Figure 2Differences in clinical variables including ( B ) liver fat , plasma levels of ( C ) ALT , ( D ) AST , ( E ) uric acid and ( F ) creatinine are presented in the CMA and placebo groups on Days 0 , 14 and 70 after weight loss adjustment . Adj . P indicates p value after weight loss adjustment . Statistical significance is defined based on paired Student ' s t test . p < 0 . 05 . The boxes show the distribution of the clinical parameters in different groups . The bottom and top of the boxes represent the 25th and 75th percentiles . The central band represents the median value . The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values . The sample sizes on Days 0 , 14 or 70 were marked in each boxplot .

Figure 2Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.


Figure 3Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B ) in both ( n = 4 ) ;

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

Figure 3Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4);


Figure 4A ) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups . Asterisks indicate statistical significance based on paired Student ' s t test . p < 0 . 05 ; B ) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine , cysteine , carnitine and nicotinamide . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM ) Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM ) C ) with the 10 most significantly correlated plasma metabolites ,

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

Figure 4A) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups. Asterisks indicate statistical significance based on paired Student's t test. p < 0.05;B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM)Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) C) with the 10 most significantly correlated plasma metabolites,


Figure 5Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A ) gut microbiome between CMA and placebo groups on Day 70 vs Day 0 ; B ) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups ; C ) oral microbiome between CMA and placebo groups on Day 70 vs Day 0 ; D ) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups . Each dot represents a species and it has been colored according to its corresponding phylum . Asterisks indicate statistical significance based on paired Wilcoxon signed - rank test . p < 0 . 05 . Log2FC : log2 ( fold change ) .

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

Figure 5Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change).


Figure 6Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , B ) plasma level of 10 most significant inflammation related proteinsHeatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , C ) the abundance of the species in gut microbiomeHeatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with D ) the abundance of the species in oral microbiome . AST , Aspartate aminotransferase ; ALT , Alanine transaminase . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Cor . Coeff : Correlation coefficient .

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

Figure 6Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteinsHeatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiomeHeatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.


Figure 1Low magnification views of a CD1 Otof - / - organ of Corti ( P23 ) transduced with otoferlin dual ‑ AAV ‑ TS vectors . IHCs : inner hair cells , OHCs : outer hair cells maximum intensity projections of confocal optical sectionsHigh magnification views of CD1B6F1 Otof - / - IHCs transduced with otoferlin dual ‑ AAV ‑ TS ( P26 ) ( B ) and dual ‑ AAV ‑ Hyb ( P26 ) ( C ) vectors . Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in ( A - C ) with warmer colors representing higher pixel intensities ) , maximum intensity projections of confocal optical sectionsPercentage of N ‑ and C ‑ terminal otoferlin labelled IHCs in dual ‑ AAV ‑ TS ( n = 10 mice ) and dual ‑ AAV ‑ Hyb ( n = 9 mice ) injected CD1B6F1 Otof - / - mice ( P18 ‑ 30 ) Average N - terminal and C ‑ terminal otoferlin immunofluorescence levels in dual ‑ AAV transduced Otof - / - and wild - type IHCs ( P23 - 30 ) . Otoferlin levels were normalized to immunofluorescence levels in non - transduced B6 wild - type IHCs for each antibody

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

Figure 1Low magnification views of a CD1 Otof-/- organ of Corti (P23) transduced with otoferlin dual‑AAV‑TS vectors. IHCs: inner hair cells, OHCs: outer hair cells maximum intensity projections of confocal optical sectionsHigh magnification views of CD1B6F1 Otof-/- IHCs transduced with otoferlin dual‑AAV‑TS (P26) (B) and dual‑AAV‑Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities ), maximum intensity projections of confocal optical sectionsPercentage of N‑ and C‑terminal otoferlin labelled IHCs in dual‑AAV‑TS (n=10 mice) and dual‑AAV‑Hyb (n=9 mice) injected CD1B6F1 Otof-/- mice (P18‑30)Average N-terminal and C‑terminal otoferlin immunofluorescence levels in dual‑AAV transduced Otof-/- and wild-type IHCs (P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately


Figure 2High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons ( CtBP2 ) from wild ‑ type ( B6 : P27 , CD1B6F1 : P29 ) , dual - AAV injected CD1B6F1 Otof - / - ( dual - AAV - TS : P26 , dual - AAV - Hyb : P28 ) , and their contralateral non - injected ears . ( * ) Transduced cells . Maximum intensity projections of optical confocal sections . Scale bars : 5 µmSynaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild ‑ type ( B6 : n = 48 IHCs , CD1B6F1 : n = 108 IHCs ) , transduced Otof - / - ( dual ‑ AAV ‑ TS : n = 59 IHCs , dual - AAV - Hyb : n = 37 IHCs ) , and non - transduced Otof - / - IHCs from injected ( - AAV injected ear , n = 65 IHCs ) and contralateral non - injected ( - AAV non - injected ear , n = 46 IHCs ) ears ( P25 - 29 ) IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild - type and Otof - / - P6 and P14 organs of Corti . Maximum intensity projections of optical confocal sections . Scale bars : 5 µm . D Synapse numbers quantified from IHCs in apical cochlear turns ( C ) of B6 wild - type ( P6 : n = 53 IHCs ; P14 : n = 73 IHCs ) and B6 Otof - / - ( P6 : n = 62 IHCs ; P14 : n = 65 IHCs ) mice at two different developmental stages ( P6 and P14 ) Ca2 + ‑ current - voltage relationship of control CD1B6F1 wild ‑ type ( n = 6 IHCs ) , dual - AAV - TS transduced ( n = 8 IHCs ) , and non ‑ transduced CD1B6F1 Otof - / - ( n = 10 IHCs ) IHCs ( P14 ‑ 18 ) Representative Ca2 + ‑ currents ( Ica ) and IHC plasma membrane capacitance increments ( ΔCm ) of a wild ‑ type control , transduced , and non ‑ transduced Otof - / - IHC in response to a 20 ms depolarization pulse at maximum Ca2 + ‑ current potentials ( typically - 14 mV ) Average exocytosis level measured as ΔCm ( G ) and corresponding Ca2 + ‑ current integrals ( QCa2 + ) ( H ) in wild ‑ type ( CD1B6F1 : n = 6 IHCs ; B6 : n = 11 IHCs ( B6 data replotted from Strenzke et al , 2016 ) ) , dual - AAV - TS transduced Otof - / - ( n = 8 IHCs ) , and non ‑ transduced ( n = 11 IHCs ) Otof - / - IHCs . Transduced IHCs that showed eGFP expression , but had almost no exocytosis are depicted as dashed lines ( not included into the average )

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

Figure 2High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild‑type (B6: P27, CD1B6F1: P29), dual-AAV injected CD1B6F1 Otof-/- (dual-AAV-TS: P26, dual-AAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 µmSynaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild‑type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof-/- (dual‑AAV‑TS: n=59 IHCs, dual-AAV-Hyb: n=37 IHCs), and non-transduced Otof-/- IHCs from injected (-AAV injected ear, n=65 IHCs) and contralateral non-injected (-AAV non-injected ear, n=46 IHCs) ears (P25-29)IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof-/- P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm. D Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n= 53 IHCs; P14: n=73 IHCs) and B6 Otof-/- (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14)Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18)Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average)


Figure 3Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual ‑ AAV ‑ TS and dual ‑ AAV ‑ Hyb injected CD1B6F1 Otof - / - animals . AAV2 / 6 . eGFP ( + AAV . eGFP ) and dual ‑ AAV ‑ TS injected CD1B6F1 wild ‑ type , and non ‑ injected control Otof - / - littermate ( - AAV ) mice served as controls . SP : summating potential ; ABR waves are indicated from I - VAverage ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks / sABR click sound ( C thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control animalsABR tone burst ( D ) thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control animals . In D , the two best animals are depicted with open circles . Animals with thresholds exceeding the maximum loudspeaker output ( arrows ) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL , respectively . Apical and basal cochlear turns are indicated as Apex and Base , respectivelySummed ABR wave I - V amplitudes at different click sound intensities in otoferlin dual ‑ AAV injected , non ‑ injected Otof - / - , and wild ‑ type control miceSummed ABR wave I ‑ V amplitudes of individual dual ‑ AAV ‑ TS treated CD1B6F1 Otof - / - animals ( n = 8 animals plotted against their full - length otoferlin IHC transduction

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

Figure 3Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-VAverage ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/sABR click sound (C thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animalsABR tone burst (D) thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals. In D, the two best animals are depicted with open circles. Animals with thresholds exceeding the maximum loudspeaker output (arrows) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL, respectively. Apical and basal cochlear turns are indicated as Apex and Base, respectivelySummed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control miceSummed ABR wave I‑V amplitudes of individual dual‑AAV‑TS treated CD1B6F1 Otof-/- animals (n=8 animals plotted against their full-length otoferlin IHC transduction rate


Figure 1B . Rev3l + / + and Rev3l - / - MEFs were pulse - labelled with EdU for 15min , permeabilized , fixed and stained for EdU incorporation ( green ) . S phase sub - stages from I to IV were evaluated by visual inspection of the cycling population ( > 300 EdU + cells , top panel ) . Scale bar = 5 μm . Dot plots and pie charts show the relative proportion ( percentage of total S ) of each sub - stage from I to IV ( middle and bottom panel , respectively ) . Each dot represents the mean of two technical replicates .

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

Figure 1B. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with EdU for 15min, permeabilized, fixed and stained for EdU incorporation (green). S phase sub-stages from I to IV were evaluated by visual inspection of the cycling population (>300 EdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of each sub-stage from I to IV (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.


Figure 2C . Analysis of the active marks H3K27ac and H3K4me3 and facultative heterochromatin mark H3K27me3 content in early , mid , late and TTR compared to disturbed replicating regions found in Rev3l - / - MEFs . Bar in boxplot represents the median , and red points represents the mean . The limit of the boxes corresponds to the 0 . 25 to 0 . 75 quartiles with whiskers extending to the maximum value of 1 . 5 times the interquartile range . Graphs show data from two biological experiments .

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

Figure 2C. Analysis of the active marks H3K27ac and H3K4me3 and facultative heterochromatin mark H3K27me3 content in early, mid, late and TTR compared to disturbed replicating regions found in Rev3l-/- MEFs. Bar in boxplot represents the median, and red points represents the mean. The limit of the boxes corresponds to the 0.25 to 0.75 quartiles with whiskers extending to the maximum value of 1.5 times the interquartile range. Graphs show data from two biological experiments.


Figure 3D . Relative mRNA levels of four genes involved in development ( Hoxb2 , 8 , 9 and WT1 ) were validated through qRT - PCR from Rev3l + / + and Rev3l - / - MEFs samples . The data were normalized to the amount of GAPDH mRNA . Error bars indicate standard error of the mean for three independent experiments . * * * , P < 0 . 001 and * * , P < 0 . 005 by Student ' s t - test . E . Relative mRNA levels of genes involved in imprinting ( paternal expressed genes : Dlk1and Slc18a4 and maternal expressed genes : Dcn1 and H19 ) were validated through qRT - PCR from Rev3l + / + and Rev3l - / - MEFs samples . The data were normalized to the amount of GAPDH mRNA . Error bars indicate standard error of the mean for three independent experiments . * * * P < 0 . 001 by Student ' s t - test .

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

Figure 3D. Relative mRNA levels of four genes involved in development (Hoxb2, 8, 9 and WT1) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. ***, P<0.001 and **, P<0.005 by Student's t-test. E. Relative mRNA levels of genes involved in imprinting (paternal expressed genes: Dlk1and Slc18a4 and maternal expressed genes: Dcn1 and H19) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. *** P<0.001 by Student's t-test.


Figure 4A . Total histones were acid extracted from Rev3l + / + and Rev3l - / - MEFs . Samples were analyzed by western - blot using indicated antibodies specific to histone marks ( right panel ) . An anti - pan H4 or H3 was used as loading control ( left panel ) . Histograms show WT / KO Rev3l fold change relative to the immunoblot shown on the top panel ( using 1X sample intensity ) . B . Histone mark levels were examined at selected genes repressed in Rev3l - / - MEFs ( Hoxb2 , Hoxb8 , WT1 ) . Histograms represent enrichment of H3K9me3 and H3K27me3 at indicated loci assessed by ChIP - qPCR in Rev3l + / + and Rev3l - / - MEFs . Error bars indicate standard error of the mean for three independent experiments . C . H3K9me3 and H3K27me3 mark levels were evaluated at selected genes that localized in genomic loci displaying replication timing delay and downregulated in Rev3l - / - MEFs ( FoxG1 , GATA6 ) . Histograms represent enrichment of H3K9me3 and H3K27me3 assessed by ChIP - qPCR at indicated loci in Rev3l + / + and Rev3l - / - MEFs . Error bars indicate standard error of the mean for three independent experiments .

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

Figure 4A. Total histones were acid extracted from Rev3l+/+ and Rev3l-/- MEFs. Samples were analyzed by western-blot using indicated antibodies specific to histone marks (right panel). An anti-pan H4 or H3 was used as loading control (left panel). Histograms show WT/KO Rev3l fold change relative to the immunoblot shown on the top panel (using 1X sample intensity).B. Histone mark levels were examined at selected genes repressed in Rev3l-/- MEFs (Hoxb2, Hoxb8, WT1). Histograms represent enrichment of H3K9me3 and H3K27me3 at indicated loci assessed by ChIP-qPCR in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.C. H3K9me3 and H3K27me3 mark levels were evaluated at selected genes that localized in genomic loci displaying replication timing delay and downregulated in Rev3l-/- MEFs (FoxG1, GATA6). Histograms represent enrichment of H3K9me3 and H3K27me3 assessed by ChIP-qPCR at indicated loci in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.


Figure 5G . MEFs cells were transfected with GFP - REV3L761 - 1029 WT PSVVL or ASAVA mutant and fixed with 4 % formaldehyde . The distribution of GFP - REV3L constructs was detected by autofluorescence ( green ) , chromocenters were visualized by HP1α immunostaining ( red ) and DNA was counterstained with DAPI . Scale bar = 10 μmH . Human 293T cells were transfected with either FH - REV3L700 - 900 , mutant V802D F - H - REV3L700 - 900 or empty vector . Forty - eight hours after transfection cell lysates were made and used for immunoprecipitation with HP1 antibody . Western blot was processed and membranes were immunoblotted with the indicated antibodies . I . 293T cells were co - transfected with F - H - REV3L700 - 900 or empty vector and V5 - HP1γ , mutant IY / EE V5 - HP1γ or empty vector . Forty - eight hours after transfection cell lysates were made and used for immunoprecipitation with α - V5 antibody . After electrophoresis , samples were immunoblotted with anti - HA , anti - HP1γ or anti - α - Tubulin as indicated .

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

Figure 5G. MEFs cells were transfected with GFP-REV3L761-1029 WT PSVVL or ASAVA mutant and fixed with 4% formaldehyde. The distribution of GFP-REV3L constructs was detected by autofluorescence (green), chromocenters were visualized by HP1α immunostaining (red) and DNA was counterstained with DAPI. Scale bar= 10 μmH. Human 293T cells were transfected with either FH-REV3L700-900, mutant V802D F-H-REV3L700- 900 or empty vector. Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with HP1 antibody. Western blot was processed and membranes were immunoblotted with the indicated antibodies.I. 293T cells were co-transfected with F-H-REV3L700-900 or empty vector and V5-HP1γ, mutant IY/EE V5-HP1γ or empty vector. Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with α-V5 antibody. After electrophoresis, samples were immunoblotted with anti-HA, anti-HP1γ or anti-α-Tubulin as indicated.


Figure 6B . Asynchronous Rev3l + / + and Rev3l - / - MEFs were fixed with PFA then subjected to in situ proximity ligation assay ( PLA ) using 53BP1 and HP1α antibodies then PLA foci were counted in both cell lines ( more than 150 nuclei for each condition were counted ) . p - values were calculated by Mann - Whitney test ( * * * * : p < 0 . 0001 ) . Red lines indicate the mean values . Error bars : SEM . Controls with a single antibody are also shown . Experiments were repeated three times . Representative images are shown . Scale bar = 5 μm . C . Asynchronous Rev3l + / + and Rev3l - / - MEFs were subjected to PLA as by using γH2AX and HP1β antibodies and processed as in ( B ) . p - values were calculated by Mann - Whitney test ( * : p < 0 . 05 ) . Red lines indicate the mean values . Error bars : SEM . Controls with a single antibody are also shown . Three independent experiments were performed . Rev3l + / + and Rev3l - / - MEFs were incubated with or without 0 . 23 μM aphidicolin for 24h before metaphase spreading . FISH was performed using major satellite probe to quantify breaks in pericentromeric regions ( F ) . Representative images of chromosomes showing abnormalities ( see arrows ) in pericentromeric regions from Rev3l - / - MEFs . Chromosomes were labelled with DAPI Error bars indicate standard error of the mean for three independent experiments . Mann - Whitney test ( ns : not significant , * p < 0 . 05 ; * * * p < 0 . 001 ) .

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

Figure 6B. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were fixed with PFA then subjected to in situ proximity ligation assay (PLA) using 53BP1 and HP1α antibodies then PLA foci were counted in both cell lines (more than 150 nuclei for each condition were counted). p-values were calculated by Mann-Whitney test ( ****: p<0.0001). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Experiments were repeated three times. Representative images are shown. Scale bar= 5 μm.C. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were subjected to PLA as by using γH2AX and HP1β antibodies and processed as in (B). p-values were calculated by Mann-Whitney test ( *: p<0.05). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Three independent experiments were performed.Rev3l+/+ and Rev3l-/- MEFs were incubated with or without 0.23 μM aphidicolin for 24h before metaphase spreading. FISH was performed using major satellite probe to quantify breaks in pericentromeric regions (F). Representative images of chromosomes showing abnormalities (see arrows) in pericentromeric regions from Rev3l-/- MEFs. Chromosomes were labelled with DAPI Error bars indicate standard error of the mean for three independent experiments. Mann-Whitney test (ns: not significant, * p<0.05; *** p< 0.001).


Figure 8A . Asynchronous Rev3l + / + and Rev3l - / - MEFs were UV - irradiated at the indicated doses and harvested 6 h or 24 h later . Cell lysates were analyzed by immunoblotting with indicated antibodies . B . Asynchronous Rev3l - / - MEFs complemented with empty vector ( EV ) or REV3L were UV - irradiated at the indicated doses and harvested 6 h or 24 h later . Cell lysates were analyzed by immunoblotting with indicated antibodies . C Asynchronous Rev3l + / + and Rev3l - / - MEFs were pulse labelled for 15 min using 10 μM EdU then UV - irradiated at 4 J / m2 . Cells pre - treated with CSK buffer were fixed and 53BP1 , RPA and RAD51 were detected by immunofluorescence . Representative images in non - irradiated cells ( NI ) and quantification of 53BP1 foci in EdU positive cells at different time points are shown Scale bar = 10 μm The number of cells analyzed is indicated in ( n ) on each graph . * * * p < 0 . 001 ( Kruskal - Wallis test ) . The presented data are representative of 2 repeats ) . Bars represent the median ± interquartile rangeF - H . Asynchronous Rev3l - / - MEFs complemented with empty vector ( EV ) or REV3L were processed as in ( C ) . Quantification of 53BP1 foci in EdU positive cells at different time points is shown ( F ) . Quantification of the intensity of chromatin - bound RPA2 ( G ) and RAD51 foci ( H ) 24 hours after UV - irradiation was performed . The number of cells analyzed is indicated in ( n ) on each graph . * * * p < 0 . 001 ( Kruskal - Wallis test ) . The presented data are representative of 2 repeats . Bars represent the median ± interquartile range .

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

Figure 8A. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. B. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. C Asynchronous Rev3l+/+ and Rev3l-/- MEFs were pulse labelled for 15 min using 10 μM EdU then UV-irradiated at 4 J/m2. Cells pre-treated with CSK buffer were fixed and 53BP1, RPA and RAD51 were detected by immunofluorescence. Representative images in non-irradiated cells (NI) and quantification of 53BP1 foci in EdU positive cells at different time points are shown Scale bar= 10 μm The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile rangeF-H. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were processed as in (C). Quantification of 53BP1 foci in EdU positive cells at different time points is shown (F). Quantification of the intensity of chromatin-bound RPA2 (G) and RAD51 foci (H) 24 hours after UV-irradiation was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats. Bars represent the median ± interquartile range.


Figure 1Agar plugs containing actively growing cultures of wild type S . sclerotiorum ( strain 1980 ) and the OA deficient A2 mutant were inoculated onto Col - 0 and ced - 9 expressing Arabidopsis leaves . ( A ) Wild type inoculations onto Col - 0 plants resulted in typical lesions for this pathogen including a rapid , spreading cell death ; however , infection was completely suppressed in ced - 9 expressing plants . The expression of this gene had no effect on the A2 phenotype . ( B ) Trypan blue staining indicates the extent of cell death for each genotype / strain combination , including an agar plug control . All images were recorded 48 hours post inoculation .

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

Figure 1Agar plugs containing actively growing cultures of wild type S. sclerotiorum (strain 1980) and the OA deficient A2 mutant were inoculated onto Col-0 and ced-9 expressing Arabidopsis leaves. (A) Wild type inoculations onto Col-0 plants resulted in typical lesions for this pathogen including a rapid, spreading cell death; however, infection was completely suppressed in ced-9 expressing plants. The expression of this gene had no effect on the A2 phenotype.(B) Trypan blue staining indicates the extent of cell death for each genotype/strain combination, including an agar plug control. All images were recorded 48 hours post inoculation.


Figure 2Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation . S . sclerotiorum A2 ( A ) and wild type ( B ) strains were inoculated onto tomato leaves using colonized agar plugs . 24 hours post inoculation ; leaves were post - fixed in osmium tetroxide , and embedded in Spurr ' s epoxy resin . A microtome was used to cut 400 nm sections . Toluidine blue stain was used to reveal fungal hyphae . H = hyphae . The dotted line represents the leading edge of the visible lesion . Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software , version 2 . 2 . 1 . 227 .

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

Figure 2Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.S. sclerotiorum A2 (A) and wild type (B) strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were post-fixed in osmium tetroxide, and embedded in Spurr's epoxy resin. A microtome was used to cut 400 nm sections. Toluidine blue stain was used to reveal fungal hyphae. H = hyphae. The dotted line represents the leading edge of the visible lesion. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227.


Figure 3S . sclerotiorum A2 strain induces autophagic structures in plants . S . sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs . 24 hours post inoculation ; leaves were stained with 100 µM final concentration of MDC ( Sigma ) in PBS for 30 min . Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope ( Olympus systems , Germany ) , with an excitation wavelength of 335 nm and an emission wavelength of 508 nm . Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software , version 2 . 2 . 1 . 227 . Scale bar = 10 µm .

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

Figure 3S. sclerotiorum A2 strain induces autophagic structures in plants.S. sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were stained with 100 µM final concentration of MDC (Sigma) in PBS for 30 min. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope (Olympus systems, Germany), with an excitation wavelength of 335 nm and an emission wavelength of 508 nm. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227. Scale bar = 10 µm.


Figure 4Transmission Electron Microscopy ( TEM ) fungal inoculated tomato leaves . Representative TEM images from four independent experiments . ( A , G ) ; Healthy non - inoculated leaf tissue . ( B - F ) Tomato leaves inoculated with the OA deficient A2 strain . ( H , I ) Tomato leaves inoculated with wild type S . sclerotiorum . Arrows , autolysosomal / autophagosomal - like structures ; C , chloroplast ; V , vacuole ; N , nucleus ; Circle , active dismantlement of chloroplast ; Rectangle , chromatin condensation within the nucleus . Black scale bars = 2 µm , white scale bars = 1 µm . Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV . Digital images were recorded with a MegaViewIII digital camera operated with iTEM software .

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

Figure 4Transmission Electron Microscopy (TEM) fungal inoculated tomato leaves.Representative TEM images from four independent experiments. (A, G); Healthy non-inoculated leaf tissue. (B-F) Tomato leaves inoculated with the OA deficient A2 strain. (H,I) Tomato leaves inoculated with wild type S. sclerotiorum. Arrows, autolysosomal/autophagosomal-like structures; C, chloroplast; V, vacuole; N, nucleus; Circle, active dismantlement of chloroplast; Rectangle, chromatin condensation within the nucleus. Black scale bars = 2 µm, white scale bars = 1 µm. Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were recorded with a MegaViewIII digital camera operated with iTEM software.


Figure 5 ( A , B ) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col - 0 and select Arabidopsis autophagy mutant plants . These mutants showed enhanced susceptibility to the normally non - pathogenic A2 strain . Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation . ( C ) Tomato leaves were either pre - infiltrated with water ( control ) or autophagy inhibitors Wortmannin , LY294002 , Chloroquine ( CQ ) , and 3 - methyladenine ( 3 - MA ) . Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection . ( B ) 48 hours post inoculation ; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol ( 1 : 3 , v / v ) . Images were taken 48 hours post inoculation .

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

Figure 5(A,B) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col-0 and select Arabidopsis autophagy mutant plants. These mutants showed enhanced susceptibility to the normally non-pathogenic A2 strain. Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation.(C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.


Figure 6 ( A ) NBT treated Arabidopsis ( Col - 0 and two independent atg8a mutant lines ) following agar plug inoculation with the A2 mutant . Images were collected 48 hours post inoculation . Dotted lines represent the edge of the observable legion . ( B ) RT - PCR was used to evaluate the transcript levels of three catalases ( CAT1 , 2 , and 3 ) and three superoxide dismutases in Col - 0 plants following inoculation with wild type S . sclerotiorum ( black bars ) and the A2 mutant strain ( grey bars ) . * > 2 fold change , * * > 5 fold change .

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

Figure 6(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion.(B) RT-PCR was used to evaluate the transcript levels of three catalases (CAT1, 2, and 3) and three superoxide dismutases in Col-0 plants following inoculation with wild type S. sclerotiorum (black bars) and the A2 mutant strain (grey bars). *>2 fold change, **>5 fold change.


Figure 1 ( C ) TNFemARE / ARE mice show significantly elevated serum levels of TNF ( wild - type n = 5 , TNFemARE / + n = 5 , TNFemARE / ARE n = 5 ) . Data information : data are represented as Mean + / - SEM , n = biological replicates . one - way ANOVA test was used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 . ( D ) Histologic H & E sections of ileum and hind paw indicate severe ileitis and arthritis in 20 - 30 w / o SPF TNFemARE / ARE mice ( Scale bars upper panel : 100 μm , scale bars lower panel : 500 μm ) . ( G ) tSNE analysis performed on CD45 + lineage ( excluding CD3 + , CD19 + , NK1 . 1 + fractions ) of lamina propria of ileum samples ( n = 5 / genotype ) . ( H ) Ileal flow cytometry data of 25 w / o TNFemARE mice show a highly activated innate immune system ( n = 5 / genotype ) . Data information : data are represented as Mean + / - SEM , n = biological replicates . one - way ANOVA test was used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 . ( J ) Ileal flow cytometry data show an increase of CD4 + cells in transgenic mice , which can be designated to the enrichment of the CD4 + RORgt + ( Th17 ) cell population ( n = 5 / genotype ) . Data information : data are represented as Mean + / - SEM , n = biological replicates . one - way ANOVA test was used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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

Figure 1(C) TNFemARE/ARE mice show significantly elevated serum levels of TNF (wild-type n=5, TNFemARE/+ n=5, TNFemARE/ARE n=5). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(D) Histologic H&E sections of ileum and hind paw indicate severe ileitis and arthritis in 20-30 w/o SPF TNFemARE/ARE mice (Scale bars upper panel: 100 μm, scale bars lower panel: 500 μm).(G) tSNE analysis performed on CD45+ lineage (excluding CD3+, CD19+, NK1.1+ fractions) of lamina propria of ileum samples (n=5/genotype).(H) Ileal flow cytometry data of 25 w/o TNFemARE mice show a highly activated innate immune system (n=5/genotype). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(J) Ileal flow cytometry data show an increase of CD4+ cells in transgenic mice, which can be designated to the enrichment of the CD4+RORgt+ (Th17) cell population (n=5/genotype). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


Figure 2 ( C ) Serum levels of TNF are elevated in hetero - and homozygous transgenic mice , in both SPF and GF conditions ( n = 5 mice / group , except for GF wild - type n = 4 ) . Data information : data are represented as Mean + / - SEM , n = biological replicates , two - way ANOVA test used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 . ( D ) Quantitative real - time PCR reveals increased expression of the tnf gene in the ileum in homozygous SPF and GF mice ( SPF wild - type n = 10 , SPF TNFemARE / + n = 10 , SPF TNFemARE / ARE n = 13 ; GF wild - type n = 3 , GF TNFemARE / + n = 4 , GF TNFemARE / ARE n = 4 ) . Data information : data are represented as Mean + / - SEM , n = biological replicates , two - way ANOVA test used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 . ( F ) Comparing SPF versus GF H & E sections of the ileum shows complete rescue of gut inflammation under axenic conditions in 20 - 30 w / o TNFemARE / ARE mice ( Scale bars : 100 μm ) .

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

Figure 2(C) Serum levels of TNF are elevated in hetero- and homozygous transgenic mice, in both SPF and GF conditions (n=5 mice/group, except for GF wild-type n=4). Data information: data are represented as Mean +/- SEM, n=biological replicates, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(D) Quantitative real-time PCR reveals increased expression of the tnf gene in the ileum in homozygous SPF and GF mice (SPF wild-type n=10, SPF TNFemARE/+ n=10, SPF TNFemARE/ARE n=13; GF wild-type n=3, GF TNFemARE/+ n=4, GF TNFemARE/ARE n=4). Data information: data are represented as Mean +/- SEM, n=biological replicates, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.(F) Comparing SPF versus GF H&E sections of the ileum shows complete rescue of gut inflammation under axenic conditions in 20-30 w/o TNFemARE/ARE mice (Scale bars: 100 μm).


Figure 3 ( C ) 20 - 30 w / o SPF TNFemARE / ARE mice clearly suffer from inflammation in the synovio - entheseal complex , which is not rescued in age - matched GF mice . ( Scale bars : 200 μm ) ( F ) < em > μ < / em > CT images of tails of wild - type versus SPF and GF TNFemARE / ARE mouse display fusion of sacral vertebrae in homozygous SPF and GF mice , as indicated by yellow arrows .

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

Figure 3(C) 20-30 w/o SPF TNFemARE/ARE mice clearly suffer from inflammation in the synovio-entheseal complex, which is not rescued in age-matched GF mice. (Scale bars: 200 μm)(F) μCT images of tails of wild-type versus SPF and GF TNFemARE/ARE mouse display fusion of sacral vertebrae in homozygous SPF and GF mice, as indicated by yellow arrows.


Figure 4 ( B ) Gait analysis of TNFemARE mice reveals distinct moving patterns of SPF and GF TNFemARE / ARE mice . Homozygous mice tend to have smaller footprints , place their feet with lower mean intensity and generally have a smaller stride length . ( SPF wild - type n = 12 , SPF TNFemARE / + n = 12 , SPF TNFemARE / ARE n = 12 ; GF wild - type n = 8 , GF TNFemARE / + n = 20 , GF TNFemARE / ARE n = 12 ) Data information : For panel ( B ) , boxplots represent the 25 - and 75 - percentile with median values as central band , whiskers span min to max value range , n = biological replicates with every datapoint representing 1 paw , two - way ANOVA test used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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

Figure 4(B) Gait analysis of TNFemARE mice reveals distinct moving patterns of SPF and GF TNFemARE/ARE mice. Homozygous mice tend to have smaller footprints, place their feet with lower mean intensity and generally have a smaller stride length. (SPF wild-type n=12, SPF TNFemARE/+ n=12, SPF TNFemARE/ARE n=12; GF wild-type n=8, GF TNFemARE/+ n=20, GF TNFemARE/ARE n=12) Data information: For panel (B), boxplots represent the 25- and 75- percentile with median values as central band, whiskers span min to max value range, n=biological replicates with every datapoint representing 1 paw, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


Figure 5 ( C ) H & E sections of the calcaneo - cuboid joint of 5 - 15 w / o A20myel - KO mice indicate immune cell infiltrates in the synovium and the fat pad and severe bone marrow edema . ( Scale bars : 100 μm ) ( E ) Histologic images of hind paws of old ( 44 w / o ) GF wild - type ( left ) versus A20myel - KO ( right ) mice . The morphology of the paw is completely lost in the transgenic mouse due to severe inflammation . ( Scale bars : 200 μm ) ( F ) Splenomegaly in GF A20myel - KO mice of 15 w / o indicates systemic inflammation in this mouse model ( wild - type n = 7 , A20myel - KO n = 8 ) . Data information : For panels F ) , data are represented as Mean + / - SEM , n = biological replicates . For graph ( F ) , two - tailed unpaired t - test was used . ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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

Figure 5(C) H&E sections of the calcaneo-cuboid joint of 5-15 w/o A20myel-KO mice indicate immune cell infiltrates in the synovium and the fat pad and severe bone marrow edema. (Scale bars: 100 μm)(E) Histologic images of hind paws of old (44 w/o) GF wild-type (left) versus A20myel-KO (right) mice. The morphology of the paw is completely lost in the transgenic mouse due to severe inflammation. (Scale bars: 200 μm)(F) Splenomegaly in GF A20myel-KO mice of 15 w/o indicates systemic inflammation in this mouse model (wild-type n=7, A20myel-KO n=8). Data information: For panels F), data are represented as Mean +/- SEM, n=biological replicates. For graph (F), two-tailed unpaired t-test was used. ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


Figure 1 ( D , E ) representative confocal micrographs and quantification of LC3 - II accumulation in CTNSWT and CTNS - / - cells in presence and absence of 25 nM bafilomycin ( BafA1 ) for 4 hrs , respectively ( n = 3 ) . Scale bars are 20 µm . ( F , G , H ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio and SQSTM1 protein levels in CTNSWT , CTNS - / - , and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h , respectively ( n = 3 ) . ( I , J ) Representative confocal micrographs and quantification of DQ - BSA and BSA in CTNSWT , CTNS - / - , and CTNSPatient cells , respectively ( n = 7 - 14 quantified images ) . Scale bars are 20 µm .

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

Figure 1(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.


Figure 2 ( D ) List of metabolites that were shared and significantly altered in both the CTNS - / - and CTNSPatient cells compared to control cells ( n = 6 ) . ( I ) List of proteins that were significantly upregulated and downregulated in CTNS - / - cells compared to control cells ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , LIPA ; Lysosomal acid lipase , ACP2 ; Lysosomal acid phosphatase , CTSS ; Cathepsin S , CTSC ; Dipeptidyl peptidase , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , SORT1 ; Sortilin , CAT ; Catalase , CTSA ; Lysosomal protective protein , and SOD ; Superoxide dismutase .

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

Figure 2(D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6).(I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.


Figure 3 ( B , C , D ) ROS production ( ROS / mg protein ) in CTNSWT , CTNS ‑ / - , and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG ( 4 hrs ) , respectively ( n = 3 ) . Representative confocal micrographs ( scale bars are 20 µm ) of caspase 3 / 7 activation in DMKG ( 2 mM ) - treated CTNSWT , CTNS ‑ / - , and CTNSPatient cells for 24 hrs ( n = 3 ) . ( H , I ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio in CTNSWT , CTNS ‑ / - , and CTNSPatient cells cultured in the presence or in the absence of BafA1 ( 25 nM ) and DMKG ( 2 mM ) for 4 hrs , respectively ( n = 3 ) .

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

Figure 3(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).


Figure 4 ( E ) , Proteomic analysis of CTNS - / - cells treated with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide , respectively ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , GLUD1 ; GLUD2 ; Mitochondrial glutamate dehydrogenase 1 / 2 , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , GSTK1 ; Glutathione S - transferase kappa 1 , COX6B1 ; Cytochrome c oxidase subunit 6B1 , ACACA ; Acetyl - CoA carboxylase 1 - Biotin carboxylase , GLS ; Glutaminase kidney isoform , mitochondrial , CASP3 ; Caspase - 3 , NPC1 ; Niemann - Pick C1 protein .

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

Figure 4(E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.


Figure 5 ( D , E ) Representative confocal micrographs and quantification of DQ - BSA in CTNSWT , and CTNS - / - cells upon treatment with bicalutamide . Scale bars are 20 µm ( n = 8 - 14 quantified images ) . ( F ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in CTNS - / - in the absence of the drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 6 ) , bicalutamide ( n = 6 ) , and a combination of cysteamine and bicalutamide ( n = 6 ) . ( G ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in CTNSPatient in the absence of the drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 4 ) , bicalutamide ( n = 4 ) , and a combination of cysteamine and bicalutamide ( n = 6 ) .

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

Figure 5(D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images).(F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6). (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).


Figure 6 ( A ) Immunocytochemistry of patient - derived cystinotic tubuloids ( CNTSPatient - 1 and CTNSPatient - 2 ) and healthy kidney tissue - derived control tubuloids ( CNTSWT - 1 and CTNSWT - 2 ) for PAX8 , TP63 and F - actin . Scale bar = 100 µm . ( C , D ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in two different patient - derived cystinotic tubuloids in the absence of the drugs ( NT ) or upon treatment with cysteamine ( 100 μM ) , bicalutamide ( 35 μM ) or cysteamine ( 100 μM ) - bicalutamide ( 35 μM ) combination treatment ( n = 3 ) . ( E ) αKG levels measured in patient - derived cystinotic tubuloids ( CNTSPatient - 1 and CTNSPatient - 2 ) in the absence of the drugs ( NT ) or upon treatment with cysteamine , bicalutamide or cysteamine - bicalutamide combination treatment using metabolomics ( n = 3 ) . ( I ) Survival rates in ctns - / - zebrafish upon treatment with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos per group ) . ( K ) Hatching rates in surviving ctns - / - zebrafish evaluated at 72 and 96 hrs post fertilization ( hpf ) with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos per group ) .

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

Figure 6(A) Immunocytochemistry of patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) and healthy kidney tissue-derived control tubuloids (CNTSWT-1 and CTNSWT-2) for PAX8, TP63 and F-actin. Scale bar = 100 µm.(C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3). (E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3). (I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).(K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).


Figure 1A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito - QC . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 5 µm in ( A ) B N2a cells expressing mito - QC and myc - Parkin together with hTau - V5 , hP301L - V5 or an empty vector control . Treatment with CCCP ( 8 µM , 17 h ) induces mitophagy in control cells , as indicated by a decrease in the GFP / mCherry fluorescence ratio , but not in hTau or hP301L cells . Deconvolution ( Classic Maximum Likelihood Estimation ) was applied to images for display only . C Quantification of GFP / mCherry fluorescence intensity per cell . Data were analysed by 2 - way ANOVA , showing significant main effects of CCCP treatment , F ( 1 , 314 ) = 15 . 66 , p < 0 . 0001 , and tau expression , F ( 2 , 314 ) = 12 . 26 , p < 0 . 0001 , and a significant interaction effect , F ( 2 , 314 ) = 6 . 137 , p = 0 . 0024 , n = 40 - 73 cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 10 µmD Cells expressing mito - QC and myc - Parkin together with hTau - V5 , hP301L - V5 or an empty vector control , treated with a mixture of antimycin and oligomycin ( AO , 15 µM , 17 h ) . E Quantification of GFP / mCherry fluorescence intensity per cell . Data were analysed by 2 - way ANOVA , showing no significant main effects of AO treatment , F ( 1 , 212 ) = 0 . 564 , p = 0 . 4534 , or tau expression F ( 2 , 212 ) = 1 . 579 , p = 0 . 2087 , but a significant interaction effect , F ( 2 , 212 ) = 9 . 854 , p < 0 . 0001 , n = 16 - 50 cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 10

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

Figure 1A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito-QC. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 5 µm in (A)B N2a cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control. Treatment with CCCP (8 µM, 17 h) induces mitophagy in control cells, as indicated by a decrease in the GFP/mCherry fluorescence ratio, but not in hTau or hP301L cells. Deconvolution (Classic Maximum Likelihood Estimation) was applied to images for display only. C Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing significant main effects of CCCP treatment, F (1, 314) = 15.66, p < 0.0001, and tau expression, F (2, 314) = 12.26, p < 0.0001, and a significant interaction effect, F (2, 314) = 6.137, p = 0.0024, n = 40-73 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µmD Cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control, treated with a mixture of antimycin and oligomycin (AO, 15 µM, 17 h). E Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing no significant main effects of AO treatment, F (1, 212) = 0.564, p = 0.4534, or tau expression F (2, 212) = 1.579, p = 0.2087, but a significant interaction effect, F (2, 212) = 9.854, p < 0.0001, n = 16-50 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm


Figure 2A Illustration of the image analysis process . The inverted " Union Jack " look - up table ( LUT ) was used to better visualise GFP - Parkin clusters , which were then manually selected as regions of interest ( ROIs ) and the corresponding mitochondrial fluorescence intensity was measured . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm . B Representative images of N2a cells expressing GFP - Parkin and hTau , hP301L or empty vector ( Control ) for 24 h , that were then treated with 10 μM CCCP for 4 h . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm . C Quantification of Parkin clusters / cell at 24 h post - transfection . Data were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 8 . 18 , p = 0 . 0168 , followed by Dunn ' s post - hoc analysis , n = 33 - 38 cells / group . D Quantification of Parkin clusters / cell at 48 h post - transfection . Data were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 19 . 2 , p < 0 . 0001 , followed by Dunn ' s post - hoc analysis , n = 37 - 55 cells / group cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm . E To assess colocalisation of Parkin clusters and mitochondria at 24 h post - transfection , Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated . Control r = 0 . 616 , p = 0 . 0001 ; hTau r = 0 . 217 , p = 0 . 2771 ; hP301L r = 0 . 548 , p = 0 . 0014 ( n = 27 - 34 cells / group ) . F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post - transfection . Control r = 0 . 434 , p = 0 . 0167 ; hTau r = 0 . 128 , p = 0 . 4941 ; hP301L r = - 0 . 299 , p = 0 . 2794 ( n = 15 - 31 cells / group ) . Note the different intensity scales between ( E ) and ( F ) reflect different bit depths of the images . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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

Figure 2A Illustration of the image analysis process. The inverted "Union Jack" look-up table (LUT) was used to better visualise GFP-Parkin clusters, which were then manually selected as regions of interest (ROIs) and the corresponding mitochondrial fluorescence intensity was measured. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.C Quantification of Parkin clusters/cell at 24 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 8.18, p = 0.0168, followed by Dunn's post-hoc analysis, n = 33-38 cells/group. D Quantification of Parkin clusters/cell at 48 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 19.2, p < 0.0001, followed by Dunn's post-hoc analysis, n = 37-55 cells/group cells/group. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.E To assess colocalisation of Parkin clusters and mitochondria at 24 h post-transfection, Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated. Control r = 0.616, p = 0.0001; hTau r = 0.217, p = 0.2771; hP301L r = 0.548, p = 0.0014 (n = 27-34 cells/ group). F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post-transfection. Control r = 0.434, p = 0.0167; hTau r = 0.128, p = 0.4941; hP301L r = -0.299, p = 0.2794 (n = 15-31 cells/ group). Note the different intensity scales between (E) and (F) reflect different bit depths of the images. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


Figure 3A FACS analysis of cells loaded with Rhodamine 123 . The geometric mean fluorescence was calculated and expressed as % of the empty vector control . A CCCP - treated cohort was included as a positive control , but not included in the statistical analysis . Data were analysed by one - way ANOVA , F ( 2 , 14 ) = 0 . 196 , p = 0 . 8245 . B FACS analysis of cells treated with CCCP after 24 h of tau expression . Data were analysed by one - way ANOVA , F ( 2 , 15 ) = 0 . 434 , p = 0 . 6555 . The DMSO negative control is shown but was not analysed statistically . Data information : Data are given as mean and SEM ( n = 5 - 6 samples / experimental group from two independent experiments ) . C Representative dot plots of cells loaded with Rhodamine 123 . The gates ( black line ) were set in unstained cells to distinguish Rhodamine - positive cells ( right half ) from Rhodamine - negative cells ( left half ) . Data information : Data are given as mean and SEM ( n = 5 - 6 samples / experimental group from two independent experiments ) .

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

Figure 3A FACS analysis of cells loaded with Rhodamine 123. The geometric mean fluorescence was calculated and expressed as % of the empty vector control. A CCCP-treated cohort was included as a positive control, but not included in the statistical analysis. Data were analysed by one-way ANOVA, F (2, 14) = 0.196, p = 0.8245. B FACS analysis of cells treated with CCCP after 24 h of tau expression. Data were analysed by one-way ANOVA, F (2, 15) = 0.434, p = 0.6555. The DMSO negative control is shown but was not analysed statistically. Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).C Representative dot plots of cells loaded with Rhodamine 123. The gates (black line) were set in unstained cells to distinguish Rhodamine-positive cells (right half) from Rhodamine-negative cells (left half). Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).


Figure 4A Immunostaining for microtubules after transient expression of hTau and hP301L . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm . B Taxol induces microtubule bundling to a similar extent to tau expression . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm . C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment ( Mann - Whitney test , U = 22448 , p < 0 . 0001 , n = 224 , 273 cells / group for CCCP only and CCCP + Taxol , respectively ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm . D Immunoblot analysis of acetylated tubulin ( Ace - Tubulin ) following expression of hTau and hP301L for 24 h ( One - way ANOVA , F ( 2 , 9 ) = 0 . 0432 , p = 0 . 9579 ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm . E Immunoblot analysis of tyrosinated tubulin ( Tyr - Tubulin ) following expression of hTau and hP301L for 24 h ( One - way ANOVA , F ( 2 , 9 ) = 0 . 0402 , p = 0 . 9608 ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm . F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide ( JPL ) ( Mann - Whitney test , U = 16069 , p < 0 . 0001 , n = 224 , 199 cells / group for CCCP only and CCCP + JPL , respectively ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

Figure 4A Immunostaining for microtubules after transient expression of hTau and hP301L. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.B Taxol induces microtubule bundling to a similar extent to tau expression. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment (Mann-Whitney test, U = 22448, p < 0.0001, n = 224, 273 cells/group for CCCP only and CCCP + Taxol, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.D Immunoblot analysis of acetylated tubulin (Ace-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0432, p = 0.9579). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.E Immunoblot analysis of tyrosinated tubulin (Tyr-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0402, p = 0.9608). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide (JPL) (Mann-Whitney test, U = 16069, p <0.0001, n = 224, 199 cells/group for CCCP only and CCCP + JPL, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


Figure 5A Co - immunoprecipitations of V5 - tagged tau and GFP - Parkin . The V5 tag was used to pull down tau and the membrane was probed for V5 ( using an antibody raised in a different species ) and GFP . IP = immunoprecipitation , Flow = flow - through lysate following removal of beads . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . B Maximum projection images of proximity ligation assays ( PLAs ) for tau and endogenous Parkin in cells expressing hTau - GFP , hP301L - GFP or GFP . Inset = negative control ( antibodies to tau and histone H3 , which are not known binding partners ) . C Quantification of PLA signals in maximum projections normalised to the GFP signal . Results were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 27 . 2 , p < 0 . 0001 , followed by Dunn ' s post hoc analysis to test for differences from the GFP control , n = 17 cells in GFP condition , 14 cells in hTau condition and 27 cells in hP301L condition . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . F , G PLA for Parkin and ∆ Tau , revealing interactions via the projection domain of tau . Data were analysed with a Mann - Whitney test ( U = 185 , p = 0 . 9224 , n = 21 , 18 cells / group for hTau and ∆ Tau , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . H , I PLA for Parkin and the microtubule - binding domain construct ( MTBD ) . Data were analysed with a t - test ( t = 5 . 226 , p < 0 . 0001 , n = 16 , 17 cells / group for hTau and MTBD , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . J , K Cells expressing tau constructs together with GFP - Parkin ( 24 h ) with and without CCCP treatment . Parkin clusters were counted , illustrating that Parkin translocation was impaired by ∆ Tau , but not MTBD . Data were analysed with a Kruskal - Wallis test χ2 ( 2 ) = 10 . 4 , p = 0 . 0056 , followed by Dunn ' s post - hoc , n = 67 , 42 , 53 cells / group for for hTau , ∆ Tau , and MTBD , respectively . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . L The V5 fluorescence intensity in each cell analysed in ( K ) ( Mann - Whitney test , U = 953 , p = 0 . 2332 , n = 42 , 53 cells / group for ∆ Tau and MTBD , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . M Cells were transfected with GFP - Parkin and hTau - V5 , hP301L - V5 or an empty vector control , and treated with CCCP . PLAs were performed for tau and Parkin . Notably , at Parkin clusters ( arrow ) , no interactions between tau and Parkin were evident . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 . N 3D reconstructions in IMARIS , illustrating a lack of PLA signals in Parkin clusters . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

Figure 5A Co-immunoprecipitations of V5-tagged tau and GFP-Parkin. The V5 tag was used to pull down tau and the membrane was probed for V5 (using an antibody raised in a different species) and GFP. IP = immunoprecipitation, Flow = flow-through lysate following removal of beads. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.B Maximum projection images of proximity ligation assays (PLAs) for tau and endogenous Parkin in cells expressing hTau-GFP, hP301L-GFP or GFP. Inset = negative control (antibodies to tau and histone H3, which are not known binding partners). C Quantification of PLA signals in maximum projections normalised to the GFP signal. Results were analysed with a Kruskal-Wallis test, χ2 (2) = 27.2, p < 0.0001, followed by Dunn's post hoc analysis to test for differences from the GFP control, n = 17 cells in GFP condition, 14 cells in hTau condition and 27 cells in hP301L condition. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.F, G PLA for Parkin and ∆Tau, revealing interactions via the projection domain of tau. Data were analysed with a Mann-Whitney test (U = 185, p = 0.9224, n = 21, 18 cells/group for hTau and ∆Tau, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.H, I PLA for Parkin and the microtubule-binding domain construct (MTBD). Data were analysed with a t-test (t = 5.226, p < 0.0001, n = 16, 17 cells/group for hTau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.J, K Cells expressing tau constructs together with GFP-Parkin (24 h) with and without CCCP treatment. Parkin clusters were counted, illustrating that Parkin translocation was impaired by ∆Tau, but not MTBD. Data were analysed with a Kruskal-Wallis test χ2 (2) = 10.4, p = 0.0056, followed by Dunn's post-hoc, n = 67, 42, 53 cells/ group for for hTau, ∆Tau, and MTBD, respectively. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.L The V5 fluorescence intensity in each cell analysed in (K) (Mann-Whitney test, U = 953, p = 0.2332, n = 42, 53 cells/group for ∆Tau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.M Cells were transfected with GFP-Parkin and hTau-V5, hP301L-V5 or an empty vector control, and treated with CCCP. PLAs were performed for tau and Parkin. Notably, at Parkin clusters (arrow), no interactions between tau and Parkin were evident. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.N 3D reconstructions in IMARIS, illustrating a lack of PLA signals in Parkin clusters. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


Figure 6Expression pattern of the mito - Rosella biosensor in the nervous system . B Analysis pipeline of mitochondria in the C . elegans ventral nerve cord . Mitochondria were selected by choosing a threshold in the dsRed channel . This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured . C , D Analysis of mitophagy in worms transgenic for mito - Rosella but not tau ( WT ) , that were treated with NaN3 ( 8 mM , 1 h ) or CCCP ( 15 µM , 2 h ) . Data were analysed by unpaired t test , NaN3 t = 5 . 56 , p < 0 . 0001 ; CCCP t = 3 . 92 p = 0 . 0002 . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral cordE , F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment ( unpaired t test , t = 1 . 62 , p = 0 . 1126 ) and CCCP treatment ( Mann - Whitney test , U = 113 , p = 0 . 002 ) . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral cordG , H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 ( Mann - Whitney test , U = 126 , p = 0 . 2711 ) or CCCP ( Mann - Whitney test , U = 375 , p = 0 . 3697 ) treatment . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral cordI Tau expression in worm lysates , as detected using the human tau - specific antibody HT7 .

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

Figure 6Expression pattern of the mito-Rosella biosensor in the nervous system.B Analysis pipeline of mitochondria in the C. elegans ventral nerve cord. Mitochondria were selected by choosing a threshold in the dsRed channel. This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured.C, D Analysis of mitophagy in worms transgenic for mito-Rosella but not tau (WT), that were treated with NaN3 (8 mM, 1 h) or CCCP (15 µM, 2 h). Data were analysed by unpaired t test, NaN3 t = 5.56, p < 0.0001; CCCP t = 3.92 p = 0.0002. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cordE, F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment (unpaired t test, t = 1.62, p = 0.1126) and CCCP treatment (Mann-Whitney test, U = 113, p = 0.002). Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cordG, H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 (Mann-Whitney test, U = 126, p = 0.2711) or CCCP (Mann-Whitney test, U = 375, p = 0.3697) treatment. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cordI Tau expression in worm lysates, as detected using the human tau-specific antibody HT7.