( A ) Immunoprecipitation analysis of HEK293T cells transfected with Flag - SQSTM1 or its I431A mutant together with HA - MAVS . Anti - Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . Cell - based studies were performed independently at least three times with comparable results .

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(A) Immunoprecipitation analysis of HEK293T cells transfected with Flag-SQSTM1 or its I431A mutant together with HA-MAVS. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( B ) Luciferase activity driven by the IFN - β promoter in HEK293T cells transfected with HA - MAVS or its 4KR mutant together with Flag - SQSTM1 or its I431A mutant . Luciferase assays were performed 24 hrs after transfection . The luciferase reporter data are presented as means ± SEM . Two - tailed Student ' s t - tests were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; and * * * , P < 0 . 001 .

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(B) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-SQSTM1 or its I431A mutant. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( C ) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing HA - MAVS or its 4KR mutant together with Flag - SQSTM1 . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

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(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing HA-MAVS or its 4KR mutant together with Flag-SQSTM1. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( D ) Luciferase activity driven by the IFN - β promoter in HEK293T cells transfected with HA - MAVS or its 4KR mutant together with Flag - NDP52 or its D439R / C443K mutant . Luciferase assays were performed 24 hrs after transfection . The luciferase reporter data are presented as means ± SEM . Two - tailed Student ' s t - tests were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; and * * * , P < 0 . 001 .

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(D) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-NDP52 or its D439R/C443K mutant. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( E ) Immunoprecipitation analysis of HEK293T cells transfected with Flag - MAVS or its 4KR mutant together with Myc - NDP52 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . Cell - based studies were performed independently at least three times with comparable results .

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(E) Immunoprecipitation analysis of HEK293T cells transfected with Flag-MAVS or its 4KR mutant together with Myc-NDP52. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( F ) Immunoprecipitation analysis of the DNP52 - RNF34 interaction in cells infected with VSV . Anti - DNP52 immunoprecipitates were analyzed using immunoblotting with an anti - DNP52 or anti - RNF34 antibody . The WCL was immunoblotted with an anti - MAVS or anti - RNF34 antibody . Cell - based studies were performed independently at least three times with comparable results .

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(F) Immunoprecipitation analysis of the DNP52-RNF34 interaction in cells infected with VSV. Anti-DNP52 immunoprecipitates were analyzed using immunoblotting with an anti-DNP52 or anti-RNF34 antibody. The WCL was immunoblotted with an anti-MAVS or anti- RNF34 antibody. Cell-based studies were performed independently at least three times with comparable results.


( G ) Immunoprecipitation analysis of the interaction between Flag - NDP52 and HA - RNF34 in WT or MAVS - / - cells . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA antibody . Cell - based studies were performed independently at least three times with comparable results .

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(G) Immunoprecipitation analysis of the interaction between Flag-NDP52 and HA-RNF34 in WT or MAVS-/- cells. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA antibody. Cell-based studies were performed independently at least three times with comparable results.


( H ) Immunoprecipitation analysis of HEK293T cells transfected with Flag - NDP52 or its D439R / C443K mutant together with HA - MAVS followed by VSV infection . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . Cell - based studies were performed independently at least three times with comparable results .

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(H) Immunoprecipitation analysis of HEK293T cells transfected with Flag-NDP52 or its D439R/C443K mutant together with HA-MAVS followed by VSV infection. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( I ) Immunoblot showing the levels of the MAVS protein in siCtrl - or siNDP52 - transfected cells expressing Flag - RNF34 or Flag - V . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

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(I) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells expressing Flag-RNF34 or Flag-V. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( J ) Immunoblot showing the levels of the MAVS protein in siCtrl - or siNDP52 - transfected cells infected with VSV ( MOI = 1 , 12 hrs ) . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

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(J) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells infected with VSV (MOI = 1, 12 hrs). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( K ) Immunoprecipitation analysis of the MAVS - LC3 interaction in siCtrl - or siNDP52 - transfected cells infected with VSV . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - LC3 antibody . NDP52 levels were analyzed by performing immunoblotting with an anti - NDP52 antibody . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

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(K) Immunoprecipitation analysis of the MAVS-LC3 interaction in siCtrl- or siNDP52-transfected cells infected with VSV. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-LC3 antibody. NDP52 levels were analyzed by performing immunoblotting with an anti-NDP52 antibody. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoblot showing MAVS , IRF3 - p , LC3 , TOM20 and HSP60 levels in siCtrl - or siRNF34 - 1 cells infected with VSV and cultured in the presence of Z - VAD ( 50 μM ) for the indicated times . α - Tubulin was used as a loading control . Cell - based studies were performed independently at least three times with comparable results .

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(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoblot showing MAVS , LC3 , TOM20 and HSP60 levels in cells transfected with Flag - RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times . Cell - based studies were performed independently at least three times with comparable results .

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(B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


Representative confocal images ( C ) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 cells infected with VSV for the indicated times . Red represents MAVS and green represents NDP52 . Scale bar , 10 μm . Cell - based studies were performed independently at least three times with comparable results .

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Representative confocal images (C) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


quantification ( D ) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 cells infected with VSV for the indicated times . Scale bar , 10 μm . Cell - based studies were performed independently at least three times with comparable results .

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quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( E ) Transmission electron microscopy images of shCtrl - and shRNF34 - 1 HEK293T cells cultured in the presence or absence of VSV . Arrows indicate mitochondria engulfed in autophagosomes . Double arrows indicate swollen mitochondria with disrupted cristae . M , mitochondria and N , nucleus . 40000 × magnification . Cell - based studies were performed independently at least three times with comparable results .

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(E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification. Cell-based studies were performed independently at least three times with comparable results.


( F ) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm . Scale bar , 10 μm . Cell - based studies were performed independently at least three times with comparable results .

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(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


B | hFwe - Lose mRNA expression is more abundant in elderly people . hFwe - Lose mRNA expression was analyzed by RT - qPCR in 86 lung tissue biopsies taken from non - COVID patients with age between 20 and 82 years . Older patients show a significant upregulation of hFwe - Lose expression . A log - Linear regression model demonstrates a positive correlation between age and hFwe - Lose expression ( R2 = 0 . 13 ; slope confidence interval of 95 % ( CI ) = [ 2 . 0 - 12 . 2 ] ; p - value of the linear regression model < 6 ⋅ 10 - 4 ) .

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B | hFwe-Lose mRNA expression is more abundant in elderly people. hFwe-Lose mRNA expression was analyzed by RT-qPCR in 86 lung tissue biopsies taken from non-COVID patients with age between 20 and 82 years. Older patients show a significant upregulation of hFwe-Lose expression. A log-Linear regression model demonstrates a positive correlation between age and hFwe-Lose expression (R2= 0.13; slope confidence interval of 95% (CI) = [2.0-12.2]; p-value of the linear regression model < 6⋅10-4).


C | hFwe - Lose expression is elevated in lung tissue biopsies from patients with comorbidities . Box plot illustrates an increased expression of hFwe - Lose in lungs of patients with hypertension ( HT ; n = 129 ) , obesity ( n = 45 ) , chronic obstructive pulmonary disease ( COPD n = 51 ) , diabetes ( n = 48 ) , cardio - vascular disease ( CVD ; n = 63 ) versus disease - free control lungs ( n = 42 ) . Patient ` s age is depicted in color . Two - sided Student ' s t - test was performed for each comorbidity ( compared to disease - free patients ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

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C | hFwe-Lose expression is elevated in lung tissue biopsies from patients with comorbidities. Box plot illustrates an increased expression of hFwe-Lose in lungs of patients with hypertension (HT; n = 129), obesity (n = 45), chronic obstructive pulmonary disease (COPD n = 51), diabetes (n = 48), cardio-vascular disease (CVD; n = 63) versus disease-free control lungs (n = 42). Patient`s age is depicted in color. Two-sided Student's t-test was performed for each comorbidity (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


D | hFwe - Lose expression is upregulated in lung tissue of COVID - 19 patients . Box plot illustrates an increased expression of hFwe - Lose in lung tissue of patients diagnosed with COVID - 19 ( n = 11 ) , individuals affected with host comorbidities ( n = 216 ) versus disease - free control lungs ( n = 42 ) . Patient ' s age is depicted in color . Two - sided Student ' s t - test was performed ( compared to disease - free patients ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

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D | hFwe-Lose expression is upregulated in lung tissue of COVID-19 patients. Box plot illustrates an increased expression of hFwe-Lose in lung tissue of patients diagnosed with COVID-19 (n = 11), individuals affected with host comorbidities (n = 216) versus disease-free control lungs (n = 42). Patient's age is depicted in color. Two-sided Student's t-test was performed (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


E | SARS - CoV - 2 - infection manifests histological changes in patients ' lungs as demonstrated by H & E staining . 1 ) For comparison , the normal lung of an elderly individual is shown , containing air - filled empty looking alveolar spaces and thin alveolar septae / gas - exchange membranes . Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested . 2 ) Diffuse ( proliferative ) alveolar damage showing evidence of cell death . An alveolar space filled with desquamated pneumocytes and macrophages , lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II . The still recognizable epithelial lining is detached . Cells and surfactant are lost creating perforations in the alveolar wall , allowing migration of blood cells and fluid to enter inside the alveolar space . The adjacent interstitial space is significantly widened , showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes . On the top and the lower right , dilated and congested capillaries can be seen ( Hematoxylin and Eosin ( H & E ) stain , 200x ) . Abbreviations : a - Alveolar structure destroyed by lymphocytes , desquamated epithelium , as well as extravasated blood ; The alveoli should be empty , but here it is filled with a combination of degenerated cells and fibrin ; b - Next alveoli ; c - Expanded pulmonary interstitium . The interstitium should be as thin as 10um , but here it is 100um ; d - Extravasation of erythrocytes ; e - Lymphocytes ; f - Congested capillaries and arterioles ; g - Multinucleated alveolocyte / pneumocyte type II ; h - Detached epithelium ; i - A histiocyte with a kidney - shaped nucleus at the bottom of the circle has ingested erythrocytes ( left - handed ) , representing the first step of cellular elimination . Round inlet with encircled karyopyknotic , karyorrhectic and " ghost - cell " figures indicative of apoptosis . 3 ) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining . The adjacent interstitial space shows analogous changes to 2A1 . At the bottom , there is a prominently dilated and congested capillary ( H & E , 200x ) . Abbreviations : a - Alveoli with detached epithelium ; b - Next alveoli ; c - E Extravasation of erythrocytes ; d - Expanded interstitium ; e - Lymphocytes ; f - Multinucleated pneumocyte type II . 4 ) Diffuse alveolar damage showing massive extravasation of fibrin ( homogeneous eosinophilic material in the center of the alveolar space ) . The lining pneumocytes are almost all apoptotic / necrotic . The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris ( H & E , 200x ) . Abbreviations : a - Alveola ; b - Congested capillaries ; c - Fibrin ; d - Remnants of degenerated / dying epithelium ; e - Expanded interstitium ; f - Cellular debris consisting of macrophages , detached epithelial cells and lymphocytes ; g - Fully degenerated / lacking epithelial coverage within the alveoli . Round inlet with encircled karyopyknotic , karyorrhectic and " ghost - cell " figures indicative of apoptosis . 5 ) Immunohistochemical stain ( IHC ) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes / lung interstitium leading to obstruction of the microcirculation ( IHC for fibrin , 200x ) . Abbreviations : a - All small alveolar septal capillaries are filled out with worm - like fibrin thrombi hampering / obstructing circulation ; b - Normal interstitium .

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

E | SARS-CoV-2-infection manifests histological changes in patients' lungs as demonstrated by H&E staining. 1)For comparison, the normal lung of an elderly individual is shown, containing air-filled empty looking alveolar spaces and thin alveolar septae/gas-exchange membranes. Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested. 2) Diffuse (proliferative) alveolar damage showing evidence of cell death. An alveolar space filled with desquamated pneumocytes and macrophages, lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II. The still recognizable epithelial lining is detached. Cells and surfactant are lost creating perforations in the alveolar wall, allowing migration of blood cells and fluid to enter inside the alveolar space. The adjacent interstitial space is significantly widened, showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes. On the top and the lower right, dilated and congested capillaries can be seen (Hematoxylin and Eosin (H&E) stain, 200x). Abbreviations: a- Alveolar structure destroyed by lymphocytes, desquamated epithelium, as well as extravasated blood; The alveoli should be empty, but here it is filled with a combination of degenerated cells and fibrin; b- Next alveoli; c- Expanded pulmonary interstitium. The interstitium should be as thin as 10um, but here it is 100um; d- Extravasation of erythrocytes; e- Lymphocytes; f- Congested capillaries and arterioles; g- Multinucleated alveolocyte/pneumocyte type II; h- Detached epithelium; i- A histiocyte with a kidney-shaped nucleus at the bottom of the circle has ingested erythrocytes (left-handed), representing the first step of cellular elimination. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 3) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining. The adjacent interstitial space shows analogous changes to 2A1. At the bottom, there is a prominently dilated and congested capillary (H&E, 200x). Abbreviations: a- Alveoli with detached epithelium; b- Next alveoli; c- E Extravasation of erythrocytes; d- Expanded interstitium; e- Lymphocytes; f- Multinucleated pneumocyte type II. 4) Diffuse alveolar damage showing massive extravasation of fibrin (homogeneous eosinophilic material in the center of the alveolar space). The lining pneumocytes are almost all apoptotic/necrotic. The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris (H&E, 200x). Abbreviations: a- Alveola; b- Congested capillaries; c- Fibrin; d- Remnants of degenerated / dying epithelium; e- Expanded interstitium; f- Cellular debris consisting of macrophages, detached epithelial cells and lymphocytes; g- Fully degenerated / lacking epithelial coverage within the alveoli. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 5) Immunohistochemical stain (IHC) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes/lung interstitium leading to obstruction of the microcirculation (IHC for fibrin, 200x). Abbreviations: a- All small alveolar septal capillaries are filled out with worm-like fibrin thrombi hampering / obstructing circulation; b- Normal interstitium.


F | Healthy lungs show very low cleaved caspase - 3 positive cells . IHC staining for cleaved caspase - 3 ( 20x ) . G | Expression of cleaved ( active ) caspase - 3 yielding apoptotic and pre - apoptotic cells : IHC for cleaved ( active ) caspase - 3 showing a brown nuclear staining signal in respective cells . 31 . 5 % cells were found to be caspase positive , 11 . 7 % with high caspase - 3 positivity and another 19 . 8 % with low . These cells are mainly located in the interstitium , but also in some alveolar epithelial and endothelial compartments . In the lower right , the edge of an alveolar space containing several ( pre - ) apoptotic pneumocytes is seen , while in the upper left a completely denuded / deepithelialized alveolus with two apoptotic remnants is observable ( IHC for cleaved caspase - 3 , 20x ) . Abbreviations : a - Apoptotic large mononuclear cells ; b - Apoptotic alveolocyte / pneumocyte ; i - Amplification of apoptotic alveolocyte / pneumocyte . As shown in the round inlets of 1E2 and 1E4 , homogeneous dark nuclear condensation up to a size of 2μm ( karyopyknosis ) , homogeneous pinkish - greyish nuclear condensation , large nuclear inclusion with marginalization of the condensed chromatin , coarse nuclear angulation , nose - like / polar body - like nuclear protrusions , the latter two being karyorrhectic debris , were all morphologically considered evidence of apoptosis . This has been correlated with and was reflected by the results of the cleaved caspase - 3 staining on step sections . All cells were counted on the 20X fields for n = 42 disease - free and n = 3 COVID - 19 autopsy patients . The expression of hFwe - Lose in COVID - 19 patients is significantly higher than in disease - free individuals , regardless of cleaved caspase - 3 staining ( Cas POS : p < 0 . 004 ; Cas NEG : p < 0 . 006 ) . In COVID - 19 patients we observed a significantly higher expression of hFwe - Lose in sections with positive cleaved caspase - 3 staining ( p < 0 . 004 ) . Two - sided Student ' s t - test was performed ( compared to disease - free samples and NEG samples , respectively ) , and p - values are presented on the plot . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

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

F | Healthy lungs show very low cleaved caspase-3 positive cells. IHC staining for cleaved caspase-3 (20x). G | Expression of cleaved (active) caspase-3 yielding apoptotic and pre-apoptotic cells: IHC for cleaved (active) caspase-3 showing a brown nuclear staining signal in respective cells. 31.5% cells were found to be caspase positive, 11.7% with high caspase-3 positivity and another 19.8% with low. These cells are mainly located in the interstitium, but also in some alveolar epithelial and endothelial compartments. In the lower right, the edge of an alveolar space containing several (pre-)apoptotic pneumocytes is seen, while in the upper left a completely denuded/deepithelialized alveolus with two apoptotic remnants is observable (IHC for cleaved caspase-3, 20x). Abbreviations: a- Apoptotic large mononuclear cells; b-Apoptotic alveolocyte / pneumocyte; i- Amplification of apoptotic alveolocyte / pneumocyte. As shown in the round inlets of 1E2 and 1E4, homogeneous dark nuclear condensation up to a size of 2μm (karyopyknosis), homogeneous pinkish-greyish nuclear condensation, large nuclear inclusion with marginalization of the condensed chromatin, coarse nuclear angulation, nose-like/polar body-like nuclear protrusions, the latter two being karyorrhectic debris, were all morphologically considered evidence of apoptosis. This has been correlated with and was reflected by the results of the cleaved caspase-3 staining on step sections. All cells were counted on the 20X fields for n = 42 disease-free and n = 3 COVID-19 autopsy patients. The expression of hFwe-Lose in COVID-19 patients is significantly higher than in disease-free individuals, regardless of cleaved caspase-3 staining (Cas POS: p < 0.004; Cas NEG: p < 0.006). In COVID-19 patients we observed a significantly higher expression of hFwe-Lose in sections with positive cleaved caspase-3 staining (p < 0.004). Two-sided Student's t-test was performed (compared to disease-free samples and NEG samples, respectively), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


A | hFwe - Lose biomarker expression is more abundant in nasopharyngeal swab probes from older adults . hFwe - Lose expression was analyzed by RT - qPCR in 283 nasopharyngeal swab samples taken from patients with age between 1 and 96 years , taken at the very beginning of the disease ( the earliest contact with physician , before the disease progression ) . The vertical axis represents relative hFwe - Lose expression normalized to the mean of non - hospitalized patients . Colors depict the outcome groups : non - hospitalized ( gray , n = 85 ) , hospitalized ( blue , n = 177 ) and deceased ( red , n = 21 ) . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The lines show the fitted curves of an asymptotic model with the same asymptotic value but different rate constants per group ( see Materials and Methods ) . Due to the comparatively low number of deceased patients in the dataset ( n = 21 ) , the curve for this group reflects the asymptotic value . Hospitalized and deceased patients show a positive correlation of hFwe - Lose expression and age with a larger rate constant for the hospitalized patients . ( R2 = 0 . 65 ) The p - value ( < 0 . 001 ) indicates that the blue curve ( for hospitalized patients ) grows faster with age , compared to the gray curve for non - hospitalized patients ) .

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

A | hFwe-Lose biomarker expression is more abundant in nasopharyngeal swab probes from older adults. hFwe-Lose expression was analyzed by RT-qPCR in 283 nasopharyngeal swab samples taken from patients with age between 1 and 96 years, taken at the very beginning of the disease (the earliest contact with physician, before the disease progression). The vertical axis represents relative hFwe-Lose expression normalized to the mean of non-hospitalized patients. Colors depict the outcome groups: non-hospitalized (gray, n = 85), hospitalized (blue, n = 177) and deceased (red, n = 21). The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The lines show the fitted curves of an asymptotic model with the same asymptotic value but different rate constants per group (see Materials and Methods). Due to the comparatively low number of deceased patients in the dataset (n = 21), the curve for this group reflects the asymptotic value. Hospitalized and deceased patients show a positive correlation of hFwe-Lose expression and age with a larger rate constant for the hospitalized patients. (R2= 0.65) The p-value (< 0.001) indicates that the blue curve (for hospitalized patients) grows faster with age, compared to the gray curve for non-hospitalized patients).


B | hFwe - Lose expression is elevated in nasopharyngeal swab probes from patients with comorbidities . Box plots illustrate an increased relative expression of hFwe - Lose in nasopharyngeal swabs of patients with diabetes ( n = 129 ) , COPD ( n = 20 ) , obesity ( BMI > 30 ; n = 152 ) , cardiomyopathy ( " CM " , n = 19 ) , heart failure ( " HF " , n = 35 ) , hypertension ( " HT " , n = 121 ) , chronic kidney disease ( " CKD " , n = 60 ) versus disease - free patients ( n = 96 ) . Two - sided Student ' s t - tests were performed ( compared to disease - free patients ) , and p - values are presented on the plot . The vertical axis represents relative hFwe - Lose expression normalized to the mean of non - hospitalized patients . The color refers to the COVID - 19 disease outcome : gray for not hospitalised , blue for hospitalised and red for deceased patients . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

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

B | hFwe-Lose expression is elevated in nasopharyngeal swab probes from patients with comorbidities. Box plots illustrate an increased relative expression of hFwe-Lose in nasopharyngeal swabs of patients with diabetes (n = 129), COPD (n = 20), obesity (BMI > 30; n = 152), cardiomyopathy ("CM", n = 19), heart failure ("HF", n = 35), hypertension ("HT", n = 121), chronic kidney disease ("CKD", n = 60) versus disease-free patients (n = 96). Two-sided Student's t-tests were performed (compared to disease-free patients), and p-values are presented on the plot. The vertical axis represents relative hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


D | Elevated hFwe - Lose expression in the nasal swab samples associates with patients ' condition severity and respective medical treatment . Box plots illustrate an increased expression of hFwe - Lose in nasal swabs of patients , who were hospitalized within 14 days of disease progression ( n = 177 ) , admitted to intensive care unit ( ICU ) ( n = 34 ) , underwent intubation ( n = 58 ) , had respiratory rate greater than 30 ( GT30 ) ( n = 76 ) , had blood oxygenation level ( SpO2 ) less than 94 % ( n = 147 ) , and who died within 30 days of disease progression ( n = 21 ) versus patients without respective conditions . Pairwise two - sided Student ' s t - tests were performed ( compared to patients without respective conditions ) , and p - values are presented on the plot . The vertical axis represents hFwe - Lose expression normalized to the mean of non - hospitalized patients . The color refers to the COVID - 19 disease outcome : gray for not hospitalised , blue for hospitalised and red for deceased patients . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

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

D | Elevated hFwe-Lose expression in the nasal swab samples associates with patients' condition severity and respective medical treatment. Box plots illustrate an increased expression of hFwe-Lose in nasal swabs of patients, who were hospitalized within 14 days of disease progression (n = 177), admitted to intensive care unit (ICU) (n = 34), underwent intubation (n = 58), had respiratory rate greater than 30 (GT30) (n = 76), had blood oxygenation level (SpO2) less than 94% (n = 147), and who died within 30 days of disease progression (n = 21) versus patients without respective conditions. Pairwise two-sided Student's t-tests were performed (compared to patients without respective conditions), and p-values are presented on the plot. The vertical axis represents hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


E | Elevated hFwe - Lose expression in nasal swab associates with patients ' disease outcome . Box plot emphasizes an increased expression of hFwe - Lose in nasal swabs of patients , who were hospitalized within 14 days of disease progression ( n = 177 ) , and who died within 30 days of disease progression ( n = 21 ) versus patients without respective conditions . Two - sided Student ' s t - tests were performed ( compared to non - hospitalised patients ) , and p - values are presented on the plot . The vertical axis represents hFwe - Lose expression normalized to the mean of non - hospitalized patients . The color refers to the COVID - 19 disease outcome : gray for not hospitalised , blue for hospitalised and red for deceased patients . The shape of data points reflects the cohorts : circles for the training cohort ( n = 203 ) and triangles for the validation cohort ( n = 80 ) . The central band shows the median , the box indicates the interquartile range , the whiskers extend to the most extreme points within the 1 . 5 - fold distance of the interquartile range above and below the box .

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

E |Elevated hFwe-Lose expression in nasal swab associates with patients' disease outcome. Box plot emphasizes an increased expression of hFwe-Lose in nasal swabs of patients, who were hospitalized within 14 days of disease progression (n = 177), and who died within 30 days of disease progression (n = 21) versus patients without respective conditions. Two-sided Student's t-tests were performed (compared to non-hospitalised patients), and p-values are presented on the plot. The vertical axis represents hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.


( C ) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with 2 , 4 , 6 , 8 or 10 % 1 , 6 hexanediol showing dissolution of NONO puncta with increasing concentration . ( D ) Dot plot of NONO foci area ( µm2 ) per nucleus at different concentrations of 1 , 6 hexanediol as in ( C ) . Bars are SD . The numbers of biological replicates are indicated in ( C ) .

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(C) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with 2, 4, 6, 8 or 10% 1,6 hexanediol showing dissolution of NONO puncta with increasing concentration. (D) Dot plot of NONO foci area (µm2) per nucleus at different concentrations of 1,6 hexanediol as in (C). Bars are SD. The numbers of biological replicates are indicated in (C).


( F ) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with PBS , RNase A or DNase I , as indicated . ( G ) Dot plot of summed green fluorescence per nucleus in ( F ) . Bars are SD . The numbers of biological replicates are indicated in ( F ) .

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

(F) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with PBS, RNase A or DNase I, as indicated. (G) Dot plot of summed green fluorescence per nucleus in (F). Bars are SD. The numbers of biological replicates are indicated in (F).


( A ) Fluorescence micrograph images of representative cells stained for NONO and NEAT1 _ 2 after transfection with plasmids expressing YFP fused NONO _ WT and NONO _ ∆ RRM1 protein . DAPI ( blue ) stain indicates cell nuclei , YFP fused NONO ( green ) and NEAT1 _ 2 RNA FISH ( red ) . ( B ) The enrichment of mean NONO fluorescence detected within RNA FISH foci is quantitatively determined as a ratio relative to mean nuclear NONO fluorescence in ( A ) . Bars are SD . The numbers of biological replicates are indicated in ( A ) .

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

(A) Fluorescence micrograph images of representative cells stained for NONO and NEAT1_2 after transfection with plasmids expressing YFP fused NONO_WT and NONO_∆RRM1 protein. DAPI (blue) stain indicates cell nuclei, YFP fused NONO (green) and NEAT1_2 RNA FISH (red). (B) The enrichment of mean NONO fluorescence detected within RNA FISH foci is quantitatively determined as a ratio relative to mean nuclear NONO fluorescence in (A). Bars are SD. The numbers of biological replicates are indicated in (A).


Sphericity per NONO segment , NONO foci number per nucleus and NONO foci size between YFP fused NONO _ WT and NONO _ ∆ RRM1 plasmids . Bars are SD . Biological replicates n = 60 in ( C ) and n = 50 in ( D )

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Sphericity per NONO segment, NONO foci number per nucleus and NONO foci size between YFP fused NONO_WT and NONO_∆RRM1 plasmids. Bars are SD. Biological replicates n=60 in (C) and n=50 in (D)


( A ) Relative NONO mRNA and protein levels in KELLY cells treated with control or NONO KD siRNAs . Representative Western blot image for NONO protein .

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(A) Relative NONO mRNA and protein levels in KELLY cells treated with control or NONO KD siRNAs. Representative Western blot image for NONO protein.


( B ) Western blot quantitation analysis of GATA2 and HAND2 protein levels between control and NONO KD siRNAs . ( C ) Representative Western blot images for GATA2 and HAND2 proteins in ( B ) .

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(B) Western blot quantitation analysis of GATA2 and HAND2 protein levels between control and NONO KD siRNAs. (C) Representative Western blot images for GATA2 and HAND2 proteins in (B).


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 .

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

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 ) 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

O O O O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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

O O O O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O 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 B-MEASURED_VAR 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

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 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

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

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

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 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 )

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

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

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


D : Co - immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels . The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co - IP , LETM1 was immunoprecipitated ( left panel , IP : LETM1 ) using a LETM1 monoclonal antibody and Protein G magnetic beads ( ProtG ) . ProtG beads alone were used as a negative control for binding , immunoprecipitates were immunoblotted ( IB ) for the indicated proteins to demonstrate interaction . 10 % of the input was used for immunoblotting . Prohibitin ( PHB ) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes . The middle and right panel of the coIPs illustrates the converse experiment , in the middle in TMBIM5WT and right TMBIMKO , using TMBIM5 as bait ( right panel , IP : TMBIM5 ) . The last two right panels show blots from BN - PAGE conducted in TMBIM5WT and KO .

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

D: Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The middle and right panel of the coIPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN-PAGE conducted in TMBIM5WT and KO.


A : Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones .

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

A: Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones.


E : Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies , HSP60 and TOM40 served as mitochondrial loading controls . F : Densitometric analysis of the bands in ( E ) normalized to loading control , bar graph of three individual experiments ( biological replicates ) , mean ± SD , One - way ANOVA with Bonferroni ' s multiple comparisons test performed against TMBIM5WT * p < 0 . 05 , * * p < 0 . 008 , Two - way ANOVA with Bonferroni ' s multiple comparisons test performed for the OPA1 statistics against TMBIM5WT , * * * p = 0 . 0009 , * * * * p < 0 . 0001 .

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

E: Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. F: Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, One-way ANOVA with Bonferroni's multiple comparisons test performed against TMBIM5WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***p=0.0009, ****p<0.0001.


( A ) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods .

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

(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods.


A ) Representative nuclear spreads of wild - type pachytene and Rec8 − / − pachytene - like spermatocytes . Nuclear spreads were immunostained for SYCP3 and ACA . Magnified views are indicated by dashed areas . Schematic representation on magnified chromosomes represents tightly associated sister - AEs ( separated for easier representation ) in paired wild - type homologs , and the appearance of two distinguishable / separated sister - AEs once REC8 - mediated cohesion is lost in Rec8 − / − univalents . Bars , 10 μm in spreads and 1 μm on insets .

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

A) Representative nuclear spreads of wild-type pachytene and Rec8−/− pachytene-like spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified chromosomes represents tightly associated sister-AEs (separated for easier representation) in paired wild-type homologs, and the appearance of two distinguishable/separated sister-AEs once REC8-mediated cohesion is lost in Rec8−/− univalents. Bars, 10 μm in spreads and 1 μm on insets.


B ) Representative nuclear spread of zygotene - like Stag3 mutant spermatocytes . Nuclear spreads were immunostained for SYCP3 and ACA . Magnified views are indicated by dashed areas . Schematic representation on magnified univalents represents : separation of sister - AEs ( centre ) and close association of sister - AEs ( right ) . Bellow : graph showing the percentages of axes with : separation of AEs ( dark grey area ) and closely associated AEs ( light grey area ) . 414 axes analysed , from 11 nuclei . Bars , 10 μm in spreads and 1 μm on insets .

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

B) Representative nuclear spread of zygotene-like Stag3 mutant spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents: separation of sister-AEs (centre) and close association of sister-AEs (right). Bellow: graph showing the percentages of axes with: separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 10 μm in spreads and 1 μm on insets.


C ) Magnified views of zygotene - like Stag3 mutant univalents with LSAEs . Nuclear spreads were immunostained for SYCP3 and ACA . Schematic representation indicates sites of LSAEs . Bellow : graph showing the percentages of axes with : LSAEs ( red area ) , extensive separation of AEs ( dark grey area ) and closely associated AEs ( light grey area ) . 414 axes analysed , from 11 nuclei . Bars , 1 μm .

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

C) Magnified views of zygotene-like Stag3 mutant univalents with LSAEs. Nuclear spreads were immunostained for SYCP3 and ACA. Schematic representation indicates sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 1 μm.


D ) Representative nuclear spread of zygotene - like Smc1β − / − spermatocytes . Nuclear spreads were immunostained for SYCP3 and ACA . Magnified views are indicated by dashed areas . Schematic representation on magnified univalents represents sites of LSAEs . Bellow : graph showing the percentages of axes with : LSAEs ( red area ) , extensive separation of AEs ( dark grey area ) and closely associated AEs ( light grey area ) . 404 axes analysed , from 10 nuclei . Bars , 10 μm in spreads and 1 μm on insets .

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

D) Representative nuclear spread of zygotene-like Smc1β−/− spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 404 axes analysed, from 10 nuclei. Bars, 10 μm in spreads and 1 μm on insets.


E ) STED images of representative zygotene - like Stag3 mutant and Smc1β − / − univalents displaying LSAEs . Nuclear spreads were immunostained for SYCP3 . Bars , 1μm .

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 B-MEASURED_VAR O O O O O

E) STED images of representative zygotene-like Stag3 mutant and Smc1β−/− univalents displaying LSAEs. Nuclear spreads were immunostained for SYCP3. Bars, 1μm.


F ) Graph indicating the inter - axis distances measured in wild - type pachytene bivalents ; pachytene - like Rec8 − / − and zygotene - like Stag3 mutant and Smc1β − / − univalents . Each measurement in wild - type and Rec8 − / − corresponds to the median of 3 distances measured along one homolog ( n = 60 ) . Each measurement in Stag3 mutant and Smc1β − / − mutant axes correspond to 1 distance measured at sites of local separation of AEs ( n = 40 and n = 20 , respectively ) . Horizontal lines indicate median .

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

F) Graph indicating the inter-axis distances measured in wild-type pachytene bivalents; pachytene-like Rec8−/− and zygotene-like Stag3 mutant and Smc1β−/− univalents. Each measurement in wild-type and Rec8−/− corresponds to the median of 3 distances measured along one homolog (n=60). Each measurement in Stag3 mutant and Smc1β−/− mutant axes correspond to 1 distance measured at sites of local separation of AEs (n=40 and n=20, respectively). Horizontal lines indicate median.


G ) Graph indicating the inter - axis distances measured at terminally and centrally located sites of LSAEs in zygotene - like Stag3 mutant univalents . Each measurement corresponds to 1 distance measured at terminally ( n = 20 ) and centrally ( n = 20 ) located sites of local separation of axial elements . Horizontal lines indicate median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph indicating the inter-axis distances measured at terminally and centrally located sites of LSAEs in zygotene-like Stag3 mutant univalents. Each measurement corresponds to 1 distance measured at terminally (n=20) and centrally (n=20) located sites of local separation of axial elements. Horizontal lines indicate median.


A ) Representative wild - type pachytene bivalents ; pachytene - like Rec8 − / − , zygotene - like Stag3 mutant and Smc1β − / − univalents . Nuclear spreads were immunostained for SYCP1 , SYCP3 and ACA . Filled arrowheads indicate sites of LSAEs with detectable SYCP1 . Bars , 1 μm . Bellow : graphs showing the percentages of sites of LSAEs with detectable SC assembly along sister - AEs of Stag3 mutant and Smc1β − / − univalents ( n = 120 and n = 68 , respectively ) .

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

A) Representative wild-type pachytene bivalents; pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP1, SYCP3 and ACA. Filled arrowheads indicate sites of LSAEs with detectable SYCP1. Bars, 1 μm. Bellow: graphs showing the percentages of sites of LSAEs with detectable SC assembly along sister-AEs of Stag3 mutant and Smc1β−/− univalents (n=120 and n=68, respectively).


B ) Representative zygotene - like Stag3 mutant univalents . Nuclear spreads were immunostained for SYCP3 , ACA and : SYCE1 , SYCE2 and TEX12 . Filled arrowheads indicate SC assembly between sites of LSAEs . Bars , 1

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

B) Representative zygotene-like Stag3 mutant univalents. Nuclear spreads were immunostained for SYCP3, ACA and: SYCE1, SYCE2 and TEX12. Filled arrowheads indicate SC assembly between sites of LSAEs. Bars, 1 μm


C ) Quantification of signal distribution within sites of LSAEs , in Stag3 mutant spermatocytes . Signal distribution measured for SYCP3 and : SYCP1 , SYCE1 , SYCE2 and TEX12 . Empty arrowheads indicate signal intensity peaks that correspond to each of the two sister - chromatid axes , filled arrowheads indicate SYCP1 peaks .

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

C) Quantification of signal distribution within sites of LSAEs, in Stag3 mutant spermatocytes. Signal distribution measured for SYCP3 and: SYCP1, SYCE1, SYCE2 and TEX12. Empty arrowheads indicate signal intensity peaks that correspond to each of the two sister-chromatid axes, filled arrowheads indicate SYCP1 peaks.


A ) Representative zygotene - like Stag3 mutant and Smc1β − / − univalents . Nuclear spreads were immunostained for SYCP3 , SYCE1 and REC8 . Bellow : graph showing the percentages of Stag3 mutant axes with sites of LSAEs with and without flanking REC8 foci . 78 axes with LSAEs were analysed . Bars , 1 μm .

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

A) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and REC8. Bellow: graph showing the percentages of Stag3 mutant axes with sites of LSAEs with and without flanking REC8 foci. 78 axes with LSAEs were analysed. Bars, 1 μm.


B ) Representative pachytene - like Rec8 − / − , zygotene - like Stag3 mutant and Smc1β − / − univalents . Nuclear spreads were immunostained for SYCP3 , SYCE1 and RAD21L . Bars , 1 μm .

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B) Representative pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21L. Bars, 1 μm.


C ) Representative zygotene - like Stag3 mutant and Smc1β − / − univalents . Nuclear spreads were immunostained for SYCP3 , SYCE1 and RAD21 . Bars , 1 μm .

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C) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21. Bars, 1 μm.


A ) Representative wild - type pachytene sex chromosomes . Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 . Filled arrowheads indicate the PAR . Dashed area indicated in centre panel corresponds to the magnified view on the right panel . Bars , 10 μm in spreads and 1 μm on insets .

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A) Representative wild-type pachytene sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3. Filled arrowheads indicate the PAR. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Bars, 10 μm in spreads and 1 μm on insets.


B ) Graph indicating the inter - axis distances measured at sites of local separation of axial elements in zygotene - like Stag3 mutant univalents and along pachytene wild - type X - chromosomes . Each measurement in the X - chromosomes corresponds to the median of 3 distances ( according to the scheme in figure ) ( n = 54 ) . Each measurement in Stag3 mutant corresponds to 1 distance measured at sites with local separation of axial elements ( n = 40 ) . Horizontal lines indicate median . p < 0 . 0001 , obtained with two - tailed Mann - Whitney test . Bellow : schematic representation of the XY pair : sister - AEs are coloured in black and grey for easier visualization . Filled arrowhead indicates PAR . Purple bars in scheme indicate regions where inter - axis distances are measured .

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B) Graph indicating the inter-axis distances measured at sites of local separation of axial elements in zygotene-like Stag3 mutant univalents and along pachytene wild-type X-chromosomes. Each measurement in the X-chromosomes corresponds to the median of 3 distances (according to the scheme in figure) (n=54). Each measurement in Stag3 mutant corresponds to 1 distance measured at sites with local separation of axial elements (n=40). Horizontal lines indicate median. p < 0.0001, obtained with two-tailed Mann-Whitney test. Bellow: schematic representation of the XY pair: sister-AEs are coloured in black and grey for easier visualization. Filled arrowhead indicates PAR. Purple bars in scheme indicate regions where inter-axis distances are measured.


C ) Representative pachytene wild - type sex chromosomes . Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and SYCE2 . Dashed area indicated in centre panel corresponds to the magnified view on the right panel . Empty arrowhead indicates two discernable sister - AEs without SYCE2 . Bars , 10 μm in spreads and 1 μm on insets .

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C) Representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and SYCE2. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Empty arrowhead indicates two discernable sister-AEs without SYCE2. Bars, 10 μm in spreads and 1 μm on insets.


A ) Analysis of REC8 distribution in representative pachytene wild - type sex chromosomes . Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and REC8 . Bars , 10 μm in spreads and 1 μm on insets . Magnified XY pair is indicated by dashed area . Scatterplot showing the number of REC8 foci per X - chromosome . 225 foci identified in 14 X - chromosomes . Horizontal line indicates the mean , error bars indicate s . d . 3 . 02 .

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A) Analysis of REC8 distribution in representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and REC8. Bars, 10 μm in spreads and 1 μm on insets. Magnified XY pair is indicated by dashed area. Scatterplot showing the number of REC8 foci per X-chromosome. 225 foci identified in 14 X-chromosomes. Horizontal line indicates the mean, error bars indicate s.d. 3.02.


B ) Top : representative computationally straightened pachytene wild - type X - chromosomes . Nuclear spreads were immunostained for SYCP3 and REC8 . Chromosomes were aligned at the PAR , and subdivided into 25 regular intervals . Bar , 1 μm . Bellow : histogram showing the distribution of the percentages of REC8 foci ( n = 225 ) among 25 regular intervals along 14 X - chromosomes . Dotted line indicates mean .

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B) Top: representative computationally straightened pachytene wild-type X-chromosomes. Nuclear spreads were immunostained for SYCP3 and REC8. Chromosomes were aligned at the PAR, and subdivided into 25 regular intervals. Bar, 1 μm. Bellow: histogram showing the distribution of the percentages of REC8 foci (n=225) among 25 regular intervals along 14 X-chromosomes. Dotted line indicates mean.


C ) Histogram showing the relative frequency of inter - REC8 distances along X - chromosomes . 211 inter - REC8 distances from 14 X - chromosomes . Grey area represents 15 % of the chromosome axis length . The respective cumulative distribution function is shown .

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C) Histogram showing the relative frequency of inter-REC8 distances along X-chromosomes.211 inter-REC8 distances from 14 X-chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.


D ) Histogram showing the best fit of the observed inter - REC8 distances to the gamma distribution . Green curve indicates the best fit of the observed inter - REC8 distances .  indicates the value for which the best fit was obtained , with estimated standard error 0 . 27 .

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D) Histogram showing the best fit of the observed inter-REC8 distances to the gamma distribution. Green curve indicates the best fit of the observed inter-REC8 distances.  indicates the value for which the best fit was obtained, with estimated standard error 0.27.


E ) Graph comparing the median distance of REC8 foci with computationally predicted even and random distributions . Median distances between foci subdivided in three categories ( 11 - 14 , 15 - 18 and 19 - 22 REC8 foci per chromosome ) . Purple asterisk indicates the median distance of all REC8 foci .

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E) Graph comparing the median distance of REC8 foci with computationally predicted even and random distributions. Median distances between foci subdivided in three categories (11-14, 15-18 and 19-22 REC8 foci per chromosome). Purple asterisk indicates the median distance of all REC8 foci.


F ) Scatterplot comparing inter - REC8 distances along on X - chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant . 211 inter - REC8 distances measured along X - chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant . Grey area represents 15 % of the chromosome axis length . Horizontal lines indicate the median . p < 0 . 0001 , obtained with two - tailed Mann - Whitney test .

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F) Scatterplot comparing inter-REC8 distances along on X-chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant. 211 inter-REC8 distances measured along X-chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant. Grey area represents 15% of the chromosome axis length. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.


A ) Representative yet - unsynapsed autosome from wild - type zygotene spermatocytes . Nuclear spreads were immunostained for SYCP3 and SYCE2 . Magnified autosome is indicated by dashed area . Filled arrowheads indicate yet - unsynapsed axes regions . Bars , 10 μm in spreads and 1 μm on

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A) Representative yet-unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3 and SYCE2. Magnified autosome is indicated by dashed area. Filled arrowheads indicate yet-unsynapsed axes regions. Bars, 10 μm in spreads and 1 μm on insets


B ) Representative unsynapsed autosome from wild - type zygotene spermatocytes . Nuclear spreads were immunostained for SYCP3 , REC8 and ACA . Magnified autosome is indicated by dashed area . Bars , 10 μm in spreads and 1 μm on insets .

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B) Representative unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3, REC8 and ACA. Magnified autosome is indicated by dashed area. Bars, 10 μm in spreads and 1 μm on insets.


C ) Scatterplot comparing the average number of REC8 foci along zygotene autosomes and X - chromosomes . Number of REC8 foci were quantified along 15 zygotene autosomes and 14 X - chromosomes . Each dot represents the number of REC8 foci on each chromosome . Total number of foci identified : zygotene autosomes ( 343 ) , X - chromosomes ( 225 ) . Horizontal lines indicate the mean , error bars indicate s . d . : 7 . 42 and 3 . 02 for zygotene autosomes and X - chromosomes , respectively . p = 0 . 0060 , obtained with two - tailed Mann - Whitney test .

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C) Scatterplot comparing the average number of REC8 foci along zygotene autosomes and X-chromosomes. Number of REC8 foci were quantified along 15 zygotene autosomes and 14 X-chromosomes. Each dot represents the number of REC8 foci on each chromosome. Total number of foci identified: zygotene autosomes (343), X-chromosomes (225). Horizontal lines indicate the mean, error bars indicate s.d.: 7.42 and 3.02 for zygotene autosomes and X-chromosomes, respectively. p = 0.0060, obtained with two-tailed Mann-Whitney test.


D ) Histogram showing the relative frequency of inter - REC8 distances along yet - unsynapsed zygotene autosomes . 294 inter - REC8 distances analyzed in 14 chromosomes . Grey area represents 15 % of the chromosome axis length . The respective cumulative distribution function is shown .

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D) Histogram showing the relative frequency of inter-REC8 distances along yet-unsynapsed zygotene autosomes. 294 inter-REC8 distances analyzed in 14 chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.


E ) Scatterplot comparing the measured inter - REC8 distances on yet - unsynapsed zygotene autosomes , X - chromosomes and sites of LSAEs in Stag3 mutant . Number of inter - REC8 distances for yet - unsynapsed zygotene autosomes , X - chromosomes and sites of local separation of axial elements in Stag3 mutant , n = 294 , n = 211 and n = 10 , respectively . Horizontal lines indicate the median . p < 0 . 0001 , obtained with two - tailed Mann - Whitney test .

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E) Scatterplot comparing the measured inter-REC8 distances on yet-unsynapsed zygotene autosomes, X-chromosomes and sites of LSAEs in Stag3 mutant. Number of inter-REC8 distances for yet-unsynapsed zygotene autosomes, X-chromosomes and sites of local separation of axial elements in Stag3 mutant, n=294, n=211 and n=10, respectively. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.


( B ) Locomotor behavior , measured by thrashing rates in liquid medium , in the C . elegans strains with neuronal expression of human WT SOD1 or ALS - linked mutant SOD1G85R , in the presence ( M1 / M1 ) or absence ( + / + ) of the suppressor mutation ( n = 16 ) .

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(B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16).


( C ) Northern ( top panel ) and western ( middle , bottom panels ) blot analyses of total RNA and protein from SOD1G85R strains , with ( M1 / M1 ) or without ( + / + ) suppressor mutations , demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation ( M1 / M1 ) . The western blot lanes are from the same gel and exposure .

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(C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure.


( E ) C . elegans SPR - 5 and UFD - 2 and their mammalian homologs lysine - specific demethylase 1 ( LSD1 ) and ubiquitination factor E4 B ( UBE4B ) share all major protein domains . These include the Swi3 - Rsc8 - Moira ( SWIRM ) , amine oxidase - like ( AOL ) , and TOWER domains in LSD1 / SPR - 5 and the Ufd2 Core and U - box domains in UBE4B / UFD - 2 . Positions of the missense , nonsense , and deletion mutations in mutant C . elegans are indicated .

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(E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated.


( F ) Locomotor behavior , measured by thrashing rates , of the C . elegans carrying the SOD1G85R transgene on the normal background ( WT ) , with the null mutation of either ufd - 2 ( tm1380 ) or spr - 5 ( by134 ) , the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) , or the M1 suppressor ufd - 2 ( W824X ) ; spr - 5 ( R646Q ) ( n = 16 ) .

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(F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16).


( G ) Western blotting of the supernatant ( S ) and pellet ( P ) protein fractions from the C . elegans carrying the SOD1G85R transgene with the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) compared with controls . While SOD1G85R protein levels are unchanged in the S fraction , those in the P fraction are decreased in the double suppressor mutant . The P fraction represents only about 1 . 7 % of total SOD1G85R in the WT sample ; therefore , a higher ratio of the P fraction relative to the S fraction ( approximately 2 : 1 ) is used for the western analysis .

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(G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis.


( H ) The overexpression ( OE ) of WT UFD - 2 or SPR - 5 in the C . elegans nervous system blocks the protection conferred by the double mutation ufd - 2 ( tm1380 ) ; spr - 5 ( by134 ) ( n = 16 ) . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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(H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


( A ) Top : schematic drawing depicts pan - neuronal expression of YFP in head and ventral neurons in the context of the C . elegans body plan . Left : micrographs show the SOD1G85R - YFP proteins expressed in WT or the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) mutant background . The double - mutant worms show a marked decrease in protein aggregation in neurons . Enlarged sections of headneurons ( red framed ) and ventral cord neurons ( white framed ) are shown . Middle : quantification of locomotion in the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) and the WT C . elegans with neuronal expression of SOD1G85R - YFP ( n = 30 ) . Right : a decrease in the protein levels of SOD1G85R - YFP in the presence of spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) is shown by western blots of the supernatant ( S ) and the pellet ( P ) fractions . ( B and C ) Analyses of the spr - 5 ( by134 ) ; ufd - 2 ( tm1380 ) and the WT C . elegans with neuronal expression of TDP - 43 - c25 - YFP ( n = 30 ) or PolyQ - YFP ( n = 12 ) as in ( A ) .

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(A) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C. elegans body plan. Left: micrographs show the SOD1G85R-YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of headneurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of SOD1G85R-YFP (n = 30). Right: a decrease in the protein levels of SOD1G85R-YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. (B and C) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C. elegans with neuronal expression of TDP-43-c25-YFP (n = 30) or PolyQ-YFP (n = 12) as in (A).