Figure 2A | 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A | 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.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.


Figure 1 ( 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 ) .

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Figure 1(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).


Figure 2 ( 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 )

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

Figure 2(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)


Figure 5 ( A ) Relative NONO mRNA and protein levels in KELLY cells treated with control or NONO KD siRNAs . Representative Western blot image for NONO protein .

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

Figure 5(A) Relative NONO mRNA and protein levels in KELLY cells treated with control or NONO KD siRNAs. Representative Western blot image for NONO protein.


Figure 6 ( 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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Figure 6(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).


Figure 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 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.


Figure 3 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 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  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)


Figure 5 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 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 . 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


Figure 1D : 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D: 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.


Figure 2A : 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 .

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Figure 2A: 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.


Figure 7 ( A ) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods .

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Figure 7(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods.


Figure 1A ) 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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.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.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.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.


Figure 2A ) 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 μmC ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A) 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 μmC) 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.


Figure 3A ) 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 . C ) Representative zygotene - like Stag3 mutant and Smc1β − / − univalents . Nuclear spreads were immunostained for SYCP3 , SYCE1 and RAD21 . Bars , 1 μm .

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

Figure 3A) 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.C) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21. Bars, 1 μm.


Figure 4A ) 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 . 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 .

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

Figure 4A) 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.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.


Figure 5A ) 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 . 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 . 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 .

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

Figure 5A) 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.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.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.


Figure 6A ) 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 insetsB ) 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 . 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 .

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

Figure 6A) 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 insetsB) 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.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.


Fig 1 ( 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 . ( 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 ) . ( 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 .

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

Fig 1(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.(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).(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.


Fig 2 ( 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 ) . ( D ) Neurodegenerative rough - eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd - 2 / UBE4B and spr - 5 / LSD1 - CG9934 and Su ( Var ) 3 - 3 , respectively , compared to the control ( CTRL ) . Eye - specific expression of TDP - 43M337V , FUSR521C , and RNA interference ( RNAi ) was driven by GMR - Gal4 . ( E ) Drosophila eye pigment quantitation . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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

Fig 2(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).(D) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2/UBE4B and spr-5/LSD1-CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43M337V, FUSR521C, and RNA interference (RNAi) was driven by GMR-Gal4.(E) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


Fig 3 ( A ) Western blots of cell lysates derived from mock ( CTRL ) , single UBE4B or LSD1 , or double UBE4B and LSD1 knockdowns . Supernatant ( S ) and pellet ( P ) fractions were probed with indicated antibodies . While the LSD1 or UBE4B single - knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions , the combined knockdown produces the strongest reduction in the aggregates . ( B ) Quantification of SOD1G85R protein levels by western blotting ( A ) . n = 3 ( supernatant ) ; n = 8 ( pellet ) . ( C ) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half - lives in the double UBE4B and LSD1 knockdown cells versus controls . ( D ) Quantification of SOD1G85R clearance , as analyzed by western blotting in ( C ) . The graph indicates the relative band intensity of SOD1G85R at each chase time point . n = 5 ; Overall p = 0 . 02 ( paired t test , CTRL versus UBE4B and LSD1 double knockdown ) . Individual p = 0 . 03 ( 3 h ) , p = 0 . 003 ( 6 h ) , p = 0 . 06 ( 9 h ) , and p = 0 . 004 ( 12 - 21 h ) . ( E ) The half - life of SOD1G85R is reduced from 8 . 5 h to 5 h upon knockdown of UBE4B and LSD1 . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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

Fig 3(A) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (B) Quantification of SOD1G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet).(C) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (D) Quantification of SOD1G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12-21 h).(E) The half-life of SOD1G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


Fig 4 ( B ) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA . Gray spots represent 22 , 148 annotated transcripts . Red spots are predicted p53 - activated targets , and blue spots are predicted p53 - inhibited targets . The enrichment of red spots in the up - regulated genes ( right upper quadrant , > 1 . 2 - fold change , p ≤ 0 . 04 ) and blue spots in the down - regulated genes ( left upper quadrant ) indicates that the p53 - mediated transcription is activated by the UBE4B and LSD1 double knockdown . C ) RT - qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double - knockdown samples . n = ( 2 to 6 ) , p ≤ 0 . 04 . ( D ) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown . Left : representative western blots showing p53 levels , the knockdown of UBE4B and LSD1 , and the actin control in HEK293T cells . Right : quantification of the p53 protein levels normalized against actin ( n = 3 ) . E ) The p53 - mediated transcriptional activity is measured by a luciferase reporter under the control of a p53 - response element promoter , which was transfected into HEK293T cells 72 - 96 h after the initiation of the UBE4B and LSD1 single or double knockdowns ( n = 6 ) . ( F ) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown . Left : representative western blots of 53BP1 co - immunoprecipitation . Right : quantification of the 53BP1 co - immunoprecipitation ( n = 2 ) . Data represent means ± SEM . ( G ) RT - qPCR validation of the expression levels of FOXO3a , FOXO4 , and PSMD11 ( n = 6 ) . ( H ) The FOXO3a - mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO - response element promoter . HEK293T cells were transfected with shRNAs for control , LSD1 , UBE4B , or both LSD1 and UBE4B , followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a ( TM ) 72 - 96 h later ( n = 6 ) . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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

Fig 4(B) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown.C) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04.(D) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (n = 3).E) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72-96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (n = 6).(F) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (n = 2). Data represent means ± SEM.(G) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (n = 6).(H) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72-96 h later (n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


Fig 5 ( A ) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells . ( B ) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate , Suc - LLVY - Luciferin , whose degradation is measured in a luciferase release assay . The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns ( n = 6 ) . ( C ) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown . The in vivo activity of the ATG4B protease , which cleaves LC3 precursors , was quantified via a Gaussia luciferase release assay ( see S5D Fig . , S5E Fig . , and Materials and Methods for details ) . The HCT116 cells were analyzed 48 h after the initiation of the knockdown . ( D ) Quantification of LC3 - II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control . The cells were treated with or without 10 mM 3 - methyladenine ( 3 - MA ) for 48h before lysis . Western blots indicate an increase in LC3 levels ( top panels ) in the double UBE4B and LSD1 knockdown cells ( middle panels ) , while the actin control is unchanged ( bottom panels ) . The graph shows the quantification of LC3 - II levels as normalized to actin levels ( n = 3 ) . A one - way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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

Fig 5(A) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells. (B) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (n = 6).(C) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see S5D Fig., S5E Fig., and Materials and Methods for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown.(D) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


Fig 6 ( A ) p53 small molecule activators Tenovin - 1 and CP - 31398 reduce the levels of misfolded SOD1 proteins , as determined by the SOD1G85R solubility assay in HEK293 cells . Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions . ( B ) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay , as shown by western blots of both supernatant ( S ) ( n = 2 ) and pellet ( P ) ( n = 3 ) fractions . ( C ) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53 - / - HCT116 cells when compared with controls . Representative western blots ( left panels ) and quantification of SOD1G85R levels in the supernatant lysates are shown . The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1 . The right graph panel shows the same data as shown in the middle panel , but normalized to the average SOD1G85R level for each amount of the transfected plasmid . Data represent means ± SEM . The numerical data used to make this figure can be found in S1 Data .

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

Fig 6(A) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions.(B) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1G85R but not WT SOD1 proteins in the SOD1G85R aggregation assay, as shown by western blots of both supernatant (S) (n = 2) and pellet (P) (n = 3) fractions.(C) A complete absence of p53 increases the accumulation of SOD1G85R mutant proteins in p53-/- HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.


Fig 7 ( A ) Left : p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown . Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs . Right : quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control , double ( UBE4B / LSD1 ) , or triple ( UBE4B / LSD1 / p53 ) shRNAs ( n = 2 ) . ( B ) The degenerative TDP - 43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 ( p53 RNAi ) , or by the overexpression of a dominant - negative p53 mutant ( p53 . R155H ) . Expression of p53 RNAi , p53 . R155H , and TDP - 43M337V are driven by GMR - Gal4 . ( C ) The p53 - activating drug Tenovin - 1 ( TEN1 ) protects spinal cord motor neurons from SOD1G85R - induced proteotoxicity . Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0 . 8 μM TEN1 or vehicle ( VEH ) DMSO , followed by infection of HSV - SOD1G85R or the HSV - LacZ control after 24 h . At day 5 post - infection , cells were fixed and stained with a motor neuron - specific anti - neurofilament H ( NF - H ) antibody . Left : representative images of motor neurons in each condition . Scale bar = 50 μm . Right : quantification of motor neuron survival ( one - way ANOVA with multiple comparisons and Tukey correction ) . Data represent means ± SEM .

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Fig 7(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2).(B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4.(C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM.


Figure 1 ( C ) Representative confocal image of long propriospinal neurons expressing FGF22 ( magenta ) in cervical spinal cord . ( E ) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals ( green ) , stained for vGlut ( red ) and vGAT ( blue ) 21 days following spinal cord injury . Insets represent 3D views generated in Imaris of the deconvolved confocal image . ( F ) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice ( n = 4 - 5 mice per group ) . ( ( G ) Schematic illustration and representative confocal image of LPSN viral labeling ( magenta ) and CST collaterals ( green ) . Insets represent 3D views generated in Imaris of the deconvolved confocal image . ( H ) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs ( n = 4 - 5 mice per group ) .

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Figure 1(C) Representative confocal image of long propriospinal neurons expressing FGF22 (magenta) in cervical spinal cord.(E) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals (green), stained for vGlut (red) and vGAT (blue) 21 days following spinal cord injury. Insets represent 3D views generated in Imaris of the deconvolved confocal image. (F) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice (n = 4-5 mice per group). ((G) Schematic illustration and representative confocal image of LPSN viral labeling (magenta) and CST collaterals (green). Insets represent 3D views generated in Imaris of the deconvolved confocal image. (H) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs (n = 4-5 mice per group).


Figure 2 ( C ) Schematic illustration and representative confocal image for vGlut2 - Cre neuronal labeling ( cyan ) and hCST collaterals ( white ) . Insets represent 3D views generated in Imaris of the confocal image . Quantification of hCST contacts onto vGlut neurons ( n = 4 - 6 mice per group ) .

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Figure 2(C) Schematic illustration and representative confocal image for vGlut2-Cre neuronal labeling (cyan) and hCST collaterals (white). Insets represent 3D views generated in Imaris of the confocal image. Quantification of hCST contacts onto vGlut neurons (n = 4-6 mice per group).


Figure 3 ( C ) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control ( left panel ) and FGF22 treated ( right panel ) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury . ( n = 6 mice per group ) . ( E ) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons ( arrows ) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 ( right ) and a control ( left ) mice . Quantification of bouton density in control and FGF22 treated mice . ( n = 6 mice per group ( F ) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals ( red ) and cervical interneurons ( cyan ) . Insets represent 3D views generated in Imaris of the confocal image . Quantification of the contacts between the hCST collaterals and cervical interneurons neurons . ( n = 6 per group ) . ( G ) Confocal images showing contacts between CST collaterals ( red ) and long propriospinal neurons ( magenta ) transduced with FGF22 ( cyan ) . Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus ( DP refers to double positive cells ) . Insets on the right are magnification of the boxed area on the left . ( n = 6 mice per group ) .

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Figure 3(C) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control (left panel) and FGF22 treated (right panel) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury. (n = 6 mice per group).(E) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons (arrows) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 (right) and a control (left) mice. Quantification of bouton density in control and FGF22 treated mice. (n = 6 mice per group(F) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals (red) and cervical interneurons (cyan). Insets represent 3D views generated in Imaris of the confocal image. Quantification of the contacts between the hCST collaterals and cervical interneurons neurons. (n = 6 per group).(G) Confocal images showing contacts between CST collaterals (red) and long propriospinal neurons (magenta) transduced with FGF22 (cyan). Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus (DP refers to double positive cells). Insets on the right are magnification of the boxed area on the left. (n = 6 mice per group).


Figure 4 ( C ) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in controls ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 mice per group ) . ( D ) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in control ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " or 2 days " 2dpi for the late treatment ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ( E ) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in control ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ) . ( F ( F ) Representative confocal image of CST boutons ( green ) and synapsin staining ( red ) and quantification of the percentage of bouton synapsin positive ( n = 6 - 7 mice per group ) . ( G ) Representative confocal images of CST bouton ( green ) and bassoon staining ( magenta ) and quantification of the percentage of boutons that are bassoon positive ( n = 5 - 6 mice per group ) .

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Figure 4(C) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in controls (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 mice per group). (D) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi" or 2 days "2dpi for the late treatment), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group). (E) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group).). (F(F) Representative confocal image of CST boutons (green) and synapsin staining (red) and quantification of the percentage of bouton synapsin positive (n = 6-7 mice per group). (G) Representative confocal images of CST bouton (green) and bassoon staining (magenta) and quantification of the percentage of boutons that are bassoon positive (n = 5-6 mice per group). D


Figure 1A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population ( www . genenetwork . org ) . Adr : Adrenal Gland mRNA , Bla : Bladder mRNA , Bone : Bone Tibia mRNA , CNS : CNS mRNA , Cec : Cecum mRNA , Epi : Epididymis mRNA , Eso : Esophagus mRNA , Eye : Eye mRNA , Fat : Peritoneal Fat mRNA , GI : GI Track mRNA , Hea : Heart mRNA , Kid : Kidney mRNA , Liv : Liver mRNA , Lung : Lung mRNA , Mus : Gastrocnemius Muscle mRNA , Ova : Ovary mRNA , Pro : Prostate mRNA , Sal : Salivary Gland mRNA , Skin : Skin Back mRNA , Sn : Sciatic Nerve mRNA , Spl : Spleen mRNA , Tes : Testis mRNA , Thy : Thymus mRNA , Ton : Tongue mRNA , Tra : Trachea mRNA , and Ute : Uterus mRNA . B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population ( www . genenetwork . org ) . C Oocytes injected with cRNA encoding KCC4 were incubated with NAD + 200 μM and NAM 10 mM for 4 h . Lysates were immunoprecipitated with an anti - KCC4 antibody and then analyzed by SDS - PAGE / immunoblotting using an anti - acetylated - lysine ( AcK ) or anti - KCC4 antibody . Non - injected oocytes ( UI ) were used as a control . D Oocytes expressing KCC4 were incubated with NAM for the indicated times , and KCC4 protein levels ( upper panel ) , and activity ( lower panel ) were analyzed by SDS - PAGE / immunoblotting or Cl - - dependent 86Rb + uptake under hypotonic conditions . E Oocytes injected with H20 or KCC4 were incubated for the indicated times , and KCC4 protein levels were analyzed by SDS - PAGE / immunoblotting . F Oocytes were injected with either WT , K114R or K114Q KCC4 and incubated with vehicle ( V ) , NAD + or NAM for 48 h , and KCC4 protein levels ( upper pannel ) , and activity ( lower pannel ) were analyzed as in D .

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Figure 1A In silico analysis of Sirt7 mRNA expression in different tissues of the BXD genetic reference population (www.genenetwork.org). Adr: Adrenal Gland mRNA, Bla: Bladder mRNA, Bone: Bone Tibia mRNA, CNS: CNS mRNA, Cec: Cecum mRNA, Epi: Epididymis mRNA, Eso: Esophagus mRNA, Eye: Eye mRNA, Fat: Peritoneal Fat mRNA, GI: GI Track mRNA, Hea: Heart mRNA, Kid: Kidney mRNA, Liv: Liver mRNA, Lung: Lung mRNA, Mus: Gastrocnemius Muscle mRNA, Ova: Ovary mRNA, Pro: Prostate mRNA, Sal: Salivary Gland mRNA, Skin: Skin Back mRNA, Sn: Sciatic Nerve mRNA, Spl: Spleen mRNA, Tes: Testis mRNA, Thy: Thymus mRNA, Ton: Tongue mRNA, Tra: Trachea mRNA, and Ute: Uterus mRNA.B Correlation plot for kidney mRNA expression of Sirt7 and Slc12a7 that codifies KCC4 in the mouse BxD genetic reference population (www.genenetwork.org).C Oocytes injected with cRNA encoding KCC4 were incubated with NAD+ 200 μM and NAM 10 mM for 4 h. Lysates were immunoprecipitated with an anti-KCC4 antibody and then analyzed by SDS-PAGE/immunoblotting using an anti-acetylated-lysine (AcK) or anti-KCC4 antibody. Non-injected oocytes (UI) were used as a control.D Oocytes expressing KCC4 were incubated with NAM for the indicated times, and KCC4 protein levels (upper panel), and activity (lower panel) were analyzed by SDS-PAGE/immunoblotting or Cl--dependent 86Rb+ uptake under hypotonic conditions.E Oocytes injected with H20 or KCC4 were incubated for the indicated times, and KCC4 protein levels were analyzed by SDS-PAGE/immunoblotting.F Oocytes were injected with either WT, K114R or K114Q KCC4 and incubated with vehicle (V), NAD+ or NAM for 48 h, and KCC4 protein levels (upper pannel), and activity (lower pannel) were analyzed as in D.