Ub ( A ) or anti - M1 - Ub ( B ) . Scale bar , 5 µm . Region of interest ( red box , merge image ) shows an example of MyD88 - GFP puncta that is colocalized with K63 - Ub ( A ) or M1 - Ub ( B ) puncta . Bottom , 2D histograms of the distribution of MyD88 puncta intensity and associated K63 - Ub ( A ) or M1 - Ub ( B ) staining intensity . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient , R , of linear fit labeled on 2D histograms ) . Blue shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes . Bottom right , zoomed images of the region of interest ( red box overlaid merge image , top ) show MyD88 - GFP channel and associated K63 - Ub ( A ) and M1 - Ub ( B ) channel ( K63 / M1 - Ub images are displayed with Fire LUT ) . Red data points on the 2D histogram are from indicated puncta in the MyD88 - GFP image ( numbered red arrows ) . Scale bar , 1 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 B-MEASURED_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 O O O O O O O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) Top, TIRF images of fixed EL4-MyD88-GFP cells incubated with IL-1 functionalized SLBs for 30 minutes, and stained with anti-K63-Ub (A) or anti-M1-Ub (B). Scale bar, 5 µm. Region of interest (red box, merge image) shows an example of MyD88-GFP puncta that is colocalized with K63-Ub (A) or M1-Ub (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (A) or M1-Ub (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R, of linear fit labeled on 2D histograms). Blue shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88-GFP channel and associated K63-Ub (A) and M1-Ub (B) channel (K63/M1-Ub images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88-GFP image (numbered red arrows). Scale bar, 1 µm.


( E and F ) 2D histograms of the distribution of MyD88 puncta intensity and associated K63 - Ub ( E ) or M1 - Ub ( F ) staining intensity on 2 . 5 µm and 1 µm grids . Linear fit is shown as a blue line superimposed on 2D histograms ( Pearson correlation coefficient of linear fit labeled on 2D histograms ) .

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

(E and F) 2D histograms of the distribution of MyD88 puncta intensity and associated K63-Ub (E) or M1-Ub (F) staining intensity on 2.5 µm and 1 µm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient of linear fit labeled on 2D histograms).


( G and H ) Quantification of mean K63 - Ub ( G ) or M1 - Ub ( H ) staining intensity for puncta classifieds as single or clustered Myddosomes , and MyD88 puncta formed on 2 . 5 and 1 µm grids . The normalized mean intensity for clustered , single Myddosomes , and MyD88 puncta on 2 . 5 and 1 µm grids are the following : for K63 - Ub 0 . 444 ± 0 . 030 , 0 . 257 ± 0 . 025 , 0 . 113 ± 0 . 015 and 0 . 104 ± 0 . 008 ; for M1 - Ub 0 . 520 ± 0 . 020 , 0 . 183 ± 0 . 008 , 0 . 174 ± 0 . 022 and 0 . 153 ± 0 . 016 ( a . u . , mean ± SEM , mean value states in the order they appear on plot , left to right ) . Violin plots show the distribution of all segmented MyD88 puncta . Data points superimposed on the violin plots are the averages from independent experiments . p values are * * < 0 . 01 , * * * < 0 . 001 . Bars represent mean ± SEM ( n = 3 - 4 biological replicates for K63 , with 14571 , 27494 and 24026 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ; n = 3 - 4 biological replicates for M1 , with 3114 , 6091 and 1844 puncta off grid , on 2 . 5 µm and 1 µm grid measured in total across all replicates ) . Statistical significance is determined using unpaired two - tailed Student ' s t - test .

O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) Quantification of mean K63-Ub (G) or M1-Ub (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 µm grids. The normalized mean intensity for clustered, single Myddosomes, and MyD88 puncta on 2.5 and 1 µm grids are the following: for K63-Ub 0.444 ± 0.030, 0.257 ± 0.025, 0.113 ± 0.015 and 0.104 ± 0.008; for M1-Ub 0.520 ± 0.020, 0.183 ± 0.008, 0.174 ± 0.022 and 0.153 ± 0.016 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. p values are **<0.01, ***<0.001. Bars represent mean ± SEM (n = 3 - 4 biological replicates for K63, with 14571, 27494 and 24026 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates; n = 3 - 4 biological replicates for M1, with 3114, 6091 and 1844 puncta off grid, on 2.5 µm and 1 µm grid measured in total across all replicates). Statistical significance is determined using unpaired two-tailed Student's t-test.


( A and B ) Top : TIRF images of MyD88 - GFP and mScarlet - TRAF6 ( A ) or mScarlet - HOIL1 ( B ) . Region of interest ( yellow box , merge image ) shows an example of a MyD88 - GFP puncta colocalized with mScarlet - TRAF6 ( A ) or mScalet - HOIL1 ( B ) . Bottom : Time - series TIRF images from the region of interest ( left ) and fluorescence intensity times series ( right ) of MyD88 and TRAF6 ( A ) or HOIL1 ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Top: TIRF images of MyD88-GFP and mScarlet-TRAF6 (A) or mScarlet-HOIL1 (B). Region of interest (yellow box, merge image) shows an example of a MyD88-GFP puncta colocalized with mScarlet-TRAF6 (A) or mScalet-HOIL1 (B). Bottom: Time-series TIRF images from the region of interest (left) and fluorescence intensity times series (right) of MyD88 and TRAF6 (A) or HOIL1 (B).


( C and D ) Quantifications of percentage of MyD88 - GFP puncta that colocalized with TRAF6 ( C ) or HOIL1 ( D ) grouped for all puncta , puncta containing ≤ 1x , > 1x and ≥ 2x Myddosome complexes . The percentages for TRAF6 ( C ) in these groups are 15 . 4 ± 2 . 4 % , 6 . 1 ± 1 . 8 % , 36 . 2 ± 5 . 2 % and 58 . 4 ± 5 . 9 % , respectively ( mean ± SEM ) . The percentages for HOIL1 ( D ) in these groups are 8 . 6 ± 0 . 5 % , 0 . 4 ± 0 . 2 % , 14 . 9 ± 1 . 9 % and 25 . 9 ± 5 . 0 % , respectively ( mean ± SEM ) . Violin plots indicate the distribution of individual cell measurements . Colored dots superimposed on the violin plots are the averages from independent experiments . Bars represent mean ± SEM ( n = 6 biological replicates for TRAF6 , with 191 cells measured in total across all replicates ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Quantifications of percentage of MyD88-GFP puncta that colocalized with TRAF6 (C) or HOIL1 (D) grouped for all puncta, puncta containing ≤1x, >1x and ≥2x Myddosome complexes. The percentages for TRAF6 (C) in these groups are 15.4 ± 2.4%, 6.1 ± 1.8%, 36.2 ± 5.2% and 58.4 ± 5.9%, respectively (mean ± SEM). The percentages for HOIL1 (D) in these groups are 8.6 ± 0.5%, 0.4 ± 0.2%, 14.9 ± 1.9% and 25.9 ± 5.0%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 6 biological replicates for TRAF6, with 191 cells measured in total across all replicates,


( A and B ) TIRF images of EL4 cells expressing MyD88 - GFP and mScarlet - TRAF6 stimulated on IL - 1 functionalized SLBs at a ligand density of 1 IL - 1 / µm2 off grids ( A ) or on 1 µm grids ( B ) . Kymographs derived from dashed lines overlaid TIRF images ( left panel ) . Scale bars , 5 µm . ( C ) Quantification of percentage of MyD88 - GFP puncta that colocalized with TRAF6 off grids and on 1 µm grids at a ligand density of 1 IL - 1 / µm2 , and the percentages are 16 . 2 ± 2 . 5 % and 6 . 1 ± 1 . 5 % , respectively ( mean ± SEM ) . Violin plots indicate the distribution of individual cell measurements . Colored dots superimposed on the violin plots are the averages from independent experiments . Bars represent mean ± SEM ( n = 4 experimental replicates off grids , with a total of 24315 MyD88 puncta from 91 cells ; n = 3 biological replicates on 1 µm grids , with a total of 23161 MyD88 puncta from 70 cells ) . Statistical significance is determined using unpaired two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) TIRF images of EL4 cells expressing MyD88-GFP and mScarlet-TRAF6 stimulated on IL-1 functionalized SLBs at a ligand density of 1 IL-1/µm2 off grids (A) or on 1 µm grids (B). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 µm. (C) Quantification of percentage of MyD88-GFP puncta that colocalized with TRAF6 off grids and on 1 µm grids at a ligand density of 1 IL-1/µm2, and the percentages are 16.2 ± 2.5% and 6.1 ± 1.5%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 4 experimental replicates off grids, with a total of 24315 MyD88 puncta from 91 cells; n = 3 biological replicates on 1 µm grids, with a total of 23161 MyD88 puncta from 70 cells). Statistical significance is determined using unpaired two-tailed Student's t-test.


( A - B ) Histogram showing the lifetime of TRAF6 and HOIL1 recruitment to single Myddsomes , and clusters containing 2 - 4 and 5 - 7 Myddosome complexes , off ( blue ) and on ( red ) 1 µm grids .

O O O O O O O O O O O O O O O O O O O O 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-B) Histogram showing the lifetime of TRAF6 and HOIL1 recruitment to single Myddsomes, and clusters containing 2-4 and 5-7 Myddosome complexes, off (blue) and on (red) 1 µm grids.


A A heatmap showing the differential miRNAs expression as determined by miRNA - seq in progerin - expressing cells relative to control cells , P < 0 . 05 . Lowly expressed miRNAs were shown in blue , highly expressed ones in red .

A A heatmap showing the differential miRNAs expression as determined by miRNA-seq in progerin-expressing cells relative to control cells, P< 0.05. Lowly expressed miRNAs were shown in blue, highly expressed ones in red.


B Volcano Plot of top 10 miRNAs upregulated and 2 miRNAs downregulated in HGPS cells compared to control .

B Volcano Plot of top 10 miRNAs upregulated and 2 miRNAs downregulated in HGPS cells compared to control.


C The level of top 10 upregulated miRNAs in HGPS cells was assessed by qPCR .

C The level of top 10 upregulated miRNAs in HGPS cells was assessed by qPCR.


D The level of novel - miR - 27 , miR - 3656 and miR - 59 in HGPS patient cells ( HGAFDFN003 p21 and HGAFDFN167 p19 ) versus control ( CRL - 1474 cells p28 ) was assessed by qPCR .

O O O O O O O O O O O O O O O O O O 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 The level of novel-miR-27, miR-3656 and miR-59 in HGPS patient cells (HGAFDFN003 p21 and HGAFDFN167 p19) versus control (CRL-1474 cells p28) was assessed by qPCR.


HGAFDFN003 cells were transfected with anti - miR - ctrl , anti - miR - 3656 or anti - miR - 59 for 5 days , cells were subjected to SA - β - gal staining .

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

HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days, cells were subjected to SA-β-gal staining.


HGAFDFN003 cells were transfected with anti - miR - ctrl , anti - miR - 3656 or anti - miR - 59 for 5 days , The percentage of the Ki67 positive cells was calculated by immunofluorescence ( F ) .

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

HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days, The percentage of the Ki67 positive cells was calculated by immunofluorescence (F).


HGAFDFN003 cells were transfected with anti - miR - ctrl , anti - miR - 3656 or anti - miR - 59 for 5 days The level of cyclin A2 and lamin B1 was detected by western blot ( G ) .

HGAFDFN003 cells were transfected with anti-miR-ctrl, anti-miR-3656 or anti-miR-59 for 5 days The level of cyclin A2 and lamin B1 was detected by western blot (G).


Progerin - expressing CRL - 1474 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days . The percentage of SA - β - gal positive cells was calculated . Scale bars : 100 μm ( H ) .

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

Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The percentage of SA-β-gal positive cells was calculated. Scale bars: 100 μm (H).


Progerin - expressing CRL - 1474 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days . The percentage of the Ki67 positive cells was calculated by immunofluorescence ( I ) .

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

Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The percentage of the Ki67 positive cells was calculated by immunofluorescence (I).


Progerin - expressing CRL - 1474 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days . The level of cyclin A2 and lamin B1 was detected by western blot ( J ) .

Progerin-expressing CRL-1474 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days. The level of cyclin A2 and lamin B1 was detected by western blot (J).


K The level of miR - 59 was detected in 88 - and 92 - year - old cells versus 7 - year - old cells was assessed by qPCR .

O O O O O O O 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 The level of miR-59 was detected in 88- and 92-year-old cells versus 7-year-old cells was assessed by qPCR.


L 7 - year - old cells and 88 - year - old cells were transfected with anti - miR - ctrl or anti - miR - 59 , the level of cyclin A2 and lamin B1 was detected .

L 7-year-old cells and 88-year-old cells were transfected with anti-miR-ctrl or anti-miR-59, the level of cyclin A2 and lamin B1 was detected.


HGAFDFN003 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days , the mRNA level of miR - 59 - targeting potential genes was assessed by qPCR ( B ) .

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

HGAFDFN003 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days, the mRNA level of miR-59-targeting potential genes was assessed by qPCR (B).


HGAFDFN003 cells were transfected with anti - miR - ctrl or anti - miR - 59 for 5 days , The protein expression of HMGA1 and HMGA2 was detected by western blot ( C ) .

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

HGAFDFN003 cells were transfected with anti-miR-ctrl or anti-miR-59 for 5 days, The protein expression of HMGA1 and HMGA2 was detected by western blot (C).


D , After transfection with anti - miR - ctrl or anti - miR - 59 in progerin - expressing CRL - 1474 cells , the protein expression ( D ) of HMGAs were detected .

D, After transfection with anti-miR-ctrl or anti-miR-59 in progerin-expressing CRL-1474 cells, the protein expression (D) of HMGAs were detected.


After transfection with anti - miR - ctrl or anti - miR - 59 in progerin - expressing CRL - 1474 cells , mRNA level ( E ) of HMGAs were detected .

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

After transfection with anti-miR-ctrl or anti-miR-59 in progerin-expressing CRL-1474 cells, mRNA level (E) of HMGAs were detected.


Western blot analysis the expression of HMGAs ( F ) in miR - ctrl or miR - 59 mimics - transfected CRL - 1474 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

Western blot analysis the expression of HMGAs (F) in miR-ctrl or miR-59 mimics-transfected CRL-1474 cells.


qPCR analysis the mRNA levels ( G ) in miR - ctrl or miR - 59 mimics - transfected CRL - 1474 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

qPCR analysis the mRNA levels (G) in miR-ctrl or miR-59 mimics-transfected CRL-1474 cells.


Compared HGPS patient cells ( HGAFDFN003 p22 and HGAFDFN167 p20 ) with control ( CRL - 1474 p26 ) , the protein expression ( H ) of HMGAs were assessed .

Compared HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL-1474 p26), the protein expression (H) of HMGAs were assessed.


Compared HGPS patient cells ( HGAFDFN003 p22 and HGAFDFN167 p20 ) with control ( CRL - 1474 p26 ) , mRNA level ( I ) of HMGAs were assessed .

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Compared HGPS patient cells (HGAFDFN003 p22 and HGAFDFN167 p20) with control (CRL-1474 p26), mRNA level (I) of HMGAs were assessed.


The protein ( J ) level of HMGAs were detected in 7 , 88 and 92 - year - old cells .

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The protein (J) level of HMGAs were detected in 7, 88 and 92-year-old cells.


mRNA ( K ) level of HMGAs were detected in 7 , 88 and 92 - year - old cells .

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mRNA (K) level of HMGAs were detected in 7, 88 and 92-year-old cells.


Transfected anti - miR - ctrl or anti - miR - 59 in 88 - year - old cells , the level of HMGAs was detected by Western blot ( L )

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Transfected anti-miR-ctrl or anti-miR-59 in 88-year-old cells, the level of HMGAs was detected by Western blot (L)


Transfected anti - miR - ctrl or anti - miR - 59 in 88 - year - old cells , the level of HMGAs was detected by qPCR ( M ) .

Transfected anti-miR-ctrl or anti-miR-59 in 88-year-old cells, the level of HMGAs was detected by qPCR (M).


N , O The binding sites of miR - 59 on HMGAs 3 ′ UTR were analyzed by TargetScan and miRanda . Luciferase experiments with the wild - type and the mutated 3 ′ UTR of HMGA1 ( N ) / HMGA2 ( O ) .

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N, O The binding sites of miR-59 on HMGAs 3′ UTR were analyzed by TargetScan and miRanda. Luciferase experiments with the wild-type and the mutated 3′ UTR of HMGA1 (N) / HMGA2 (O).


In Flag - HMGA1 or Flag - HMGA2 infected CRL - 1474 and HGPS cells ( HGAFDFN003 ) , the level of cyclin A2 , lamin B1 was detected by western blot ( A ) .

In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), the level of cyclin A2, lamin B1 was detected by western blot (A).


In Flag - HMGA1 or Flag - HMGA2 infected CRL - 1474 and HGPS cells ( HGAFDFN003 ) , SA - β - gal staining were performed , the percentage of SA - β - gal positive cells was calculated .

In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), SA-β-gal staining were performed, the percentage of SA-β-gal positive cells was calculated.


In Flag - HMGA1 or Flag - HMGA2 infected CRL - 1474 and HGPS cells ( HGAFDFN003 ) , Expression of Ki67 was analyzed by immunofluorescence . Scale bars : 50 μm ( C ) .

In Flag-HMGA1 or Flag-HMGA2 infected CRL-1474 and HGPS cells (HGAFDFN003), Expression of Ki67 was analyzed by immunofluorescence. Scale bars: 50 μm (C).


Progerin - expressing CRL - 1474 cells were transfected with His - control or His - HMGA1 . SA - β - gal staining was performed . The percentage of SA - β - gal positive cells was calculated . Scale bars : 100 μm ( D ) .

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Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. SA-β-gal staining was performed. The percentage of SA-β-gal positive cells was calculated. Scale bars: 100 μm (D).


Progerin - expressing CRL - 1474 cells were transfected with His - control or His - HMGA1 . The level of cyclin A2 , lamin B1 was detected by western blot ( E ) .

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Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. The level of cyclin A2, lamin B1 was detected by western blot (E).


Infection with control shRNA ( shCtrl ) or HMGA1 / HMGA2 shRNA ( shHMGA1 and shHMGA2 ) in anti - miR - 59 - transfected HGPS cells ( HGAFDFN003 ) , cells were subjected to SA - β - gal staining . Scale bars : 100 μm ( F ) . Cells were subjected to immunofluorescence using anti - Ki67 ( red ) antibody ( left ) . The percentage of the Ki67 positive cells was calculated . Scale bars : 50 μm ( G ) .

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Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti-miR-59-transfected HGPS cells (HGAFDFN003), cells were subjected to SA-β-gal staining. Scale bars: 100 μm (F). Cells were subjected to immunofluorescence using anti-Ki67(red) antibody (left). The percentage of the Ki67 positive cells was calculated. Scale bars: 50 μm (G).


Infection with control shRNA ( shCtrl ) or HMGA1 / HMGA2 shRNA ( shHMGA1 and shHMGA2 ) in anti - miR - 59 - transfected HGPS cells ( HGAFDFN003 ) The level of cyclin A2 , lamin B1 , HMGA1 and HMGA2 was detected by western blot ( H ) .

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Infection with control shRNA (shCtrl) or HMGA1/HMGA2 shRNA (shHMGA1 and shHMGA2) in anti-miR-59-transfected HGPS cells (HGAFDFN003) The level of cyclin A2, lamin B1, HMGA1 and HMGA2 was detected by western blot (H).


A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co - IP assay using anti - HMGA1 antibody in H1299 cells . Pulled - down protein complexes were analyzed . Benzonase ( B ) was added in cell lysate . B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co - IP assay using anti - HMGA2 antibody in H1299 cells . Pulled - down protein complexes were analyzed .

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A Interaction of endogenous HMGA1 with HMGA2 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA1 antibody in H1299 cells. Pulled-down protein complexes were analyzed. Benzonase (B) was added in cell lysate. B Interaction of endogenous HMGA2 with HMGA1 and TFIIH4 complex was analyzed by Co-IP assay using anti-HMGA2 antibody in H1299 cells. Pulled-down protein complexes were analyzed.


D Interaction of endogenous CDK7 with HMGAs , XPB and TFIIH4 was analyzed by Co - IP assay using anti - CDK7 antibody in H1299 cells . Pulled - down protein complexes were analyzed .

D Interaction of endogenous CDK7 with HMGAs, XPB and TFIIH4 was analyzed by Co-IP assay using anti-CDK7 antibody in H1299 cells. Pulled-down protein complexes were analyzed.


HEK293T cells were transfected with Flag - HMGA1 ( E ) HMGA1 was IP with anti - Flag antibody and anti - CDK7 antibody .

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HEK293T cells were transfected with Flag-HMGA1 (E) HMGA1 was IP with anti-Flag antibody and anti-CDK7 antibody.


HEK293T cells were transfected with CDK7 ( F ) . HMGA1 or CDK7 was IP with anti - Flag antibody and anti - CDK7 antibody .

HEK293T cells were transfected with CDK7 (F). HMGA1 or CDK7 was IP with anti-Flag antibody and anti-CDK7 antibody.


G HEK293T cell lysates were incubated with GST or GST - HMGA1 sepharose beads . Pulled - down protein complexes were analyzed .

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G HEK293T cell lysates were incubated with GST or GST-HMGA1 sepharose beads. Pulled-down protein complexes were analyzed.


I HEK293T cells were transfected with Flag - HMGA1 deletion mutants . IP of Flag was performed . Pulled - down protein complexes were analyzed .

I HEK293T cells were transfected with Flag-HMGA1 deletion mutants. IP of Flag was performed. Pulled-down protein complexes were analyzed.


J HEK293T cell lysates were incubated with GST or GST - HMGA1 deletion mutants sepharose beads . Recombinant proteins from pull - down assays visualized by silver staining and western blot .

J HEK293T cell lysates were incubated with GST or GST-HMGA1 deletion mutants sepharose beads. Recombinant proteins from pull-down assays visualized by silver staining and western blot.


K , L Progerin - expressing H1299 cells were transfected with His - control or His - HMGA1 . Immunoprecipitation of RNA Pol II ( K ) or CDK7 ( L ) with anti - RNAPII antibody or anti - CDK7 antibody was performed , respectively . Pulled - down protein complexes were analyzed .

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K, L Progerin-expressing H1299 cells were transfected with His-control or His-HMGA1. Immunoprecipitation of RNA Pol II (K) or CDK7 (L) with anti-RNAPII antibody or anti-CDK7 antibody was performed, respectively. Pulled-down protein complexes were analyzed.


RNAPII , the RNAPII Ser2P and RNAPII Ser5P in HGAFDFN003 cells ( M ) compared to CRL - 1474 cells were detected by western blot .

RNAPII, the RNAPII Ser2P and RNAPII Ser5P in HGAFDFN003 cells (M) compared to CRL-1474 cells were detected by western blot.


RNAPII , the RNAPII Ser2P and RNAPII Ser5P in 88 , 92 - year - old cells ( N ) compared to CRL - 1474 cells were detected by western blot .

RNAPII, the RNAPII Ser2P and RNAPII Ser5P in 88, 92-year-old cells (N) compared to CRL-1474 cells were detected by western blot.


O Progerin - expressing CRL - 1474 cells were transfected with His - control or His - HMGA1 . Indicated proteins and modifications were detected by western blot .

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O Progerin-expressing CRL-1474 cells were transfected with His-control or His-HMGA1. Indicated proteins and modifications were detected by western blot.


P Infection with siCtrl or siCDK7 in Flag - HMGA1 - transfected HGPS cells ( HGAFDFN003 ) , cyclin A1 , RNAPII , the RNAPII Ser2P and RNAPII Ser5P was detected .

P Infection with siCtrl or siCDK7 in Flag-HMGA1-transfected HGPS cells (HGAFDFN003), cyclin A1, RNAPII, the RNAPII Ser2P and RNAPII Ser5P was detected.


Q Infection with control shRNA ( shCtrl ) or HMGA1 shRNA ( shHMGA1 ) in anti - miR - 59 - transfected HGAFDFN003 cells . Indicated proteins and modifications were detected by western blot .

Q Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti-miR-59-transfected HGAFDFN003 cells. Indicated proteins and modifications were detected by western blot.


The mRNA level of cell cycle genes in HGPS patient cells ( HGAFDFN003 and HGAFDFN167 ) ( D ) was assessed by qPCR .

The mRNA level of cell cycle genes in HGPS patient cells (HGAFDFN003 and HGAFDFN167) (D) was assessed by qPCR.


The mRNA level of cell cycle genes in HGPS and 88 - and 92 - year - old cells ( E ) was assessed by qPCR .

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The mRNA level of cell cycle genes in HGPS and 88- and 92-year-old cells (E) was assessed by qPCR.


F HGAFDFN003 cells were infected with His - HMGA1 and the mRNA level of cell cycle genes was assessed by qPCR .

F HGAFDFN003 cells were infected with His-HMGA1 and the mRNA level of cell cycle genes was assessed by qPCR.


G Infection with control shRNA ( shCtrl ) or HMGA1 shRNA ( shHMGA1 ) in anti - miR - 59 - transfected HGAFDFN003 cells . The mRNA level of cell cycle genes was assessed by qPCR .

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G Infection with control shRNA (shCtrl) or HMGA1 shRNA (shHMGA1) in anti-miR-59-transfected HGAFDFN003 cells. The mRNA level of cell cycle genes was assessed by qPCR.


A miR - 59 was detected in heart , liver , lung , kidney , skin , muscle , and brain of LmnaG609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group ) .

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A miR-59 was detected in heart, liver, lung, kidney, skin, muscle, and brain of LmnaG609G/G609G mice by qPCR at 12 weeks of life (n=5 for each group).


B , C The mRNA level of Hmga1 ( B ) and Hmga2 ( C ) was detected in heart , liver , lung , kidney , skin , muscle and brain of LmnaG609G / G609G mice by qPCR at 12 weeks of life ( n = 5 for each group ) .

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B, C The mRNA level of Hmga1 (B) and Hmga2 (C) was detected in heart, liver, lung, kidney, skin, muscle and brain of LmnaG609G/G609G mice by qPCR at 12 weeks of life (n=5 for each group).


D Transfected anti - miR - Ctrl or anti - miR - 59 in progerin - expressing NIH3T3 cells . Western blot analysis of HMGAs .

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D Transfected anti-miR-Ctrl or anti-miR-59 in progerin-expressing NIH3T3 cells. Western blot analysis of HMGAs.


G , H Luciferase experiments with the wild - type and the mutated 3 ′ UTR of Hmga1 and Hmga2 .

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G, H Luciferase experiments with the wild-type and the mutated 3′ UTR of Hmga1 and Hmga2.


A The in vivo therapy scheme . Eight - day - old mice were injected intraperitoneally with AAV9 - mCherry - anti - miR - 59 ( 5x1011 viral particles ) . The mCherry signals were detected 30 days post - injection ( DPI ) in LmnaG609G / G609G mice versus PBS - injected control ( dorsal ) .

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A The in vivo therapy scheme. Eight-day-old mice were injected intraperitoneally with AAV9-mCherry-anti-miR-59 (5x1011 viral particles). The mCherry signals were detected 30 days post-injection (DPI) in LmnaG609G/G609G mice versus PBS-injected control (dorsal).


B Expression of the mCherry reporter in different organs at 30DPI in LmnaG609G / G609G mice versus PBS - injected control . Hrt , Mscl , Ver , Kid , Liv : heart , Muscle , vertebrae , kidney , liver . AAV9 - mCherry - anti - miR - 59 was injected into 8 - day - old mice and detected at different points .

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B Expression of the mCherry reporter in different organs at 30DPI in LmnaG609G/G609G mice versus PBS-injected control. Hrt, Mscl, Ver, Kid, Liv: heart, Muscle, vertebrae, kidney, liver. AAV9-mCherry-anti-miR-59 was injected into 8-day-old mice and detected at different points.


C , D The gross morphology of the wild - type and LmnaG609G / G609G mice which injected with anti - ctrl or anti - miR - 59 for 14 weeks .

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C, D The gross morphology of the wild-type and LmnaG609G/G609G mice which injected with anti-ctrl or anti-miR-59 for 14 weeks.


E Progression of body weight of male mice transduced with anti - Ctrl versus anti - miR - 59 was detected from 4 weeks of life . The sample size ( n ) indicates the initial number of mice at week 4 , initial n = 10 WT ; n = 9 anti - Ctrl - transduced mice ; n = 7 anti - miR - 59 - transduced mice . Shown are mean values ± standard deviations , two - sided t - test was used when comparing anti - Ctrl - transduced mice and anti - miR - 59 - transduced mice from week 6 - 16 .

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E Progression of body weight of male mice transduced with anti-Ctrl versus anti-miR-59 was detected from 4 weeks of life. The sample size (n) indicates the initial number of mice at week 4, initial n=10 WT; n=9 anti-Ctrl-transduced mice; n=7 anti-miR-59-transduced mice. Shown are mean values ± standard deviations, two-sided t-test was used when comparing anti-Ctrl-transduced mice and anti-miR-59-transduced mice from week 6-16.


F Body weight comparison at 7 - and 11 - week - old male mice ( n = 9 anti - Ctrl - transduced mice ; n = 7 anti - miR - 59 - transduced mice ) . Data are presented as the mean ± SD ( * P < 0 . 05 , * * P < 0 . 01 ) . P - values were calculated by unpaired Student ' s t - test .

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F Body weight comparison at 7- and 11-week-old male mice (n=9 anti-Ctrl-transduced mice; n=7 anti-miR-59-transduced mice). Data are presented as the mean ± SD (*P<0.05, **P<0.01). P-values were calculated by unpaired Student's t-test.


G Progression of body weight of female mice transduced with anti - Ctrl versus anti - miR - 59 was detected from four weeks of life . The sample size ( n ) indicates the initial number of mice at week 4 , initial n = 5 WT ; n = 6 anti - Ctrl - transduced mice ; n = 6 anti - miR - 59 - transduced mice . Shown are mean values ± standard deviations , two - sided t - test was used when comparing anti - Ctrl - transduced mice and anti - miR - 59 - transduced mice from week 6 - 16 .

G Progression of body weight of female mice transduced with anti-Ctrl versus anti-miR-59 was detected from four weeks of life. The sample size (n) indicates the initial number of mice at week 4, initial n=5 WT; n=6 anti-Ctrl-transduced mice; n=6 anti-miR-59-transduced mice. Shown are mean values ± standard deviations, two-sided t-test was used when comparing anti-Ctrl-transduced mice and anti-miR-59-transduced mice from week 6-16.


H Body weight comparison at 7 - and 11 - week - old female mice ( n = 6 for each group ) . Data are presented as the mean ± SD ( * P < 0 . 05 ) . P - values were calculated by unpaired Student ' s t - test .

H Body weight comparison at 7- and 11-week-old female mice (n=6 for each group). Data are presented as the mean ± SD (*P<0.05). P-values were calculated by unpaired Student's t-test.


I Kaplan - Meier survival plot of the wild - type and LmnaG609G / G609G mice which injected with anti - Ctrl or anti - miR - 59 ( n = 12 WT ; n = 13 anti - Ctrl - transduced mice ; n = 13 anti - miR - 59 - transduced mice , two - sided log - rank test ) .

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I Kaplan-Meier survival plot of the wild-type and LmnaG609G/G609G mice which injected with anti-Ctrl or anti-miR-59 (n=12 WT; n=13 anti-Ctrl-transduced mice; n=13 anti-miR-59-transduced mice, two-sided log-rank test).


A Running ability of male mice transduced with anti - Ctrl versus anti - miR - 59 at 12 weeks of life ( n = 5 for each group ) .

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A Running ability of male mice transduced with anti-Ctrl versus anti-miR-59 at 12 weeks of life (n=5 for each group).


Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle ( B ) The boxes and arrows denoted the aortic adventitia .

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Masson staining showed the perivascular interstitial fibrosis in the heart and quadriceps muscle (B) The boxes and arrows denoted the aortic adventitia.


Masson staining showed the adventitial fibrosis ( blue areas ) and the adventitial width of aorta ( C ) . The boxes and arrows denoted the aortic adventitia .

Masson staining showed the adventitial fibrosis (blue areas) and the adventitial width of aorta (C). The boxes and arrows denoted the aortic adventitia.


D H & E staining in skin showed the epidermis and fat layer of skin . Scale bars , 150 μm . The boxes and arrows denoted epidermis and vertical bars indicated fat layer .

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D H&E staining in skin showed the epidermis and fat layer of skin. Scale bars, 150 μm. The boxes and arrows denoted epidermis and vertical bars indicated fat layer.


E The level of miR - 59 was detected in heart , liver , lung , kidney , skin , and muscle of LmnaG609G / G609G mice ( n = 5 for each group ) . F , G The mRNA level of Hmga1 ( F ) and Hmga2 ( G ) was detected in heart , liver , lung , kidney , skin , and muscle of LmnaG609G / G609G mice ( n = 5 for each group ) . H Hmgas immunohistochemistry in skin of WT mice and LmnaG609G / G609G mice . Scale bars , 50 μm .

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E The level of miR-59 was detected in heart, liver, lung, kidney, skin, and muscle of LmnaG609G/G609G mice (n=5 for each group). F, G The mRNA level of Hmga1 (F) and Hmga2 (G) was detected in heart, liver, lung, kidney, skin, and muscle of LmnaG609G/G609G mice (n=5 for each group). H Hmgas immunohistochemistry in skin of WT mice and LmnaG609G/G609G mice. Scale bars, 50 μm. D


Percentage of the total signal intensity attributed to phosphopeptides ( blue bars ; 11 unique peptide ions ) and non - phosphorylated peptides ( grey bars ; 25 unique peptide ions ) following SAX fractionation at ( A ) pH 6 . 0 , ( B ) pH 6 . 8 and ( C ) pH 8 . 0 , with later fractions consisting of up to 100 % phosphopeptide ion signal . Representative pHis - containing myoglobin peptide ( LFTGHPETLEK ; solid line ) , and its non - phosphorylated counterpart ( LFTGHPETLEK ; dashed line ) , quantified across all 16 SAX fractions ; SAX was performed at pH 6 . 0 ( red ) , pH 6 . 8 ( blue ) or pH 8 . 0 ( green ) . % of the total peak area of each individual peptide across the gradient is plotted for each fraction . In order to assess pHis stability and effects of SAX separation in a complex mixture ( black ) , SAX was also repeated at pH 6 . 8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion . Data for the five pHis - containing myoglobin peptides is shown in Fig . EV3 .

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Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3.


siRNA - mediated knockdown of PHPT1 in HeLa cells ( 24 h ) with reference to GAPDH loading control . NT - non - targeting siRNA control .

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siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control.


Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) .

Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm).


Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) . Base peak chromatograms are shown for select SAX fractions following high - resolution LC - MS / MS using an Orbitrap Fusion mass spectrometer : ( D ) fraction 3 ( green ) ; ( E ) fraction 6 ( red ) ; ( F ) fraction 10 ( grey ) . Peptides were fragmented by HCD , with neutral loss of 98 amu from the precursor ions triggering EThCD ( Ferries et al , 2017 ) . Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot . The ptmRS node was used for phosphosite localisation . Analysis was performed on three independent biological replicates for each condition ( NT or PHPT1 siRNA ) .

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Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).


Comparison of triplet score ( ≥ 5 % S / N ) following HCD of unique phosphopeptides ( 5 % FDR ) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides . Site of phosphorylation ( ptmRS score ≥ 0 . 90 ) is indicated . % of phosphopeptides exhibiting either no neutral loss ( Triplet = 0 ) , or neutral loss of any 1 ( Triplet = 1 ) , 2 ( Triplet = 2 ) , or 3 ions from the precursor ( ∆ 80 , ∆ 98 and / or ∆ 116 amu ) . Distribution of triplet score following HCD fragmentation as a function of site localisation confidence ( ptmRS score ≥ 0 . 75 , ≥ 0 . 90 , ≥ 0 . 95 , ≥ 0 . 99 ) for unique singly phosphorylated peptide as a function of the residue phosphorylated . Numbers of phosphopeptides ( ptmRS ≥ 0 . 90 ) exhibiting different combinations of the three neutral loss species ( ∆ 80 , ∆ 98 and / or ∆ 116 amu from precursor ) for each of the phosphorylated residues .

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

Comparison of triplet score (≥5% S/N) following HCD of unique phosphopeptides (5% FDR) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides. Site of phosphorylation (ptmRS score ≥ 0.90) is indicated. % of phosphopeptides exhibiting either no neutral loss (Triplet =0), or neutral loss of any 1 (Triplet = 1), 2 (Triplet =2), or 3 ions from the precursor (∆80, ∆98 and/or ∆116 amu). Distribution of triplet score following HCD fragmentation as a function of site localisation confidence (ptmRS score ≥ 0.75, ≥ 0.90, ≥ 0.95, ≥ 0.99) for unique singly phosphorylated peptide as a function of the residue phosphorylated. Numbers of phosphopeptides (ptmRS ≥ 0.90) exhibiting different combinations of the three neutral loss species (∆80, ∆98 and/or ∆116 amu from precursor) for each of the phosphorylated residues.


A Immunohistochemical analysis of LONP1 expression in CKD children with mild ( n = 11 ) , moderate ( n = 10 ) , or severe fibrosis ( n = 9 ) . N = 4 in Normal group .

O O O O O O O O O O O O O O O O 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 Immunohistochemical analysis of LONP1 expression in CKD children with mild (n=11), moderate (n=10), or severe fibrosis (n=9). N=4 in Normal group.


D Western blot analysis for the expression of LONP1 in UUO models at different time points .

D Western blot analysis for the expression of LONP1 in UUO models at different time points.


F Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . G Sirius red staining in WT and cKI mice after UUO . Scale bar : 50μm .

O O O O O O O O O O O O O O O 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 Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. G Sirius red staining in WT and cKI mice after UUO. Scale bar: 50μm.


J , K Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and TGF - β1 ( monomer ) in WT and cKI mice after UUO ( n = 3 or 4 , biological replicates ) .

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

J, K Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and TGF-β1 (monomer) in WT and cKI mice after UUO (n=3 or 4, biological replicates).


M Succinate dehydrogenase ( SDH ) staining in WT and cKI mice after UUO . Scale bar : 50μm .

M Succinate dehydrogenase (SDH) staining in WT and cKI mice after UUO. Scale bar: 50μm.


N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO . Scale bar : 500nm .

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N Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO. Scale bar: 500nm.


A Deposition of total fibrosis in kidney tissues was determined by Masson ' s trichrome staining . Scale bar : 50μm . B Sirius red staining in WT and cKO mice after UUO . Scale bar :

O O O O O O O O O O O O O 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 Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50μm. B Sirius red staining in WT and cKO mice after UUO. Scale bar: 50μ


D , E Western blot and densitometric analysis for the expression of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after UUO ( n = 3 , biological replicates ) .

D, E Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after UUO (n=3, biological replicates).


I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO . Scale bar : 500nm .

I Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKO mice after UUO. Scale bar: 500nm.


C Masson ' s trichrome staining of WT and cKI mice after 5 / 6Nx . Scale bar : 100μm .

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C Masson's trichrome staining of WT and cKI mice after 5/6Nx. Scale bar: 100μm.


D qRT - PCR analysis of FN1 , Collagen I and Collagen III in WT and cKI mice after 5 / 6Nx ( n = 6 in cKI + Sham group , n = 5 in the other three groups , biological replicates )

D qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after 5/6Nx (n=6 in cKI+Sham group, n=5 in the other three groups, biological replicates)


G Masson ' s trichrome staining of WT and cKO mice after 5 / 6Nx . Scale bar : 100μm .

G Masson's trichrome staining of WT and cKO mice after 5/6Nx. Scale bar: 100μm.


H qRT - PCR analysis of FN1 , Collagen I , Collagen III and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 5 in WT + Sham group , n = 7 in cKO + Sham group , n = 6 in 5 / 6Nx groups , biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of FN1, Collagen I, Collagen III and α-SMA in WT and cKO mice after 5/6Nx (n=5 in WT+Sham group, n=7 in cKO+Sham group, n=6 in 5/6Nx groups, biological replicates).


I , J Western blot and densitometric analysis for the expression of Collagen I and α - SMA in WT and cKO mice after 5 / 6Nx ( n = 4 , biological replicates ) .

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

I, J Western blot and densitometric analysis for the expression of Collagen I and α-SMA in WT and cKO mice after 5/6Nx (n=4, biological replicates).


L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5 / 6Nx . Scale bar : 500nm .

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L Transmission electron microscopy images of the mitochondria in tubular cells of WT and cKO mice after 5/6Nx. Scale bar: 500nm.


A Quantification of mitochondrial ROS production ( n = 4 , biological replicates ) .

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

A Quantification of mitochondrial ROS production (n=4, biological replicates).


D qRT - PCR analysis of FN1 , Collagen I , Collagen III , Collagen IV , α - SMA and Vimentin ( n = 3 , biological replicates ) .

O O O O O O O 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 qRT-PCR analysis of FN1, Collagen I, Collagen III, Collagen IV, α-SMA and Vimentin (n=3, biological replicates).


Measurement of OCR using an XF96 Extracellular Flux Analyzer ( n = 8 or 10 in each group , biological replicates ) .

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

Measurement of OCR using an XF96 Extracellular Flux Analyzer (n=8 or 10 in each group, biological replicates).


Western blot analysis for the expression of Collagen I , α - SMA , and Vimentin ( n = 3 , two independent experiments were carried out ) .

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

Western blot analysis for the expression of Collagen I, α-SMA, and Vimentin (n=3, two independent experiments were carried out).


C Western blot validation of the up - regulated proteins in mitochondria ( n = 2 , biological replicates ) .

C Western blot validation of the up-regulated proteins in mitochondria (n=2, biological replicates).


E GST pull down analysis of interaction between LONP1 and HMGCS2 .

E GST pull down analysis of interaction between LONP1 and HMGCS2.


G Immunofluorescence co - localization analysis of LONP1 , HMGCS2 and mitochondria in mPTC ( up , scale bar : 20μm ) and human kidney tissue ( down , scale bar : 200μm ) .

O O O O O O O O O O O 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 Immunofluorescence co-localization analysis of LONP1, HMGCS2 and mitochondria in mPTC (up, scale bar: 20μm) and human kidney tissue (down, scale bar: 200μm).


A Immunohistochemical analysis of HMGCS2 expression in CKD children . Scale bar : 50μm . B Immunohistochemical semi - quantitative IOD analysis of HMGCS2 ( n = 8 in Control group , n = 15 in CKD group ) .

A Immunohistochemical analysis of HMGCS2 expression in CKD children. Scale bar: 50μm. B Immunohistochemical semi-quantitative IOD analysis of HMGCS2 (n=8 in Control group, n=15 in CKD group).


Western blot analysis of HMGCS2 in WT and cKI mice of UUO model ( n = 3 or 4 , biological replicates ) .

Western blot analysis of HMGCS2 in WT and cKI mice of UUO model (n=3 or 4, biological replicates).


western blotting analysis of Hmgcs2 expression in WT and Hmgcs2 + / - mice ( n = 3 , biological replicates ) .

western blotting analysis of Hmgcs2 expression in WT and Hmgcs2+/- mice (n=3, biological replicates).


G , H Representative immunoblot and densitometric analysis of HMGCS2 , FN1 , Collagen III , α - SMA and Vimentin in mPTCs treated as indicated . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H Representative immunoblot and densitometric analysis of HMGCS2, FN1, Collagen III, α-SMA and Vimentin in mPTCs treated as indicated. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group).


J - N Representative Western blot and densitometric analysis of LONP1 , FN1 , α - SMA and Vimentin of each group was shown . Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel ( n = 3 in each group ) . mPTCs were transfected with siNC or siLONP1 , and then pre - treated with 84 - B10 ( 10 μM ) 2h before TGF - β1 ( 10ng / mL ) treatment for another 24h .

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

J-N Representative Western blot and densitometric analysis of LONP1, FN1, α-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 μM) 2h before TGF-β1 (10ng/mL) treatment for another 24h.


A Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model . Scale bar : 50μm . B Quantification of fibrotic area in UUO model ( n = 10 in each group , biological replicates ) .

O O O O O O O O O O O O O O O O O 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 Fibrosis deposition of kidney tissues was determined by Sirius red staining in UUO model. Scale bar: 50μm. B Quantification of fibrotic area in UUO model (n=10 in each group, biological replicates).


G , H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model ( n = 10 in each group , biological replicates ) . Scale bar : 50μm .

G, H Fibrosis deposition of kidney tissues was determined by Sirius red staining and quantified in UIRI model (n=10 in each group, biological replicates). Scale bar: 50μm.


A Activity of an NF - κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 ( hTLR4 ) or an isogenic control cell line that does not express TLR4 ( null2 ) and were stimulated with vehicle ( veh ) , 1 ng / mL LPS , 400 μM nickel chloride , or 25 , 50 or 100 μM platinum ( II ) chloride and platinum ( IV ) chloride ( n = 3 independent biological replicates ) . B As per panel ( A ) but secreted IL - 8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg / mL LPS , 200 μM nickel chloride , 100 μM platinum ( II ) chloride or 100 μM platinum ( IV ) chloride ( n = 4 independent biological replicates ) . Data Information : For all panels actual individual data from each experiment are plotted as box ( 25th and 75th percentile borders ; median central band ) with Tukey whiskers . Statistical analyses were assessed by 2 - way ANOVA : in A ) hTLR4 compared to null2 cells ; in B ) agonist treatment compared to non - treated ( nil ) ; ns , not significant ; * * , P < . 01 ; ; * * * * , P < . 0001 ( Dunnett ' s test , A ,

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-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 O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Activity of an NF-κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/mL LPS, 400 μM nickel chloride, or 25, 50 or 100 μM platinum(II) chloride and platinum(IV) chloride (n=3 independent biological replicates). B As per panel (A) but secreted IL-8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/mL LPS, 200 μM nickel chloride, 100 μM platinum(II) chloride or 100 μM platinum(IV) chloride (n=4 independent biological replicates). Data Information: For all panels actual individual data from each experiment are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2-way ANOVA: in A) hTLR4 compared to null2 cells; in B) agonist treatment compared to non-treated (nil); ns, not significant ; **, P<.01; ; ****, P<.0001 (Dunnett's test, A, B


A IL - 8 secretion in HEK cells stably expressing hTLR4 but not MD - 2 ( HEK - isoTLR4 ) , transfected with empty vector ( EV ) or MD - 2 and left untreated ( nil ) or treated with 1 ng / mL LPS , 200 μM nickel chloride , 2 μg / ml HMGB1 , 100 μM platinum ( II ) chloride , 100 μM platinum ( IV ) chloride or 25 μM cisplatin ( n = 3 or 4 independent biological replicates ) . C IL - 8 secretion in HeLa cells transfected with non - targeting ( siNT ) or TLR4 - targeting ( siTLR4 ) siRNA and left untreated ( nil ) , or treated with 30 μM cisplatin ( n = 3 independent biological replicates ) . Mock cells were not subject to siRNA treatment prior . Data Information : Actual individual data are plotted as box ( 25th and 75th percentile borders ; median central band ) with Tukey whiskers ( A , C ) Statistical analyses were determined in comparison to nil treatments using 2 - way ( A , C ) ns , not significant ; * , P < . 05 ; * * * * , P < . 0001 ( Dunnett ' s test ) .

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

A IL-8 secretion in HEK cells stably expressing hTLR4 but not MD-2 (HEK-isoTLR4), transfected with empty vector (EV) or MD-2 and left untreated (nil) or treated with 1 ng/mL LPS, 200 μM nickel chloride, 2 μg/ml HMGB1, 100 μM platinum(II) chloride, 100 μM platinum(IV) chloride or 25 μM cisplatin (n=3 or 4 independent biological replicates). C IL-8 secretion in HeLa cells transfected with non-targeting (siNT) or TLR4-targeting (siTLR4) siRNA and left untreated (nil), or treated with 30 μM cisplatin (n=3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25th and 75th percentile borders; median central band) with Tukey whiskers (A, C) Statistical analyses were determined in comparison to nil treatments using 2-way (A, C) ns, not significant; *, P<.05; ****, P<.0001 (Dunnett's test).


A - HEI - OC1 cells containing a Tlr4 deletion ( Tlr4 - / - ) were compared to HEI - OC1 non - targeting ( NT ) control cells and assessed for cell viability ( A ) , AnnexinV / propidium iodide staining ( B ) , following cisplatin treatment at the indicated concentrations ( n = 3 independent biological replicates ) . Data Information : In all panels data are presented as mean and standard deviation . Statistical comparisons to NT at the same cisplatin concentration were assessed by 2 - way ( A , B ) * , P < . 05 ; * * , P < . 0001 ( Bonferroni test ) .

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A- HEI-OC1 cells containing a Tlr4 deletion (Tlr4-/-) were compared to HEI-OC1 non-targeting (NT) control cells and assessed for cell viability (A), AnnexinV/propidium iodide staining (B), following cisplatin treatment at the indicated concentrations (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2-way (A, B) *, P<.05; **, P<.0001(Bonferroni test).


A IL - 6 secretion in HEI - OC1 cells treated with 100 pg / mL LPS or 20μM cisplatin ( n = 3 or 4 independent biological replicates ) . B IL - 6 secretion in TLR4 - / - cells transfected with empty vector ( EV ) or mouse Tlr4 ( mTlr4 ) following treatment with 20μM cisplatin ( n = 6 independent biological replicates ) . Data information : In all panels data are presented as mean and standard deviation . Statistical comparisons to 0 hr time point were assessed by 2 - way ANOVA . ns , not significant ; * , P < . 05 ; * * , P < . 01 ; * * * , P < . 001 ; * * * * , P < . 0001 ( Dunnett ' s test ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IL-6 secretion in HEI-OC1 cells treated with 100 pg/mL LPS or 20μM cisplatin (n=3 or 4 independent biological replicates). B IL-6 secretion in TLR4-/- cells transfected with empty vector (EV) or mouse Tlr4 (mTlr4) following treatment with 20μM cisplatin (n=6 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).


C Il6 and Tlr4 transcript levels in HEI - OC1 cells following treatment with 20μM cisplatin for the indicated times ( n = 3 independent biological replicates ) . Data information : In all panels data are presented as mean and standard deviation . Statistical comparisons to 0 hr time point were assessed by 2 - way ANOVA . ns , not significant ; * , P < . 05 ; * * , P < . 01 ; * * * , P < . 001 ; * * * * , P < . 0001 ( Dunnett ' s test ) .

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C Il6 and Tlr4 transcript levels in HEI-OC1 cells following treatment with 20μM cisplatin for the indicated times (n=3 independent biological replicates). Data information: In all panels data are presented as mean and standard deviation. Statistical comparisons to 0 hr time point were assessed by 2-way ANOVA. ns, not significant; *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 (Dunnett's test).


B Hair cell viability in larval zebrafish pre - treated with control - , tlr4ba - and / or tlr4bb - targeting morpholino oligonucleotides ( MO ) and subsequently treated with 15 μM cisplatin . Data information : In both panels each data point represents a score of hair cell integrity in an individual animal ( taken from multiple samples per animal ) with lines representing mean and standard deviation . Statistical comparisons to control morpholino ( B , except as indicated in blue ) were assessed by one - way ANOVA . * , P < . 05 ; * * , P < . 01 ; * * * * , P < . 0001 ( Tukey test ) .

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B Hair cell viability in larval zebrafish pre-treated with control-, tlr4ba- and/or tlr4bb-targeting morpholino oligonucleotides (MO) and subsequently treated with 15 μM cisplatin. Data information: In both panels each data point represents a score of hair cell integrity in an individual animal (taken from multiple samples per animal) with lines representing mean and standard deviation. Statistical comparisons to control morpholino (B, except as indicated in blue) were assessed by one-way ANOVA. *, P<.05; **, P<.01; ****, P<.0001 (Tukey test).


A IL - 6 secretion in HEI - OC1 cells pretreated with DMF vehicle ( veh ) , 4μM TAK242 ( TAK ) or left untreated ( nil ) following treatment with 10 ng / mL LPS or 20μM cisplatin ( n = 5 - 7 independent biological replicates ) . Data Information : In all panels data are presented as mean and standard deviation . Statistical comparisons to nil treatment were assessed by 2 - way ( A ) or one - way * , P < . 05 ; * * * * , P < . 0001 ( Dunnett ' s test ) .

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 B-CONTROLLED_VAR 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

A IL-6 secretion in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 10 ng/mL LPS or 20μM cisplatin (n=5-7 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by 2-way (A) or one-way *, P<.05 ; ****, P<.0001(Dunnett's test).


B ROS generation in HEI - OC1 cells pretreated with DMF vehicle ( veh ) , 4μM TAK242 ( TAK ) or left untreated ( nil ) following treatment with 20μM cisplatin ( n = 3 independent biological replicates ) . Data Information : In all panels data are presented as mean and standard deviation . Statistical comparisons to nil treatment were assessed by one - way ( B ) ANOVA . ; * * , P < . 01 P < . 0001 ( Dunnett ' s test ) .

O B-MEASURED_VAR 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 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

B ROS generation in HEI-OC1 cells pretreated with DMF vehicle (veh), 4μM TAK242 (TAK) or left untreated (nil) following treatment with 20μM cisplatin (n=3 independent biological replicates). Data Information: In all panels data are presented as mean and standard deviation. Statistical comparisons to nil treatment were assessed by one-way (B) ANOVA. ; **, P<.01 P<.0001(Dunnett's test).


A - F Brain MRI of the proband at 23 years . ( A ) Sagittal fast spin echo ( FSE ) T1 - weighted image shows thinning of the corpus callosum , dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem ; two lacunar lesions are evident in the dorsal pons . ( B ) T2 - weighted axial fluid - attenuated inversion recovery ( FLAIR ) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami . ( C ) MR angiography 3D TOF ( time of flight ) reconstruction shows two saccular aneurisms in the right M1 ( ø 4 mm ) and left M2 ( ø 2 . 5 mm ) segments of middle cerebral arteries ( arrows ) . ( D ) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging ( DTI ) in the right side of the dorsal pons . Severe cavitations and lateral ventricles dilation ( left > right ) on FLAIR images ( B , E ) and diffuse microbleeds as small hypointense foci on SWI are visible ( F ) in the same slices of ( E ) . R = right , L = left .

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A-F Brain MRI of the proband at 23 years. (A) Sagittal fast spin echo (FSE) T1-weighted image shows thinning of the corpus callosum, dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem; two lacunar lesions are evident in the dorsal pons. (B) T2-weighted axial fluid-attenuated inversion recovery (FLAIR) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami. (C) MR angiography 3D TOF (time of flight) reconstruction shows two saccular aneurisms in the right M1 (ø 4 mm) and left M2 (ø 2.5 mm) segments of middle cerebral arteries (arrows). (D) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging (DTI) in the right side of the dorsal pons. Severe cavitations and lateral ventricles dilation (left > right) on FLAIR images (B, E) and diffuse microbleeds as small hypointense foci on SWI are visible (F) in the same slices of (E). R = right, L = left.


G , H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2 - weighted images in the periventricular and subcortical cerebral white matter , expression of gliosis secondary to chronic small vessel ischemic changes , more evident in the father ( G ) , 56 years , than in the mother ( H ) , 54 years .

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G, H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2-weighted images in the periventricular and subcortical cerebral white matter, expression of gliosis secondary to chronic small vessel ischemic changes, more evident in the father (G), 56 years, than in the mother (H), 54 years.


Gene expression of NOTCH3 , HES1 , HEY1 , HEYL and the 17 recently identified targets were established by a real - time PCR panel assay in skeletal muscle of controls ( n = 6 ) , proband ( II : 1 ) , parents ( mother I : 1 , father I : 2 ) and CADASIL patients ( n = 3 ) . Graph shows gene expression fold changes relative to controls and normalized on GAPDH ( reference gene ) , expressed as mean of two experiments ± SEM . Among the 17 novel assayed target genes , RCAN2 , ANGPT4 , HP and SORBS were not detected in any sample and were therefore not reported . Green and red lines indicate 2 . 0 and 0 . 5 fold changes , respectively . Statistical significances and P - values are reported in Supplementary Table S1 .

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Gene expression of NOTCH3, HES1, HEY1, HEYL and the 17 recently identified targets were established by a real-time PCR panel assay in skeletal muscle of controls (n = 6), proband (II:1), parents (mother I:1, father I:2) and CADASIL patients (n = 3). Graph shows gene expression fold changes relative to controls and normalized on GAPDH (reference gene), expressed as mean of two experiments ± SEM. Among the 17 novel assayed target genes, RCAN2, ANGPT4, HP and SORBS were not detected in any sample and were therefore not reported. Green and red lines indicate 2.0 and 0.5 fold changes, respectively. Statistical significances and P-values are reported in Supplementary Table S1.


A - C Histopathological examination of skin ( A ) and skeletal muscle biopsies ( B , C ) of control ( left ) , proband ( middle ) and CADASIL patient ( right ) . ( A ) Collagen IV staining ( in green ) in skin vessels : Collagen wall appears compact in the control , while it is disorganized in the skin vessels of the proband and of the CADASIL patient . Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient , where collagen wall is quite compact . Vessel ' s endothelium is delineated by ULEX staining ( in red ) . Collagen IV staining in skeletal muscle vessels ( B ) recapitulates the skin picture . Smooth muscle actin immunostaining of skeletal muscle biopsies ( C ) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient , with foci characterized by a complete SMCs loss ( particularly in the CADASIL patient ) and thinning of the vessel wall ( arrows ) .

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A-C Histopathological examination of skin (A) and skeletal muscle biopsies (B, C) of control (left), proband (middle) and CADASIL patient (right). (A) Collagen IV staining (in green) in skin vessels: Collagen wall appears compact in the control, while it is disorganized in the skin vessels of the proband and of the CADASIL patient. Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient, where collagen wall is quite compact. Vessel's endothelium is delineated by ULEX staining (in red). Collagen IV staining in skeletal muscle vessels (B) recapitulates the skin picture. Smooth muscle actin immunostaining of skeletal muscle biopsies (C) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient, with foci characterized by a complete SMCs loss (particularly in the CADASIL patient) and thinning of the vessel wall (arrows).


D , E Ultrastructural analysis of skin biopsies . ( D ) Skin biopsy of the proband : ( left panel ) At low magnification , the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect ( arrows ) ; SMCs are identified by the presence of " focal adhesions " ( square boxes ) ; ( middle panel ) collagen fibrils appear organized in a parallel pathway along SMC ( arrow ) , note the " focal adhesions " ( square box ) ; ( right panel ) collagen fibrils ( " CF " ) are more represented in dense bundles than elastic ones ( " E " ) . No GOM is detected , in contrast to CADASIL patient ( E ) , showing deposits of granular osmiophilic material ( arrows ) located between SMC plasmalemma and basal lamina .

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D, E Ultrastructural analysis of skin biopsies. (D) Skin biopsy of the proband: (left panel) At low magnification, the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect (arrows); SMCs are identified by the presence of "focal adhesions" (square boxes); (middle panel) collagen fibrils appear organized in a parallel pathway along SMC (arrow), note the "focal adhesions" (square box); (right panel) collagen fibrils ("CF") are more represented in dense bundles than elastic ones ("E"). No GOM is detected, in contrast to CADASIL patient (E), showing deposits of granular osmiophilic material (arrows) located between SMC plasmalemma and basal lamina.


F KCNA5 immunostaining of skeletal muscle biopsies of control ( left ) , proband ( middle ) and CADASIL patient ( right ) . A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient . Note multiple areas in which KCNA5 is barely detectable ( arrows ) .

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F KCNA5 immunostaining of skeletal muscle biopsies of control (left), proband (middle) and CADASIL patient (right). A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient. Note multiple areas in which KCNA5 is barely detectable (arrows).


Selected BioID REV7 results , shown as dot plots . The spectral counts for each indicated prey protein are shown as AvgSpec . Proteins were selected based on and iProphet probability of > 0 . 95 , BFDR of < 0 . 05 and ≥ 10 peptide count . The circle size represents the relative abundance of preys over baits .

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Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of >0.95, BFDR of <0.05 and ≥10 peptide count. The circle size represents the relative abundance of preys over baits.


U2OS EJ5 - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) .

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U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


Quantification of the Neutral Comet assay . U2OS cells stably expressing shCtrl , shREV7 or shSHLD2 were exposed to IR ( 10 Gy ) and run in low melting agarose under neutral conditions . Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment .

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Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment.


U2OS cells were transfected with the indicated siRNA . 48h post - transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0 , 1 , 2 , 4 and 24 hr post NCS treatment . Flow Cytometry analysis of phosphorylated - H2AX signal was used to measure γ - H2AX endogenous signal .

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U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal.


Sensitivity to IR monitored by colony formation assay . MCF - 7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR . 24 h post - irradiation , the cells were re - seeded to allow colony formation , fixed and stained with 0 . 4 % crystal violet . Shown is the quantification of colonies per condition which possessed more than 50 colonies .

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Sensitivity to IR monitored by colony formation assay. MCF-7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR. 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies.


MCF - 7 cells stably expressing the the indicated shRNAs 24 h post - irradiation , the cells were re - seeded to allow colony formation , fixed and stained with 0 . 4 % crystal violet . Shown is the quantification of colonies per condition which possessed more than 50 colonies . cells were exposed to increasing doses of UV radiation .

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MCF-7 cells stably expressing the the indicated shRNAs 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. cells were exposed to increasing doses of UV radiation.


U2OS cells stably expressing HA - REV7 ( Top ) or HA - SHLD2 ( Bottom ) were pre - sensitized with 10ug / mL Hoescht 33342 before exposed to UV micro - irradiation . Immunofluorescence against endogenous HA and γ - H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage . Shown are representative micrographs . Scale bar = 5µm .

U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.


U2OS LacR - Fok1 cells were transfected with GFP or GFP - SHLD2 and 24 h later DNA damage was induced using Shield - 1 and 4 - OHT . The cells were then processed for GFP and mCherry immunofluorescence . Shown are representative micrographs . Scale bar = 5µm . Quantification of the experiments shown in ( C ) . Data are represented as the mean ± SD ( n = 3 ) . At least 100 cells per condition were counted .

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U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm. Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted.


Quantification of the experiments shown in ( C ) . Shown is the quantification of the GFP signal at the mCherry - LacR - Fok1 focus .

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Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus.


293T cell lines expressing ER - AsiSI with Flag - SHLD2 and treated with 1 µM of 4 - OHT . 6h later the cells were processed and immunoprecipitated with Anti - FLAG Magnetic Beads and anti - γ - H2AX . x / Protein A / G magnetic beads . DNA was purified and subjected to qPCR detection . Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input ( Bottom ) .

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293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom).


U2OS cells stably expressing HA - SHLD2 ∆ 720 - 904 ( Left ) or HA - SHLD2 ∆ 1 - 680 ( Right ) were processed as in ( B ) . Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage . Shown are representative micrographs . Scale bar = 5µm .

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U2OS cells stably expressing HA-SHLD2∆720-904 (Left) or HA-SHLD2∆1-680 (Right) were processed as in (B). Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.


U2OS mCherry - LacR - Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP - SHLD2 construct . 24 h post - transfection DNA damage was induced using Shield - 1 and 4 - OHT . The cells were then fixed and analyzed for the intensity of the GFP - SHLD2 signal at mCherry - LacR - Fok1 focus . Shown is the quantification of the GFP - SHLD2 signal at the Fok1 focus . Data are represented as a box - and - whisker plot where the whiskers represent the 10 - 90 percentile . At least 75 cells were counted per condition .

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U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition.


293T cells were transfected with Flag - REV7 and GFP - SHLD2 expression vectors as indicated . 24h post - transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU - 60019 for 1 h prior to irradiation . 1h post - irradiation ( 10 Gy ) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti - Flag ( M2 ) Resin and then analyzed by immunoblotting using GFP , REV7 and p - Chk1 antibodies .

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293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies.


CH12F3 - 2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines ( CIT ) to induce class switching to IgA . The percentage of IgA + cells was monitored 24 and 48h post - stimulation by staining with an anti - IgA antibody followed by flow cytometry analysis .

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CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.


U2OS DR - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) .

U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


U2OS cells were transfected with the indicated siRNAs and then treated with CldU , IdU and NCS 48h post - transfection as indicated . The slides were stained , dehydrated , mounted and visualized and shown is the quantification of CldU / IdU tract length in order to visualize DNA end - resection ( Right ) . At least 500 DNA tracks were measured per condition .

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U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition.


Genes significantly enriched or dropped out after a 14 - day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated . Ranking was determined based on the Log2 fold score ( Left ) . The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change ( Fc ) in Log2 .

Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2.