C - E ) of interaction of DRP1 with variants of BAX on % with detectable RA - BAX / GB - DRP1 signal ( interacting ) normalized to number of mito - BFP positive . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) of n = 3 independent experiments ( with n = 100 each ) . of significance were determined by paired two - Student ´ s t - ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) compared to BAX wild type ( WT ) .

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C-E) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to BAX wild type (WT).


G ) Representative confocal microscopy of U2OS BAX / BAK DKO of interactions between RA - BAX GB - DRP1 variants , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm .

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G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.


H ) of interaction of DRP1 variants with BAX on % with detectable RA - BAX / GB - DRP1 signal ( interacting ) normalized to number of mito - BFP positive . Box plots represent interquartile ( box ) , median ( ) SD ( whiskers ) of n = 3 independent experiments ( with n = 100 each ) . Significance was tested using paired two - Student ´ s t - ( n . s . p > 0 . 05 ) compared to DRP1 WT .

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H) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Significance was tested using paired two-tailed Student´s t-test (n.s. p > 0.05) compared to DRP1 WT.


C ) Forced dimerization of BAX DRP1 in healthy induces their translocation to mitochondria foci formation . Confocal microscopy of U2OS BAX / BAK DKO transfected with FKBP - mCherry - DRP1 ( red ) FRB - EGFP - BAX ( green ) stained with MitoTracker Deep Red FM ( cyan ) were acquired before ( 0 min ) after induction of BAX / DRP1 dimerization ( 5 - 60 min ) . From top to down , rows correspond to signal of BAX DRP1 merge of both together with MitoTracker signal , respectively . Scale bar 20 µm .

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C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm.


E ) Induced dimerization of BAX DRP1 causes mitochondrial depolarization . U2OS BAX / BAK DKO were transfected as described in C ) stained with mitochondrial potential - sensitive dye MitoSpy NIR ( cyan ) . Confocal microscopy were acquired before ( 0 min ) after induced dimerization of BAX DRP1 ( 10 min ) . Scale Bar 20 µm . F ) of mitochondrial depolarization normalized to number of transfected before ( Ctrl ) after induced dimerization of BAX DRP1 ( Dim ) . Significance was tested using paired two - Student ´ s t - ( * p < 0 . 05 ) compared to non - induced control .

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E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm. F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control. G


G - K ) Induced dimerization of DRP1 with itself ( G ) , BAX with itself ( H ) , BAX with TOM20 ( I ) in U2OS BAX / BAK DKO , or BAX with DRP1 in HCT OctaKO ( J , K ) , transected with FKBP - FRB - mCherry DRP1 ( G , red ) , FKBP - FRB - EGFP - BAX ( H , green ) , TOM20 - mCherry - FKBP ( red ) FRB - EGFP - BAX ( green , I ) , respectively . Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR ( cyan ) as indicated . Scale bar 20 µm ( G - I ) 10 µm ( J , K ) . are representative of n = 3 independent experiments .

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G-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.


Representative H & E immunohistochemical stainings of CK5 / 6 p63 in pulmonary head - - neck squamous - carcinomas . Scale bar : 500 µm .

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Representative H&E and immunohistochemical stainings of CK5/6 and p63 in pulmonary and head-and-neck squamous-cell carcinomas. Scale bar: 500 µm.


showing somatic mutations in defined pathways HPV status for HNSCC SQCLC cases analysed . ( Blue , no mutation / no HPV detected ; red , mutation / HPV detected ; , no data available )

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Heatmap showing somatic mutations in defined pathways and HPV status for all HNSCC and SQCLC cases analysed. (Blue, no mutation/ no HPV detected; red, mutation/HPV detected; white, no data available)


Comparison of mutation rates in HNSCC ( n = 279 ) SQCLC ( n = 178 ) cases from TCGA . Genes exhibiting a mutation rate > 0 . 25 in both cancer types are labelled .

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Comparison of mutation rates in HNSCC (n=279) and SQCLC (n=178) cases from TCGA. Genes exhibiting a mutation rate >0.25 in both cancer types are labelled.


Representative histogram showing SILAC distribution between Super - SILAC standard SQCLC ( left ) HNSCC ( right ) samples .

Representative histogram showing the SILAC ratio distribution between the Super-SILAC standard and SQCLC (left) and HNSCC (right) samples.


Boxplot showing numbers of quantified , derived from 44 SQCLC 30 HNSCC tissue samples . central in boxes represents median number of over samples , upper lower borders of boxes correspond to 25 % 75 % quantiles . Whiskers indicate minimum maximum .

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Boxplot showing the numbers of quantified proteins, derived from 44 SQCLC and 30 HNSCC tissue samples. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum.


Distribution of MS - signal intensities in 74 tissue samples of training cohort .

Distribution of MS-based protein signal intensities in all 74 tissue samples of the training cohort.


Principal - component of SQCLC ( n = 44 ; blue ) HNSCC ( n = 30 ; red ) expression . Shown are first two principal components , accounting for 8 . 51 % 8 . 03 % of total variance in data , respectively .

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Principal-component analysis of SQCLC (n=44; blue) and HNSCC (n=30; red) protein expression. Shown are the first two principal components, accounting for 8.51% and 8.03% of the total variance in the data, respectively.


Volcano plot adjusted p values for differential expression to averaged normalised SILAC ratios of two replicates . Red ( higher expression in HNSCC ) blue ( higher expression in SQCLC ) dots indicate significantly regulated ( p < 0 . 05 ) .

Volcano plot adjusted p values for differential protein expression to averaged normalised SILAC ratios of two replicates. Red (higher expression in HNSCC) and blue (higher expression in SQCLC) dots indicate significantly regulated proteins (p < 0.05).


Two - class comparison of genetic dependencies from a publically available genome - scale CRISPR - Cas9 screen of HNSCC ( 12 ) versus SQCLC ( 10 lines ) identified a subset of differentially expressed that were also differential dependencies in these types . x - axis represents effect of mean difference of dependency scores in HNSCC compared to SQCLC lines . Positive effect indicates a greater mean dependency in HNSCC ; negative effect indicates a greater mean dependency in SQCLC . y - axis represents statistical significance of differential enrichment calculated as - log10 ( p - value ) from a two - sided t - . p - values used for this plot are uncorrected for multiple hypothesis testing . Highlighted in green are genetic dependencies that were also identified as differentially expressed in our study .

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Two-class comparison of genetic dependencies from a publically available genome-scale CRISPR-Cas9 screen of HNSCC (12 cell line) versus SQCLC (10 cell lines) identified a subset of differentially expressed proteins that were also differential dependencies in these tumor types. The x-axis represents the effect size of the mean difference of dependency scores in HNSCC compared to SQCLC cell lines. Positive effect size indicates a greater mean dependency in HNSCC; negative effect size indicates a greater mean dependency in SQCLC. The y-axis represents the statistical significance of differential enrichment calculated as -log10(p-value) from a two-sided t-test. The p-values used for this plot are uncorrected for multiple hypothesis testing. Highlighted in green are genetic dependencies that were also identified as differentially expressed proteins in our study.


Immunohistochemical of expression of HMGCS - 1 ( p = 0 . 0014 ) , FTL ( p = 0 . 0001 ) ( C ) , LGALS7 ( p = 0 . 0001 ) FAM83H ( p = 0 . 0001 ) ( D ) in an independent cohort of 212 SQCLC 343 HNSCC cases . Scale bar indicates 100µm . Shown are mean values standard deviation . Statistical significance was assessed using Wilcoxon - Mann - Whitney .

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Immunohistochemical analysis of the expression of HMGCS-1 (p=0.0014),FTL (p=0.0001) (C),LGALS7 (p=0.0001) and FAM83H (p=0.0001)(D) in an independent cohort of 212 SQCLC and 343 HNSCC cases. Scale bar indicates 100µm. Shown are mean values and standard deviation. Statistical significance was assessed using Wilcoxon-Mann-Whitney test.


Boxplot showing number of quantified derived from 19 SQCLC , 19 HNSCC 51 squamous - lung tumours of unknown origin derived from HNSCC . central in boxes represents median number of over samples , upper lower borders of boxes correspond to 25 % 75 % quantiles . Whiskers indicate minimum maximum .

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Boxplot showing the number of quantified proteins derived from 19 SQCLC, 19 HNSCC and 51 squamous-cell lung tumours of unknown origin derived from HNSCC patients. The central line in the boxes represents the median number of proteins over all samples, upper and lower borders of boxes correspond to 25% and 75% quantiles. Whiskers indicate minimum and maximum.


Representative H & E immunohistochemical stainings of CK5 / 6 p63 in squamous - lung tumours of unknown origin derived from HNSCC . Scale bar : 500 µm .

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Representative H&E and immunohistochemical stainings of CK5/6 and p63 in squamous-cell lung tumours of unknown origin derived from HNSCC patients. Scale bar: 500 µm.


Kaplan - Meier of overall survival ( OS ) in which with lung tumours were grouped according to clinical classification ( left , median survival SQCLC : 55 months ; metHNSCC : 31 months ) or proteomic classification ( right , median survival SQCLC : 55 months ; metHNSCC : 8 months ) of tumour as metHNSCC or SQCLC . p value is from a log - rank . Kaplan - Meier of overall survival ( OS ) in which only that were independent from training cohort were grouped according to clinical classification ( left , median survival SQCLC : 84 months ; metHNSCC : 35 months ) or proteomic classification ( right , median survival SQCLC : 84 months ; metHNSCC : 5 months ) of tumour as metHNSCC or SQCLC . p value is from a log - rank .

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Kaplan-Meier analysis of overall survival (OS) in which all patients with lung tumours were grouped according to the clinical classification (left, median survival SQCLC: 55 months; metHNSCC: 31 months) or proteomic classification (right, median survival SQCLC: 55 months; metHNSCC: 8 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test. Kaplan-Meier analysis of overall survival (OS) in which only the patients that were independent from the training cohort were grouped according to the clinical classification (left, median survival SQCLC: 84 months; metHNSCC: 35 months) or proteomic classification (right, median survival SQCLC: 84 months; metHNSCC: 5 months) of the tumour as metHNSCC or SQCLC. The p value is from a log-rank test.


A cycle by flow cytometry ( EdU vs . TO - PRO - 3 ) in complete medium ( ENRAD ) or in absence of A83 - 01 ( 24 hours ) .

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A Cell cycle analysis by flow cytometry (EdU vs. TO-PRO-3) in complete medium (ENRAD) or in the absence of A83-01 (24 hours).


B Detection of secreted by mouse prostate organoids in culture on click - chemistry enrichment mass spectrometry ( n = 4 biological replicates ; data points are shown with crossing representing mean value ) .

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B Detection of proteins secreted by mouse prostate organoids in culture based on click-chemistry enrichment and mass spectrometry analysis (n=4 biological replicates; data points are shown with crossing line representing mean value).


C IF for selected markers in mouse prostate tissue organoid sections ( scale bar = 50 μm ) .

C IF analysis for selected markers in mouse prostate tissue and organoid sections (scale bar = 50 μm).


D Representative stereoscopic of mouse prostate organoids cultured in absence of A83 - 01 , with or without Follistatin ( Fst , 500 ng / mL , 6 days ) , or with or without Activin A neutralizing antibody MAB3381 ( 1 μg / mL , 6 days ) . Scale bar = 1

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D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83-01, with or without Follistatin (Fst, 500 ng/mL, 6 days), or with or without the Activin A neutralizing antibody MAB3381 (1 μg/mL, 6 days). Scale bar = 1 mm


E Western blot ( WB ) in mouse prostate organoids for selected canonical non - canonical Tgf - β signalling mediators , cycle inhibitor p21 ( Activin A , 50 ng / mL ; Tgfb1 , 500 ng / mL ; 24 hours ) .

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E Western blot (WB) analysis in mouse prostate organoids for selected canonical and non-canonical Tgf-β signalling mediators, and the cell cycle inhibitor p21 (Activin A, 50 ng/mL; Tgfb1, 500 ng/mL; 24 hours).


G Representative stereoscopic of mouse prostate organoids following treatment with Takinib ( Tak1 inhibitor ; 5 μM , 6 days ) , SB203580 ( p38α / β inhibitor ; 10 μM , 6 days ) or structurally unrelated Ralimetinib ( p38α / β inhibitor ; 1 μM , 7 days ) . Scale bar = 1

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G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib (Tak1 inhibitor; 5 μM, 6 days), SB203580 (p38α/β inhibitor; 10 μM, 6 days) or the structurally unrelated Ralimetinib (p38α/β inhibitor; 1 μM, 7 days). Scale bar = 1 mm


H / fractionation western blot in mouse prostate organoids for selected signalling mediators in presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 hours ) .

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H Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 hours).


I Gene set enrichment ( GSEA ) plots displaying significant enrichment for inflammatory response Necrosis Factor ( TNF ) signalling in mouse prostate organoids cultured without A83 - 01 ( 24 hours ) versus complete medium ( ENRAD ) .

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I Gene set enrichment analysis (GSEA) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor (TNF) signalling in mouse prostate organoids cultured without A83-01 (24 hours) versus complete medium (ENRAD).


J / fractionation western blot in mouse prostate organoids for selected cycle regulators , in presence or absence of SB203580 ( p38α / β inhibitor ; 10 μM , 24 or 48 hours ) .

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J Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators, in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 or 48 hours).


A Diagram depicting expansion of mouse prostate organoid cultures in absence of Tgf - β ligand inhibitor A83 - 01 ( n = 3 biological replicates per lobe - specific organoid culture ; arrow = continuous expansion ; dot = passage ; cross = culture loss ) .

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A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf-β ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific organoid culture; arrow = continuous expansion; dot = passage; cross = culture loss).


B Representative stereoscopic of C57BL / 6J DLP # 1 # 3 mouse prostate organoid lines during adaptation in absence of A83 - 01 . Scale bar = 1

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B Representative stereoscopic images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mm


D Gene set enrichment ( GSEA ) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines ( C57 # 1 DLP , top ; C57 # 3 DLP , bottom ) adapted to grow in absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) .

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D Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD).


E Western blot in C57 # 1 C57 # 3 DLP mouse prostate organoid lines adapted to grow in absence of A83 - 01 vs . normal control organoids cultured in complete medium ( ENRAD ) . Immunoblots are displayed for Chek1 ( ATR signalling mediator ) , Trp53 p21 .

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E Western blot analysis in C57#1 and C57#3 DLP mouse prostate organoid lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). Immunoblots are displayed for Chek1 (ATR signalling mediator), Trp53 and p21.


F Representative spectral karyotype ( SKY ) of metaphase spreads obtained from C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread genomic instability is observed following adaptation . Scale bars = 5 µm . G - H Representative telomere FISH - ( n = 2 biological replicates , 20 metaphases per condition ) - in C57 # 1 DLP mouse prostate organoids cultured in normal conditions ( ENRAD ) or upon adaptation without A83 - 01 . Widespread telomeric instability is observed following adaptation ( arrows indicate telomere doublets , One - way ANOVA , scale bars = 10 µm ) .

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F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm. G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).


Plasma of 13 biomarkers belonging to clusters 3 , 4 , 6 ( see Table 2 ) in healthy subjects ( n = 16 ) PDAC ( n = 25 ) , * p < 0 . 01 ( Mann - Whitney ) . Data are expressed as a scatter - plot , mean ± . p - values ( for each cluster ) were calculated with Wilcoxon Rank Sum indicate a significant association between each cluster PDAC .

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Plasma levels of the 13 biomarkers belonging to clusters 3, 4, 6 (see Table 2) in healthy subjects (n=16) and PDAC patients (n=25), *p<0.01 (Mann-Whitney). Data are expressed as a scatter-plot, mean ± SEM. p-values (for each cluster) were calculated with the Wilcoxon Rank Sum Test and indicate a significant association between each cluster and PDAC.


A ) of TIMP1 , MMP7 TSP2 in plasma of KC mice ( n = 7 - 9 ) at 60 , 120 , 180 , 240 330 days of KPC mice ( n = 3 - 8 ) at 30 , 90 150 days of ( mean ± ) . * p < 0 . 05 ( Mann - Whitney ) . exact n p - values are indicated in Appendix Table S3

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A) Levels of TIMP1, MMP7 and TSP2 in plasma of KC mice (n=7-9) at 60, 120, 180, 240 and 330 days of age and KPC mice (n=3-8) at 30, 90 and 150 days of age (mean ± SEM). *p<0.05 (Mann-Whitney). The exact n and p-values are indicated in Appendix Table S3


B ) of TIMP1 , MMP7 TSP2 in plasma of healthy mice ( n = 15 ) , mice with chronic pancreatitis at 150 days of ( n = 19 ) , KC mice at 330 days of ( control PdxCre n = 3 - 4 ; KC n = 7 - 8 ) , KPC mice at 150 days of ( control PdxCre n = 4 - 7 ; KPC n = 4 ) . exact n is indicated in Appendix Table S3B . Box plots extends from 25th to 75th percentiles , whiskers extends from min to max horizontal lines indicate median . p - values were calculated with 1 - way ANOVA with Tukey " s multiple comparison

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

B) Levels of TIMP1, MMP7 and TSP2 in plasma of healthy mice (n=15), mice with chronic pancreatitis at 150 days of age (n=19), KC mice at 330 days of age (control PdxCre n=3-4; KC n=7-8), KPC mice at 150 days of age (control PdxCre n=4-7; KPC n=4). The exact n is indicated in Appendix Table S3B. Box plots extends from 25th to 75th percentiles, whiskers extends from min to max and horizontal lines indicate median. p-values were calculated with 1-way ANOVA with Tukey"s multiple comparison test


C ) Histological of pancreas from PdxCre KC mice with different grades of PanIN lesions at 330 days of . Anti - TIMP1 , anti - MMP7 , anti - TSP2 anti - CCN2 of a representative KC PanIN lesion ( 200X , scale bars : 100μm )

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

C) Histological analysis of pancreas from PdxCre and KC mice with different grades of PanIN lesions at 330 days of age. Anti-TIMP1, anti-MMP7, anti-TSP2 and anti-CCN2 staining of a representative KC PanIN lesion (200X, scale bars: 100μm)


Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) . A ) of TIMP1 , MMP7 TSP2 in plasma of mice bearing orthotopic HuPa8 at 30 , 90 150 days after transplantation ( mean ± ; n ≥ 4 for each group ) . * p < 0 . 05 , ( Mann - Whitney ) . exact n p - values are indicated in Table S5A

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

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments). A) Levels of TIMP1, MMP7 and TSP2 in plasma of mice bearing orthotopic HuPa8 at 30, 90 and 150 days after tumor transplantation (mean ± SEM; n≥4 for each group). *p<0.05, (Mann-Whitney). The exact n and p-values are indicated in Table S5A


Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) B ) Magnetic resonance imaging of HuPa8 . Representative are shown , dotted lines indicate masses

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

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) B) Magnetic resonance imaging of HuPa8. Representative images are shown, the white dotted lines indicate tumor masses


. Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) C ) growth over time measured by MRI ; each black arrow indicates one treatment ( mean ± ; n = 4 for each group ; 2 - way ANOVA with Tukey " s multiple comparison )

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

. Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) C) Tumor growth over time measured by MRI; each black arrow indicates one treatment (mean ± SEM; n=4 for each group; 2-way ANOVA with Tukey"s multiple comparison test)


Responsiveness to gemcitabine ( GEM ) , either alone or combined with Nab - paclitaxel ( NAB - P ) , was evaluated in mice bearing orthotopic HuPa8 in pancreas . Nab - paclitaxel ( 25mg / kg ) gemcitabine ( 150mg / kg ) were given i . v . on days 1 8 of each 21 - day cycle ( for a total of 8 treatments ) D ) of 3 selected biomarkers in plasma of HuPa8 bearing mice , prior to ( day 80 ; at randomization ) , during ( day 120 ) after ( day 165 ) treatments ( mean ± ; n ≥ 4 for each group ; 2 - way ANOVA with Tukey " s multiple comparison ) . exact n is indicated in Appendix Table S5B

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

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments) D) Levels of the 3 selected biomarkers in plasma of HuPa8 bearing mice, prior to (day 80; at randomization), during (day 120) and after (day 165) the treatments (mean ± SEM; n≥4 for each group; 2-way ANOVA with Tukey"s multiple comparison test). The exact n is indicated in Appendix Table S5B


Immunoprecipitation was performed with GFP - binding immobilized on magnetic beads using extracts of Arabidopsis seedlings , expressing either YFP - MKKK7 or Lti6B . Seedlings were treated with 1 μM flg22 for indicated time . YFP - MKKK7 Lti6B - GFP were detected with an anti - GFP antibody while FLS2 was detected with an FLS - specific antibody . Upper panel shows ( co ) - immunoprecipated , lower panel shows input of . Arrowheads indicate position of of interest .

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

Immunoprecipitation was performed with GFP-binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings, expressing either YFP-MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated time. YFP-MKKK7 and Lti6B-GFP were detected with an anti-GFP antibody while FLS2 was detected with an FLS-specific antibody. Upper panel shows (co)-immunoprecipated proteins, lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.


Application of selected reaction monitoring ( SRM ) mass spectrometry to quantify phosphorylated peptides in extracts treated with 1 μM flg22 . Bars represent mean of endogenous phosphopeptide versus spiked - in synthetic phosphopeptide normalized to t = 0 with error bars ± ( n = 3 . ) . Asterisks indicate significant difference level compared to t = 0 ( student t - , * > 0 . 05 , * * > 0 . 01 * * * > 0 . 001 ) . color of each bar corresponds to different time points ( 0 minutes = blue , 5 minutes = red , 10 minutes = green , 20 minutes = purple , 30 minutes = blue ) . Above each graph is name phosphorylated residue ( in brackets ) is indicated below corresponding phosphopeptide is shown with Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation site indicated by " [ + 80 ] " .

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

Application of selected reaction monitoring (SRM) mass spectrometry to quantify phosphorylated peptides in cell extracts treated with 1 μM flg22. Bars represent the mean ratio of endogenous phosphopeptide versus spiked-in synthetic phosphopeptide normalized to t = 0 with error bars ± SEM (n=3.). Asterisks indicate significant difference level compared to t = 0 (student t-test, *>0.05, **>0.01 and ***>0.001). The color of each bar corresponds to the different time points (0 minutes = dark blue, 5 minutes = red, 10 minutes = green, 20 minutes = purple, 30 minutes = light blue). Above each graph is the protein name and the phosphorylated residue (in brackets) is indicated and below the corresponding phosphopeptide is shown with the Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation site indicated by "[+80]".


A ) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col - 0 in mkkk7 . extracts were made from seedlings treated with 1 μM flg22 samples were taken at t = 0 , 10 30 min post induction . p44 / 42 antibody was used to detect phosphorylated MAPKs . Position of phosphorylated MAPKs is indicated at right . Equal loading of is shown with an α - Actin antibody as a loading control ( bottom panel ) . Three biological replicates were done with identical

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

A) Immunoblot analyses showing MAPK phosphorylation after flg22 induction in Col-0 and in mkkk7. Protein extracts were made from seedlings treated with 1 μM flg22 and samples were taken at t=0, 10 and 30 min post induction. The p44/42 antibody was used to detect phosphorylated MAPKs. Position of the individual phosphorylated MAPKs is indicated at the right. Equal loading of proteins is shown with an α-Actin antibody as a loading control (bottom panel). Three biological replicates were done with identical results


B ) MPK6 phosphorylation is specifically enhanced in mkkk7 . Comparison of phosphopeptide abundances from selected MAP kinases in Col - 0 ( blue ) mkkk7 ( red ) seedlings at t = 0 min t = 10 min after 1 μM flagellin treatment by selected reaction monitoring ( SRM ) mass spectrometry . Phosphopeptide corresponding to MKKK7 are only detectable in Col - 0 seedlings are non - detectable ( ND ) in mkkk7 . Bars represent means of measured peptide areas ( sum of transition areas ) for three biological replicates , with error bars ± ( n = 3 ) . Asterisks indicate significant difference between Col - 0 mkkk7 at time points ( student t - , * > 0 . 05 , * * > 0 . 01 * * * > 0 . 001 ) . ND indicates integration of an without transitions significantly above background . Above each graph name phosphorylated residue ( in brackets ) is indicated as well as corresponding phosphopeptide . Serine ( S ) , Threonine ( T ) or Tyrosine ( Y ) phosphorylation is indicated by " [ + 80 ] " .

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

B) MPK6 phosphorylation is specifically enhanced in mkkk7. Comparison of phosphopeptide abundances from selected MAP kinases in Col-0 (blue) and mkkk7 (red) seedlings at t = 0 min and t = 10 min after 1 μM flagellin treatment by selected reaction monitoring (SRM) mass spectrometry. Phosphopeptide corresponding to MKKK7 are only detectable in Col-0 seedlings and are non-detectable (ND) in mkkk7. Bars represent means of measured peptide areas (sum of all transition areas) for three biological replicates, with error bars ± SEM (n=3). Asterisks indicate significant difference between Col-0 and mkkk7 at individual time points (student t-test, *>0.05, **>0.01 and ***>0.001). ND indicates integration of an area without transitions significantly above background. Above each graph the protein name and the phosphorylated residue (in brackets) is indicated as well as the corresponding phosphopeptide sequence. Serine (S), Threonine (T) or Tyrosine (Y) phosphorylation is indicated by "[+80]".


A ) Transient expression in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment . Protoplasts were isolated from 4 week old transfected with pWRKY29 : fLUC ( WRKY29 ) or pFRK1 : fLUC ( FRK1 ) constructs together with 35S : rLUC , as indicated in graph . Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated . horizontal axis indicates treatment while vertical axis represents expression relative to mock treated control sample , as fold induction . measurements were normalized to rLUC activity . Bars represent means ± STDEV ( n = 2 ) . Experiment was repeated 6 times with similar results .

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

A) Transient expression analysis in Arabidopsis mesophyll protoplasts shows enhanced defense gene expression in mkkk7 protoplasts after flg22 treatment. Protoplasts were isolated from 4 week old plants and transfected with pWRKY29:fLUC (WRKY29) or pFRK1:fLUC(FRK1) constructs together with 35S:rLUC, as indicated in the graph. Protoplasts were treated for 4 hrs with 10 µM flg22 or mock treated as indicated. The horizontal axis indicates the treatment while the vertical axis represents expression levels relative to the mock treated control sample, as fold induction. All measurements were normalized to the rLUC activity. Bars represent means ± STDEV (n=2). Experiment was repeated 6 times with similar results.


B ) WRKY29 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 mkkk7 were treated with 1 µM flg22 for t = 0 , 1 , 2 , 4h . WRKY29 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - ) .

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

B) WRKY29 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 for t=0, 1, 2, and 4h. WRKY29 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


C ) FRK1 transcripts measured by qRT - PCR in flg22 treated leaf material . Leaf strips of Col - 0 mkkk7 were treated with 1 µM flg22 fort = 0 , 1 , 2 , 4h . FRK1 transcripts were normalized against Ubiquitin transcript as described before [ 62 ] . Bars represent mean value error bars show SE ( n = 3 ) . ( * p < 0 . 05 , * * p < 0 . 01 , student t - ) .

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

C) FRK1 transcripts measured by qRT-PCR in flg22 treated leaf material. Leaf strips of Col-0 and mkkk7 were treated with 1 µM flg22 fort=0, 1, 2, and 4h. FRK1 transcripts were normalized against Ubiquitin transcript as described before [62]. Bars represent mean value and error bars show SE (n=3). (* p<0.05, ** p<0.01, student t-test).


A ) Transient co - expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22 - induced WRKY29 gene expression . Protoplasts were transfected with pWRKY29 : fLUC , 35S : rLUC indicated overexpression constructs of MKKK7 ( OE - MKKK7 , OE - MKKK7AA or OE - MKKK7DD ) as indicated on horizontal axis . Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs . measurements were normalized to rLUC activity expression is relative to mock treated control sample , shown as fold induction on vertical axis . Results shown are means ± STDEV ( n = 2 ) . At least two biological replicates were done with similar

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

A) Transient co-expression of MKKK7 in Arabidopsis mesophyll protoplasts shows suppression of flg22-induced WRKY29 gene expression. Protoplasts were transfected with pWRKY29:fLUC, 35S:rLUC and indicated overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7AA or OE-MKKK7DD) as indicated on the horizontal axis. Protoplasts were treated with 10 μM flg22 or mock treated for 4 hrs. All measurements were normalized to the rLUC activity and expression is relative to the mock treated control sample, shown as fold induction on the vertical axis. Results shown are means ± STDEV (n=2). At least two biological replicates were done with similar results


A ) Four - week - old seedlings were dipped into a suspension containing virulent Pst DC3000 72 h later disease symptoms were scored . Data represents mean values ± ( n = 20 ; * * * , p < 0 . 001 ; paired t - ) . Three biological experiments were done showing similar results .

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A) Four-week-old seedlings were dipped into a suspension containing virulent Pst DC3000 and 72 h later the disease symptoms were scored. Data represents mean values ± SEM (n=20;***, p<0.001; paired t-test). Three biological experiments were done showing similar results.


B ) of growth in Arabidopsis lines Col - 0 , mkkk7 p35S : MKKK7 - GFP in mkkk7 background . Four to five - week - old were pressure - infiltrated with virulent Pst DC3000 at indicated time points 6 samples were harvested re - isolated on selective media . number of colony forming units ( cfu / cm2 ) was determined at t = 0 , 2 3days post inoculation ( dpi ) . Data represents mean values ± ( n = 6 ; * * , p < 0 . 01 ; paired t - ) . Experiments were done at least twice with similar results .

O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of bacterial growth in Arabidopsis lines Col-0, mkkk7 and p35S:MKKK7-GFP in the mkkk7 background. Four to five-week-old plants were pressure-infiltrated with virulent Pst DC3000 and at indicated time points 6 samples were harvested and bacteria re-isolated on selective media. The number of colony forming units (cfu/cm2) was determined at t=0, 2 and 3days post inoculation (dpi). Data represents mean values ± SEM (n=6; **, p<0.01; paired t-test). Experiments were done at least twice with similar results.


C ) Disease symptom development in Pst - infected lines with estradiol inducible constructs of ind - MKKK7AA L8 , ind - MKKK7AA L10 , ind - MKKK7DD L1 ind - MKKK7DD L3 . Two independent transgenic lines for each construct were grown under short - day conditions disease symptoms were scored 3 dpi . Data represents mean values ± ( n = 20 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; paired t - ) . vertical axis represents disease symptoms . Experiments were done at least twice with similar results .

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

C) Disease symptom development in Pst-infected lines with estradiol inducible constructs of ind-MKKK7AA L8, ind-MKKK7AA L10, ind-MKKK7DD L1 and ind-MKKK7DD L3. Two independent transgenic lines for each construct were grown under short-day conditions and disease symptoms were scored 3 dpi. Data represents mean values ± SEM (n=20; *, p<0.05; **, p<0.01; paired t-test). The vertical axis represents the percentage disease symptoms. Experiments were done at least twice with similar results.


A ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old of Col - 0 mkkk7 .

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A) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old plants of Col-0 and mkkk7.


B ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 week old of Col - 0 two independent inducible MKKK7AA transgenic lines .

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

B) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 week old plants of Col-0 and two independent inducible MKKK7AA transgenic lines.


C ) Effect of 100 nM flg22 treatment on ROS burst measured in 5 old of Col - 0 two independent inducible MKKK7DD transgenic lines . Graphs represent means with error bars ± ( n = 24 ) . vertical axis represents relative increase in ROS production ( photon ) after PAMP treatment . At least three biological replicate experiments were done with similar results .

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

C) Effect of 100 nM flg22 treatment on the ROS burst measured in 5 weeks old plants of Col-0 and two independent inducible MKKK7DD transgenic lines. Graphs represent means with error bars ± SEM (n=24). The vertical axis represents the relative increase in ROS production (photon counts) after PAMP treatment. At least three biological replicate experiments were done with similar results.


( A ) Kaplan - Meier survival curves ( n = 12 - 16 ) .

(A) Kaplan-Meier survival curves (n = 12 - 16).


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(C) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40).


( F ) Relative expression of cardiac hypertrophy markers ( Nppa Nppb ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t ; * * p < 0 . 01 , * * * * p < 0 . 0001 ) .

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(F) Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5). Bars represent mean ± S.E.M. (Student's t test; **p < 0.01, ****p < 0.0001).


( G ) Enzyme histochemistry double for COX SDH activities . ( n = 4 ) .

(G) Enzyme histochemistry double staining for COX and SDH activities. (n = 4).


( H ) Assessment of cardiac fibrosis by Masson ' s trichrome ( n = 4 ) . scale bars , 100μm ; black scale bars , 1 mm .

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

(H) Assessment of cardiac fibrosis by Masson's trichrome staining (n = 4). White scale bars, 100μm; black scale bars, 1 mm.


( A ) Western blot ( B ) Relative of UPRmt markers in heart extracts . HSC70 is used as a loading control ( CTRL ) , ( n = 3 ) .

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(A) Western blot analysis and (B) Relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL), (n = 3).


( C D ) Relative expression normalized to WT of ( C ) established UPRmt markers in 3 - 4 week old mice ( n = 3 - 4 ) .

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(C and D) Relative expression levels normalized to WT of (C) established UPRmt markers in 3 - 4 week old mice (n = 3 - 4).


( C D ) Relative expression normalized to WT of ( D ) mitochondrial chaperones , proteases TFAM in 3 - 4 week old mice ( n = 3 - 4 ) .

(C and D) Relative expression levels normalized to WT of (D) mitochondrial chaperones, proteases and TFAM in 3 - 4 week old mice (n = 3 - 4).


( E ) Western blot of stress markers . HSC70 is used as a loading control ( CTRL ) , ( n = 3 )

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(E) Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL), (n = 3)


( F ) Relative expression of transcription factors involved in UPRmt ISR ( left ) Fgf21 ( right ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

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(F) Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).


( A ) Western blot of OXPHOS subunits in heart extracts . HSC70 Calnexin are used as loading controls , ( n = 3 ) . Antibodies against OXPHOS subunits ( on right ) were used to detect OXPHOS complexes ( on left ) .

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(A) Western blot analysis of individual OXPHOS subunits in heart extracts. HSC70 and Calnexin are used as loading controls, (n = 3). Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).


( B ) BN - PAGE subsequent Western blot for OXPHOS complexes . Antibodies against OXPHOS subunits ( on right ) were used to detect OXPHOS complexes ( on left ) .

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

(B) BN-PAGE and subsequent Western blot analysis for OXPHOS complexes. Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).


( C , D ) In gel activity of complexes I ( C ) IV ( D ) performed after BN - PAGE .

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(C, D) In gel activity of complexes I (C) and IV (D) performed after BN-PAGE.


( E ) Oxygen consumption rates in intact heart mitochondria in presence of pyruvate - glutamate - malate ( C I ) pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) ; State 4 ( + oligomycin ) ; Uncoupled ( + FCCP ) . ( n = 3 ) . Bars represent mean ± S . E . M . ( Student ' s t , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

(E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).


( A ) Representative gel of In organello translation of heart mitochondria . De novo synthetized are isolated after labeling with 35S - met ( 1 hour ) or after cold chase ( 3h Chase ) . Positions of are indicated on left . Position of full - ( Proficient ) low molecular polypeptides ( Abortive ) are indicated on right . Note that in WT ClpP KO low molecular polypeptides include ATP8 / ND4L .

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

(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins.


( B ) Relative overall synthesis turnover rate .

(B) Relative overall protein synthesis and turnover rate.


( C ) Western blot of DARS2 . HSC70 is used as a loading control ( CTRL ) .

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

(C) Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL).


( D ) Relative of de novo synthesized OXPHOS subunits in DKO mitochondria normalized to corresponding Dars2 KO polypeptide level .

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

(D) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level.


( E ) Relative of synthesis of Proficient Abortive polypeptides . ( F ) of turnover rate of full - ( Proficient ) de novo synthesized . Bars represent mean ± S . E . M . ( Student ' s t , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) , ( n = 3 - 4 ) .

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

(E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).


( A - C ) Western blot of THP - 1 infected with ( A ) HSV - 1 at MOI 5 for 3 h , 6 h , 12 h , 18 h , 24 h ( B ) CHIKV at MOI 5 for 3 h , 6 h , 12 h , 18 h , 24 h ( C ) JEV at MOI 5 for 3 h , 6 h , 12 h , 24 h .

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

(A-C) Western blot analysis of THP-1 cells infected with (A) HSV-1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h.


( I - L ) Control IRGM knockdown THP - 1 IFN reporter were treated with ( I ) Poly I : C ( 1 μg / ml ) for 8 h , 12 h 24 h or ( J ) Poly dA : dT ( 1 μg / ml ) for 12 h 24 h or ( K ) IFN - β ( 500 ng / ml ) for 8 h 12 h or ( L ) cGAMP ( 1 μg / ml ) for 12 h 24 h supernatant was subjected to luciferase reporter using QuantiLuc

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

(I-L) Control and IRGM knockdown THP-1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN-β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QuantiLuc reagent


( M - P ) Control IRGM knockdown THP - 1 were untreated treated with Poly I : C ( 1 μg / ml ) for 8 h total was subjected to qRT PCR with primers of ( M ) MX2 ( N ) ISG15 ( O ) OAS1 ( P ) IFN - β . ( Q - T ) Control IRGM knockdown THP - 1 were untreated treated with Poly dA : dT ( 1 μg / ml ) for 8 h total was subjected to qRT PCR with primers of ( Q ) MX2 ( R ) ISG15 ( S ) OAS1 ( T ) IFN - β .

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

(M-P) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN-β. (Q-T) Control and IRGM knockdown THP-1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN-β.


( D ) was isolated from irgm1 + / + irgm1 - / - BMDMs subjected to qRT - PCR with indicated viral restriction factor

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(D) RNA was isolated from irgm1+/+ and irgm1-/- BMDMs and subjected to qRT-PCR with indicated viral restriction factor genes


( H - J ) isolated from control IRGM knockdown HT - 29 subjected to qRT - PCR with indicated genes of ( H ) Immunoproteasome ( I ) TAP ( J ) human leukocyte antigen ( HLA ) system .

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(H-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system.


( L , M ) Antigen processing shown by representative confocal of control IRGM siRNA transfected THP - 1 treated with DQ - OVA ( green ) ( 10 µg / ml , 30 min ) . Scale Bar , 10 µm . ( M ) Graph depicts of control IRGM knockdown THP - 1 with DQ - OVA '

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

(L, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's


( P ) Representative confocal of H - 2Kb - SIINFEKL ( red ) on surface of Irgm1 + / + irgm1 - / - mouse BMDMs treated with OVA ( 2 mg / ml , 3 h ) , Scale bar 5 µm or 8 µm ( as indicated ) .

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(P) Representative confocal images of H-2Kb-SIINFEKL (red) on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated).


( C ) Representative of plaque in Vero performed from culture supernatant of CHIKV ( MOI 1 , 24 h ) infected HT - 29 control IRGM knockdown .

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(C) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( F ) Representative of plaque in Vero performed from culture supernatant of HSV - 1 ( MOI 1 , 24 h ) infected HT - 29 control IRGM knockdown .

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(F) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV-1 (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( I ) Representative of plaque in Vero performed from culture supernatant of JEV ( MOI 1 , 24 h ) infected HT - 29 control IRGM knockdown .

(I) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.


( L ) Representative of plaque in Vero were performed from culture supernatant of WNV ( MOI 0 . 3 , 16 h ) infected HuH7 control IRGM knockdown .

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(L) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV (MOI 0.3, 16 h) infected HuH7 control and IRGM knockdown cells.


( O ) Representative of plaque in Vero were performed from culture supernatant of ZIKV ( MOI 5 , 48 h ) infected Huh7 control IRGM knockdown .

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(O) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells.


( S ) Representative of plaque in Vero E6 were performed from culture supernatant of SARS - CoV2 ( MOI 1 , 24 h ) infected THP - 1 control IRGM knockdown .

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(S) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS-CoV2 (MOI 1, 24 h) infected THP-1 control and IRGM knockdown cells.


( D ) Kaplan - Meier survival graph depicts survival of mock CHIKV infected irgm1 + / + irgm1 - / - neonates during course of

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(D) Kaplan-Meier survival graph depicts percentage survival of mock and CHIKV infected irgm1+/+ and irgm1-/- neonates during the course of infection


( H , I ) Total isolated from brains of mock CHIKV infected ( MOI 1x107 PFU / mouse ) ( 6 dpi ) irgm1 + / + irgm1 - / - mice was subjected to qRT PCR with MX2 total used for qRT PCR with brain is four times more than .

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(H, I) Total RNA isolated from muscles and brains of mock and CHIKV infected (MOI 1x107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1-/-mice and was subjected to qRT PCR with MX2 The total RNA used for qRT PCR with the brain is four times more than muscles.


( A - D ) Control si - IRGM transfected THP - 1 IFN reporter were kept uninfected ( Mock ) or infected with ( A ) JEV ( MOI 5 ) or ( B ) CHIKV ( MOI 5 ) or ( C ) HSV - 1 ( MOI 2 . 5 ) or ( D ) VSV - eGFP ( MOI 1 ) supernatant collected 8 hpi were subjected to luciferase . graphs depict fold change in interferon response .

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

(A-D) Control and si-IRGM transfected THP-1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV-1 (MOI 2.5) or (D) VSV-eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response.


( L ) Western blot with lysates of mock CHIKV ( MOI 5 , 24 h ) infected control IRGM knockdown HT - 29 probed with indicated antibodies . ( M ) Western blot with lysates of mock JEV ( MOI 5 , 24 h ) infected control si - IRGM transfected HT - 29 probed with indicated antibodies .

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(L) Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT-29 cells and probed with the indicated antibodies. (M) Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si-IRGM transfected HT-29 cells and probed with the indicated antibodies.


( Q ) Western blot with lysates of mock CHIKV ( MOI 1 , 24 h ) infected control IRGM knockdown HT - 29 transfected with indicated siRNA probed with indicated antibodies .

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(Q) Western blot analysis with cell lysates of mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells transfected with indicated siRNA and probed with the indicated antibodies.


( B , C ) Representative immunoblots ( B ) of Tau degradation ( C ) from 14 DIV neurons transduced with EGFP or shTSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , probed for Tau tubulin . shTSG101 - expressing neurons exhibit markedly decreased Tau degradation compared to EGFP - expressing controls ( n = 18 - 19 per condition , unpaired student ' s t - , * p = 0 . 0108 )

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(B, C) Representative immunoblots (B) and quantification of Tau degradation (C) from 14 DIV primary neurons transduced with EGFP or shTSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shTSG101-expressing neurons exhibit markedly decreased Tau degradation compared to EGFP-expressing controls (n=18-19 per condition, unpaired student's t-test, *p=0.0108)


( D , E ) Representative immunoblots ( D ) of Tau degradation ( E ) from 14 DIV neurons transduced with mCh or mCh - TSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , probed for Tau tubulin . Overexpression of TSG101 increases Tau degradation ( n = 17 / condition , unpaired student ' s t - , * p = 0 . 0115 )

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

(D, E) Representative immunoblots (D) and quantification of Tau degradation (E) from 14 DIV neurons transduced with mCh or mCh-TSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. Overexpression of TSG101 increases Tau degradation (n=17/condition, unpaired student's t-test, *p=0.0115)


( F , G ) Representative immunoblots ( F ) of Tau degradation ( G ) from 14 DIV neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , probed for Tau tubulin . shRab35 - expressing neurons exhibit markedly decreased Tau degradation compared to mCh - expressing controls ( n = 23 - 26 per condition , unpaired student ' s t - , * * * p = 0 . 0008 )

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

(F, G) Representative immunoblots (F) and quantification of Tau degradation (G) from 14 DIV neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shRab35-expressing neurons exhibit markedly decreased Tau degradation compared to mCh-expressing controls (n=23-26 per condition, unpaired student's t-test, ***p=0.0008)


( H , I ) Representative immunoblots ( H ) of Tau degradation ( I ) from 14 DIV neurons transduced with mCh or mCh - Rab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , probed for Tau or tubulin . Rab35 overexpression increases Tau degradation ( n = 18 per condition , unpaired student ' s t - , * p = 0 . 0126 )

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(H, I) Representative immunoblots (H) and quantification of Tau degradation (I) from 14 DIV neurons transduced with mCh or mCh-Rab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau or tubulin. Rab35 overexpression increases Tau degradation (n=18 per condition, unpaired student's t-test, *p=0.0126)


( J ) Flow cytometry distribution of N2a cotransfected with either HA vector ( black ) or HA - Rab35 ( green ) medium fluoresence timer - tagged wild - type Tau ( FT - Tau ) ; blue ( y axis ) indicates " younger " Tau while red ( x axis ) indicates " older "

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(J) Flow cytometry distribution of N2a cells cotransfected with either HA vector (black) or HA-Rab35 (green) and medium fluoresence timer-tagged wild-type Tau (FT-Tau); blue fluorescence (y axis) indicates "younger" Tau protein while red fluorescence (x axis) indicates "older" Tau


( K ) of expressing red to blue ( older : younger ) FT - Tau is reduced in overexpressing Rab35 , indicating faster Tau turnover ( n = 9 / condition , unpaired student ' s t - , * * * * p < 0 . 0001 )

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(K) The ratio of cells expressing red to blue (older:younger) FT-Tau is reduced in cells overexpressing Rab35, indicating faster Tau turnover (n=9/condition, unpaired student's t-test , ****p<0.0001)


( A , B ) Representative immunoblots ( A ) of Tau degradation ( B ) from 14 DIV neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , probed for pSer396 / 404 - Tau ( PHF1 ) , pSer262 - Tau , or pSer202 - Tau ( CP13 ) tubulin . shRab35 - expressing neurons exhibit markedly decreased pSer262 - p396 / 404 - Tau degradation compared to mCh - expressing controls , while pSer202 - Tau degradation is unaffected ( n = 4 per condition for pSer396 / 404 - Tau pSer202 - Tau , n = 3 for pSer262 - Tau , unpaired student ' s t - , * * p = 0 . 0027 , * p = 0 . 0120 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120)


( C ) of PLA signal ( green ) for Hrs / Tau interaction in N2a co - transfected with FLAG - Hrs either mCh or mCh - Rab35 ( red ) , probed with antibodies against FLAG total ( DA9 ) or phospho - Tau species ( pSer396 / 404 - Tau , pSer262 - Tau , or pSer202 - Tau ) ; scale bar : 5 μm . ( D ) Rab35 overexpression significantly increases PLA for total , pSer396 / 404 - Tau , pSer262 - Tau but not pSer202 - Tau ( n = 121 - 132 for total Tau , 178 - 195 for pSer396 / 404 - Tau , 71 - 98 for pSer262 - Tau , 25 - 29 for pSer202 - Tau ; Mann - Whitney , * * * * p < 0 . 0001 )

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

(C) Images of PLA signal (green) for Hrs/Tau interaction in N2a cells co-transfected with FLAG-Hrs and either mCh or mCh-Rab35 (red), and probed with antibodies against FLAG and total (DA9) or phospho-Tau species (pSer396/404-Tau, pSer262-Tau, or pSer202-Tau); scale bar: 5 μm. (D) Rab35 overexpression significantly increases PLA puncta for total, pSer396/404-Tau, pSer262-Tau but not pSer202-Tau (n=121-132 cells for total Tau, 178-195 cells for pSer396/404-Tau, 71-98 cells for pSer262-Tau, 25-29 cells for pSer202-Tau; Mann-Whitney test, ****p<0.0001)


( A , B ) Representative immunoblots ( A ) of Hrs , TSG101 , CHMP2b , Rab35 ( B ) from 14 DIV neurons treated with either DMSO ( CON ) or glucocorticoids ( GC ) . GC treatment selectively decreases Rab35 without affecting of Hrs , TSG101 , or CHMP2b ( n = 9 - 10 / condition , unpaired student ' s t - , * p = 0 . 0221 )

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(A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221)


( C ) Rab35 mRNA are decreased by GC in 14 DIV hippocampal neurons N2a ( for neurons , n = 9 / condition , unpaired student ' s t - , * * p = 0 . 0036 ; for N2a , n = 6 / condition , unpaired student ' s t - , * p = 0 . 0138 )

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(C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138)


( E , F ) Representative immunoblots ( E ) of of different Rab ( F ) in hippocampus of GC - treated CON . of Rab35 , but not other Rabs , are decreased in GC - treated compared to CON ones ( n = 5 / condition , unpaired student ' s t - , * p = 0 . 0257 )

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(E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257)


( G , H ) Immunofluorescence of Rab35 ( green ) DAPI ( blue ) ( G ) showing that Rab35 is reduced in hippocampal ( CA1 ) of GC - treated aniamls ( H ) ( n = 15 slices / condition ; unpaired student ' s t - , * * * p = 0 . 0003 ) . Data information : numeric data represent mean ±

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

(G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM


( A , B ) Representative immunoblots ( A ) of Tau degradation ( B ) in 14 DIV neurons expressing mCh - Rab35 or mCh , treated for 24 h with cycloheximide ( CHX ) or DMSO ( CON ) under GC conditions . GC significantly decreases Tau degradation , whereas Rab35 overexpression blocks this effect ( n = 12 - 14 / condition ; 1 - way ANOVA , Dunnet post - hoc , * pmCh vs mCh GC = 0 , 0319 , * pmCh vs Rab35 GC = 0 , 047 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047)


( D , E ) Representative immunoblots ( D of Tau ( E ) in hippocampal synaptosomes reveal that GC increases total Tau in expressing EGFP , but not Rab35 ( n = 5 - 6 / group , 2 replicates , 2 - way ANOVA , GC x Rab35 interaction F1 , 39 = 10 , 51 p = 0 . 002 ; Sidak posthoc * p = 0 . 0168 )

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(D, E) Representative immunoblots (D of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168)


( F ) Golgi - 3D neuronal reconstruction of hippocampal pyramidal neurons ( CA1 )

(F) Golgi-based 3D neuronal reconstruction of hippocampal pyramidal neurons (CA1 area)


( G ) GC treatment reduces of apical dendrites in expressing EGFP , but not EGFP - Rab35 ( n = 6 - 7 / group ; 6 - 8 neurons / , 2 - way ANOVA , GC x Rab35 interaction F1 , 155 = 3 . 969 p = 0 . 0481 , overall GC effect F1 , 155 = 8 . 998 p = 0 . 0031 , Sidak posthoc * * p = 0 . 0021 )

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(G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021)


( H ) GC treatment reduces mature spine density in expressing EGFP , but not EGFP - Rab35 ( 2 - way ANOVA , GC x Rab35 interaction F1 , 499 = 12 . 33 p = 0 . 0005 , overall GC effect F1 , 499 = 4 . 373 p = 0 . 0370 , Sidak posthoc * * * p = 0 . 0003 ; n = 6 - 7 / group ; 6 - 8 neurons per )

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

(H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal)


( I , J ) Sholl of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP - expressing ; however , this effect is not seen in EGFP - Rab35 - expressing ( 3 - way ANOVA , GC x Rab35 interaction F1 , 4212 = 14 , 926 p < 0 . 0001 , Simple effect , Sidak for multiple comparisons * p120 = 0 . 012 , * * p140 = 0 . 001 , * * p220 = 0 . 002 , * * * p240 < 0 . 001 , * * * p260 < 0 . 001 , * * p280 = 0 . 001 , * * p300 = 0 . 001 , * p320 = 0 . 021 , * p340 = 0 . 047 , n = 6 - 7 / group , 6 - 8 neurons per )

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

(I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal)


( A ) Volcano plot representation of PARL interaction partners . Mitochondria isolated from PARL - / - FITR293Tcells expressing PARL - FLAG were subjected to co - immunoprecipitation ( IP ) . Co - purifying were separated by SDS - PAGE identified by quantitative MS ( n = 3 ) ( Dataset EV1 ) .

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

(A) Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL-/- FITR293Tcells expressing PARL-FLAG were subjected to co-immunoprecipitation (IP). Co-purifying proteins were separated by SDS-PAGE and identified by quantitative MS (n=3)(Dataset EV1).


( B ) Heat map of Log2 transformed LFQ intensities for three independent experiments . significant ( FDR < 0 . 05 ) showing a positive between PARLIP control in ( A ) are shown . Grey color shows missing quantitative information . Clustering was performed using complete method with Euclidean .

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(B) Heat map of Log2 transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARLIP and control in (A) are shown. Grey color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance.


( C ) Complexome of MEFmitochondria ( n = 3 ) . Heat maps of relative abundances of significantly enriched in eluate of PARLimmunoprecipitates at a FDR level of 0 . 05 ( A ) identified by mass spectrometry after BN - PAGE are shown after hierarchical clustering .

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(C) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.


( D ) BN - PAGE of mitochondria lacking SLP2 , YME1L or PARL . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a of 2 . 5 mg / ml . Solubilized were separated by a 3 - 13 % blue native gradient gel analyzed using SLP2 - , YME1L - PARL - specific antibodies .

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(D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.


( E ) High resolution complexome of SPY members SLP2 , YME1L PARL using MEFmitochondria ( n = 3 ) . Heat maps migration profiles are shown after separation using a 3 - 9 % BN - PAGE . SLP2 , YME1L PARL migrate in a high molecular ( 2 MDa ) , whereas SLP2 additionally is present in complexes of 1 . 6 MDa ) .

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(E) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEFmitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).


( A ) Co - immunoprecipitation of endogenous SLP2 , PARL YME1L in mitochondria isolated from human FITR293T using either SLP2 - or PARL - specific antibodies . IgG was used as a negative control . In , input ( 10 % ) .

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(A) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%).


( B , C ) Co - immunoprecipitation of endogenous SLP2 YME1L with PARL - specific antibodies in mitochondria isolated from wild type MEFs ( WT ) MEFs lacking YME1L , SLP2 or PARL . In , input ( 10 % ) .

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(B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%).


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( D ) Assembly of SLP2 , PARL YME1L into a high molecular . Mitochondria isolated from FITR293T ( Con ) inducibly expressing SLP2 - FLAG ( FLAG ) were solubilized in digitonin subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of precipitate were analyzed by BN - PAGE immunoblotting using SLP2 - , PARL - , YME1L - PHB2 - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) .

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(D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).


( E ) High resolution BN - PAGE of mitochondria lacking PARL , YME1L or SLP2 . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a of 2 . 5 mg / ml . Solubilized were separated by a 3 - 9 % gradient gel containing 0 - 10 % glycerol analyzed using SLP2 - specific antibodies .

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(E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.


( F , G ) Submitochondrial localization of SLP2 . ( F ) Mitochondria were isolated from MEFs fractionated in presence or absence of proteinase K as indicated . Fractions were analyzed by SDS - PAGE immunoblotting . MFN2 served as OM marker , TIMM23 , SMAC / DIABLO , YME1L as IMS markers , AFG3L1 AFG3L2 as matrix marker .

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(F, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins.


( F , G ) Submitochondrial localization of SLP2 . ( G ) Mitochondrial membranes were extracted with sodium carbonate at indicated pH separated into pellet ( P ) supernatant ( S ) fraction by centrifugation . Fractions were analyzed by SDS - PAGE immunoblotting .

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(F, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting.


( A ) Steady state of proteolytic substrates of YME1L . Whole extracts of Yme1l - / - , Parl - / - , Slp2 - / - MEFs were analyzed by SDS - PAGE immunoblotting using indicated antibodies .

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(A) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies.


( B ) PGAM5 processing depends on proteolytically active PARL . PARL catalytic inactive PARLS277A harboring C - terminal FLAG - epitopes were expressed under control of a tetracycline ( tet ) - inducible promoter in wild type ( WT ) PARL - / - FITR293T as indicated . Processing of L - PGAM5 to S - PGAM5 was monitored by immunoblotting .

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(B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.


( C ) Accelerated processing of L - PGAM5 in Slp2 - / - . Processing of L - PGAM5 was analyzed by immunoblotting in wild type ( WT ) , Yme1l - / - , Parl - / - Slp2 - / - MEFs after inhibition of synthesis by cycloheximide ( CHX ) . A of L - PGAM5 at different time points is shown in lower panel . Two - way ANOVA ( n = 3 ; * * * * , p < 0 . 0001 ) .

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(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).


( D ) accelerated processing of L - PGAM5 in Slp2 - / - is mediated by PARL . PARL was depleted from Slp2 - / - by RNAi prior to CHX treatment . A of L - PGAM5 at different time points is shown . Two - way ANOVA ( n = 3 ; * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) .

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

(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).


( E ) PGAM5 associates with SPY harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T ( WT ) or PARL - / - FITR293T expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin were analyzed by BN - PAGE immunoblotting using FLAG - PGAM5 - specific antibodies .

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

(E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.