Effect of TFL457 , TFL482 and TFL541 on TrkB - FL levels . Cultures preincubated with TrkB - FL peptides or TMyc ( 25 µM , 30 min ) , followed by chronic NMDA treatment ( 2 h ) , were compared to cells without peptide . Levels of full - length ( FL ) TrkB were established with panTrkB , an antibody for the extracellular domain that also detects the truncated forms ( tTrkB ) . Quantitation of peptide interference of TrkB - FL downregulation . Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA . Means ± SEM are represented and analysis was performed by ANOVA test followed by post - hoc Tukey HSD test ( * p = 0 . 02 ; n = 4 ) .

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Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB). Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4).


Effect of TFL457 , TFL482 and TFL541 on neuronal viability after chronic excitotoxicity . Cultures were preincubated with peptides and treated with NMDA for 0 - 6 h as before . Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA . Mean ± SEM of eleven ( TFL457 ) , three ( TFL482 ) and seven ( TFL541 ) independent experiments is given .

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Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given.


Effect of TFL518 on TrkB - FL levels . Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457 .

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Effect of TFL518 on TrkB-FL levels. Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457.


TFL518 effect on neuronal viability . Cultures were treated and analyzed as above . No significant differences were found for TFL518 while neuroprotection by TFL457 was

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TFL518 effect on neuronal viability. Cultures were treated and analyzed as above. No significant differences were found for TFL518 while neuroprotection by TFL457 was confirmed


Effect of TFL457 on neuronal viability after acute excitotoxicity . Cells were induced with NMDA ( 50 μM ) and glycine ( 10 μM ) for 1 h and , after agonists removal , culture proceeded for 20 h with DL - AP5 ( 200 μM ) and TMyc or TFL457 ( 15 μM ) . Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented .

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Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented.


Effect of TFL457 on Y515 and Y816 phosphorylation . Cultures preincubated with TMyc or TFL457 ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) and analyzed with panTrkB or phosphospecific antibodies . Quantitation of pY515 and pY816 . Mean ± SEM of normalized pY515 ( n = 7 ) or pY816 ( n = 4 ) levels obtained after NMDA - treatment ( 2 h ) are represented relative to those found in cells with the same peptide but no NMDA . Statistical analysis was performed by unpaired Student t test ( * p = 0 . 046 ; n . s . = non - significant ) .

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Effect of TFL457 on Y515 and Y816 phosphorylation. Cultures preincubated with TMyc or TFL457 (25 μM, 30 min) were treated with NMDA (0-6 h) and analyzed with panTrkB or phosphospecific antibodies. Quantitation of pY515 and pY816. Mean ± SEM of normalized pY515 (n = 7) or pY816 (n = 4) levels obtained after NMDA-treatment (2 h) are represented relative to those found in cells with the same peptide but no NMDA. Statistical analysis was performed by unpaired Student t test (*p = 0.046; n.s. = non-significant).


Analysis by immunoprecipitation of TFL457 effect on pY816 levels . Cultures preincubated and treated as before with NMDA ( 2 h ) , were immunoprecipitated with antibodies specific for phosphorylated tyrosine ( pY ) . pY816 was analyzed by WB in immunoprecipates .

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Analysis by immunoprecipitation of TFL457 effect on pY816 levels. Cultures preincubated and treated as before with NMDA (2 h), were immunoprecipitated with antibodies specific for phosphorylated tyrosine (pY). pY816 was analyzed by WB in immunoprecipates.


TFL457 effects on neuronal viability after inhibition of TrkB - FL signaling . Cultures were preincubated ( 30 min ) with inhibitors specific for PI3K ( Wortmannin , 100 nM ) , MAPK / ERK ( UO126 , 300 nM ) or PLCγ ( U - 73122 , 5 μM ) before incubation with TMyc or TFL457 ( 25 μM , 30 min ) . Viability was established 4 h after NMDA treatment . Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post - hoc Tukey HSD test ( * * * p = 0 . 0001 , * * p = 0 . 009 , * p = 0 . 02 , n . s . = non - significant , respectively for TMyc vs TFL457 in untreated or Wortmannin , UO126 or U - 73122 - treated cells ; * * p = 0 . 008 for untreated vs U - 73122 - treated cells preincubated with TFL457 ; n = 5 ) .

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TFL457 effects on neuronal viability after inhibition of TrkB-FL signaling. Cultures were preincubated (30 min) with inhibitors specific for PI3K (Wortmannin, 100 nM), MAPK/ERK (UO126, 300 nM) or PLCγ (U-73122, 5 μM) before incubation with TMyc or TFL457 (25 μM, 30 min). Viability was established 4 h after NMDA treatment. Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post-hoc Tukey HSD test (***p = 0.0001, **p = 0.009, *p = 0.02, n.s. = non-significant, respectively for TMyc vs TFL457 in untreated or Wortmannin, UO126 or U-73122-treated cells; **p = 0.008 for untreated vs U-73122-treated cells preincubated with TFL457; n = 5).


Dose - dependent TFL457 effects . Cultures incubated with TMyc o TFL457 ( 5 , 15 o 25 μM , 30 min ) were treated with NMDA ( 2 h ) and compared to cells without peptide or NMDA .

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Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA.


Time - course TFL457 effects . Cells preincubated with peptides ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) .

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Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h).


Quantitation of peptide effects . Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA . Means ± SEM are given . Results were analyzed by a two - way ANOVA test followed by post - hoc Bonferroni test , comparing TMyc or TFL457 - treated cells for each time - point ( TrkB - FL , * * * p = 0 . 0009 and * p = 0 . 02 ; CREB , * p = 0 . 0149 and * p = 0 . 0148 ; pCREB , * * * p = 0 . 0007 and * * p = 0 . 0012 ; MEF2D , * * p = 0 . 0023 and * * p = 0 . 0047 . All values are respectively for 2 or 4 h ; n = 6 ) .

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Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).


Effect of TFL457 on MEF2 - promoter activity . Cultures transfected with pMEF2 ( two minimal wild type MEF2 elements ) or pMEF2mut ( mutant ) were preincubated with CPPs ( 25 μM , 30 min ) and treated with NMDA ( 100 µM , 2 h ) . Means ± SEM of luciferase activities obtained in excitotoxicity , relative to values found in cells treated with same peptide and no NMDA , are presented .

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Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented.


Effect on CRE - promoter activity . pCRE contains two minimal CREs . Peptide preincubation was as above with or without KG - 501 ( 10 µM ) . Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG - 501 .

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Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501.


Effect on NMDAR - subunit promoters . Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A ( pGluN1 , * p = 0 . 016 ; pGluN2A , * p = 0 . 029 ; n = 14 ) .

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Effect on NMDAR-subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A (pGluN1, *p = 0.016; pGluN2A, *p = 0.029; n = 14).


Effects on BDNF promoter III or TrkB promoter . Cells transfected with pIIIBDNF ( n = 9 ) , pTrkB ( n = 7 ) or pCRE ( n = 5 ) , as a control , were processed as above but using 20 µM NMDA for 4 h . Data are presented and analyzed as in panel A ( pCRE , * * p = 0 . 0098 ; pIIIBDNF , * p = 0 . 029 ; pTrkB , * p = 0 . 033 ) .

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Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF (n = 9), pTrkB (n = 7) or pCRE (n = 5), as a control, were processed as above but using 20 µM NMDA for 4 h. Data are presented and analyzed as in panel A (pCRE, **p = 0.0098; pIIIBDNF, *p = 0.029; pTrkB, *p = 0.033).


Effects of TFL457 on mRNA levels of CREB / MEF2 - regulated genes . Total RNA was extracted from cultures preincubated with CPPs ( 25 μM , 30 min ) and treated or not with NMDA ( 100 µM , 4 h ) . Levels of mRNA were normalized to NSE ( genes expressed in neurons , left panel ) or GAPDH ( neuronal and glial expression , right panel ) . Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented . Differences found in excitotoxicity were analyzed by ANOVA test followed by post - hoc Tukey HSD test ( GluN1 , * * * p = 0 . 00096 ; GluN2A , p = 0 . 98 ; TrkB - FL , * * * p = 0 . 00037 ; BDNF , * p = 0 . 045 ; n = 8 ) .

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Effects of TFL457 on mRNA levels of CREB/MEF2-regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 µM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post-hoc Tukey HSD test (GluN1, ***p = 0.00096; GluN2A, p = 0.98; TrkB-FL, ***p = 0.00037; BDNF, *p = 0.045; n = 8).


TFL457 interference of RIP and calpain - processing in excitotoxicity . Cultures were preincubated with peptides ( 25 µM , 30 min ) or left untreated before NMDA addition ( 2 h ) . Different exposures are shown to facilitate visualization of FL , f42 / 39 or f32 . Quantitation of normalized f32 and f42 / 39 levels . Means ± SEM are shown relative to cultures treated with NMDA and no peptide .

TFL457 interference of RIP and calpain-processing in excitotoxicity. Cultures were preincubated with peptides (25 µM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39 or f32. Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide.


Effect of TFL457 on neuronal viability after calpain inhibition . After 30 min preincubation with calpeptin ( Calp , 10 µM ) and calpain inhibitor III ( CiIII , 10 µM ) , cultures were treated with CPPs and NMDA ( 4 h ) as before . Means ± SEM relative to those obtained in untreated cultures are given .

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Effect of TFL457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 µM) and calpain inhibitor III (CiIII, 10 µM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given.


Effect of TFL457 on neuronal viability after metalloproteinase inhibition . Cultures preincubated with GM6001 ( 10 µM , 30 min ) were treated with CPPs and NMDA as before . Analysis was performed as above ( without GM6001 , * * * p = 0 . 0007 ; with GM6001 , * * * p = 0 . 0006 ; n = 4 ) .

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Effect of TFL457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 µM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, ***p = 0.0007; with GM6001, ***p = 0.0006; n = 4).


Effect of NMDA on total and cell - surface TrkB - FL levels . Cultures were incubated with NMDA ( 100 µM ) or BDNF ( 100 ng / ml ) for 1 h . Membrane proteins were labeled and purified and compared to corresponding total lysates . TrkB - FL levels are expressed relative to untreated cells .

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Effect of NMDA on total and cell-surface TrkB-FL levels. Cultures were incubated with NMDA (100 µM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB-FL levels are expressed relative to untreated cells.


Effect of TFL457 on the decrease of TrkB - FL surface levels induced by NMDA . Analysis was performed as before in cells incubated with CPPs ( 25 μM , 30 min ) and NMDA - treated ( 1 h ) . Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA .

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Effect of TFL457 on the decrease of TrkB-FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA-treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA.


Effect of endocytosis inhibition on TrkB - FL downregulation . The infarcted area of the ipsilateral hemisphere ( I ) was compared to the corresponding region of the contralateral area ( C ) . Results from representative mice injected with dynasore or vehicle are shown . Different exposures are presented to facilitate visualization of dynasore effects on TrkB - FL and f32 . Quantitation of TrkB - FL and TrkB - T1 in the infarcted area . Normalized protein levels are expressed relative to those of the corresponding contralateral region .

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Effect of endocytosis inhibition on TrkB-FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB-FL and f32. Quantitation of TrkB-FL and TrkB-T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region.


Confirmation of Bio - TFL457 delivery to mice cortex . Biotinylated TFL457 ( Bio - TFL457 , 4 nMole / g , retro - orbitally injected ) was detected in coronal sections stained with NeuN and DAPI . Representative confocal microscopy images of cortical areas correspond to single sections . Scale bar , 10 µm .

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Confirmation of Bio-TFL457 delivery to mice cortex. Biotinylated TFL457 (Bio- TFL457, 4 nMole/g, retro-orbitally injected) was detected in coronal sections stained with NeuN and DAPI. Representative confocal microscopy images of cortical areas correspond to single sections. Scale bar, 10 µm.


Representative analysis of TrkB - FL at 3 h of damage . Comparison of the infarcted area ( I ) and the corresponding contralateral region ( C ) is shown . Quantitation of TrkB - FL levels . Means ± SEM of normalized TrkB - FL levels in the infarcted area relative to the corresponding contralateral

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Representative analysis of TrkB-FL at 3 h of damage. Comparison of the infarcted area (I) and the corresponding contralateral region (C) is shown. Quantitation of TrkB-FL levels. Means ± SEM of normalized TrkB-FL levels in the infarcted area relative to the corresponding contralateral region


Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457 .

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Representative 1 mm brain coronal slices stained with TTC after 24 h of insult corresponding to animals injected with MTMyc or MTFL457.


Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume . Means ± SEM are given . Differences were analyzed by Student ' s t - test ( * * * p = 0 . 00008 ; n = 14 ) . Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc .

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Infarct volume of animals injected with MTMyc or MTFL457 expressed as a percentage of the hemisphere volume. Means ± SEM are given. Differences were analyzed by Student's t-test (***p = 0.00008; n = 14). Results for MTFL457 are also expressed as percentage of the infarct volume in animals injected with MTMyc.


Evaluation of balance and motor coordination . Number of contralateral hind paw slips were measured after 24 h of damage . Data spread is presented by box and whisker plots showing interquartile range , median , minimum and maximum values .

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Evaluation of balance and motor coordination. Number of contralateral hind paw slips were measured after 24 h of damage. Data spread is presented by box and whisker plots showing interquartile range, median, minimum and maximum values.


( B ) Migration efficiency ( % of cells that migrated through filter pores in each round ) of A375 cells during 5 μm constricted migration and 12 μm migration ( left panels ) . Biological replicates shown . Phase contrast imaging of the final cell populations after the 10 rounds of migration , cells cultured on 2D plastic ( right panels , Scale bar = 50 µm ) .

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(B) Migration efficiency (% of cells that migrated through filter pores in each round) of A375 cells during 5 μm constricted migration and 12 μm migration (left panels). Biological replicates shown. Phase contrast imaging of the final cell populations after the 10 rounds of migration, cells cultured on 2D plastic (right panels, Scale bar = 50 µm).


( C ) A375 nuclei stained with DAPI ( blue ) , Lamin A / C ( green ) and H3K9me3 ( red ) in the indicated migration conditions . Images shown represent a maximum projection of z - stacks ( Scale bar = 5 µm ) .

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(C) A375 nuclei stained with DAPI (blue), Lamin A/C (green) and H3K9me3 (red) in the indicated migration conditions. Images shown represent a maximum projection of z-stacks (Scale bar= 5 µm).


( D ) Cumulative distribution plot of Lamin A / C coefficient of variation in all indicated A375 conditions ( Control ( n = 59 ) , Top - 5 ( n = 64 ) , Bottom - 5 ( n = 62 ) nuclei ) . Kolmogorov - Smirnov test p - values indicated in plot .

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(D) Cumulative distribution plot of Lamin A/C coefficient of variation in all indicated A375 conditions (Control (n = 59), Top-5 (n = 64), Bottom-5 (n = 62) nuclei). Kolmogorov-Smirnov test p-values indicated in plot.


( E ) Radial intensity distribution of H3K9me3 signal in the maximum projected nuclei of Control ( n = 59 ) , Top - 5 ( n = 63 ) , Bottom - 5 ( n = 64 ) and Bottom - 12 ( n = 80 ) nuclei ( * * p = 0 . 0069 , two - tailed t - test ) . Error bars = mean ± SD .

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(E) Radial intensity distribution of H3K9me3 signal in the maximum projected nuclei of Control (n = 59), Top-5 (n = 63), Bottom-5 (n = 64) and Bottom-12 (n = 80) nuclei (** p = 0.0069, two-tailed t-test). Error bars = mean ± SD.


( F ) Maximum projection confocal images of Top - 5 and Bottom - 5 cells embedded in collagen and stained with DAPI ( blue ) and Phalloidin ( red ) . ( Inset ) Zoom in of nucleus of Bottom - 5 cell . Top - 5 and Bottom - 5 zoomed inset scale = 5 μm ; Bottom - 5 zoomed out image scale = 20 μm .

(F) Maximum projection confocal images of Top-5 and Bottom-5 cells embedded in collagen and stained with DAPI (blue) and Phalloidin (red). (Inset) Zoom in of nucleus of Bottom-5 cell. Top-5 and Bottom-5 zoomed inset scale = 5 μm; Bottom-5 zoomed out image scale = 20 μm.


( G ) Fraction of Control , Top - 5 and Bottom - 5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices . Each biological replicate is connected by a line .

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(G) Fraction of Control, Top-5 and Bottom-5 cells displaying projections at indicated timepoints after cells were embedded in 3D collagen matrices. Each biological replicate is connected by a line.


( H ) Nuclei of Top - 5 ( left ) and Bottom - 5 cells ( right ) embedded in 3D collagen matrix and stained with DAPI ( blue ) and Lamin A / C ( green ) . Scale = 5μm . For 3D reconstruction visualization , see Movies EV5 and EV6 .

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(H) Nuclei of Top-5 (left) and Bottom-5 cells (right) embedded in 3D collagen matrix and stained with DAPI (blue) and Lamin A/C (green). Scale = 5μm. For 3D reconstruction visualization, see Movies EV5 and EV6.


( I ) Percent of cells embedded in collagen that had elongated nuclei ( Aspect Ratio > 2 . 5 ) for A375 Control ( n = 54 ) , Top - 5 ( n = 52 ) and Bottom - 5 ( n = 53 ) .

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(I) Percent of cells embedded in collagen that had elongated nuclei (Aspect Ratio > 2.5) for A375 Control (n = 54), Top-5 (n = 52) and Bottom-5 (n = 53).


( J ) Cumulative distribution plot of Lamin A / C coefficient of variation for Control ( n = 70 ) , Top - 5 ( n = 78 ) and Bottom - 5 ( n = 109 ) cells embedded in 3D collagen matrices . Kolmogorov - Smirnov test p - values indicated in plot .

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(J) Cumulative distribution plot of Lamin A/C coefficient of variation for Control (n = 70), Top-5 (n = 78) and Bottom-5 (n = 109) cells embedded in 3D collagen matrices. Kolmogorov-Smirnov test p-values indicated in plot.


( E ) Metaphase spread labeled with chromosome specific FISH probes for chr5 ( green ) and chr13 ( red ) . Bottom graphs represent frequency of t ( 5 ; 13 ) quantified by FISH ( left ) and copy number quantification for chr5 and chr13 ( right ) . n = 1328 Control and 2370 metaphases analyzed for Bottom - 5 . ( Scale = 10 μm )

(E) Metaphase spread labeled with chromosome specific FISH probes for chr5 (green) and chr13 (red). Bottom graphs represent frequency of t(5;13) quantified by FISH (left) and copy number quantification for chr5 and chr13 (right). n = 1328 Control and 2370 metaphases analyzed for Bottom-5. (Scale = 10 μm)


A . Expression profiling of differentially expressed circRNAs between the tumor and normal groups . Rows represent circRNAs , and columns represent samples . Rows were ordered by fold change , and columns were ordered by their group . The sample of N8 was not included due to low sequencing library size .

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A. Expression profiling of differentially expressed circRNAs between the tumor and normal groups. Rows represent circRNAs, and columns represent samples. Rows were ordered by fold change, and columns were ordered by their group. The sample of N8 was not included due to low sequencing library size.


B . Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups . Both the row and column were unsupervised and clustered with the hierarchical clustering method .

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B. Expression profiling of differentially expressed circRNAs between the recurrence and nonrecurrence groups. Both the row and column were unsupervised and clustered with the hierarchical clustering method.


C . The 4 of 22 differentially expressed circRNAs were confirmed by qRT - PCR , which were retained after marker selection procedure . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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C. The 4 of 22 differentially expressed circRNAs were confirmed by qRT-PCR, which were retained after marker selection procedure. **P < 0.01, Student's t-test, mean ± SD.


E . Time - dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset . P - values are shown for the indicated comparison of AUC between each marker and cirScore . Student ' s t - test , AUC = area under the curve .

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E. Time-dependent AUC analysis of individual circle RNA and cirScore for predicting recurrence in the training dataset. P-values are shown for the indicated comparison of AUC between each marker and cirScore. Student's t-test, AUC=area under the curve.


A - F . Kaplan - Meier curves of DFS ( A ) and OS ( B ) in 249 patients in the training set . Kaplan - Meier curves of DFS ( C ) and OS ( D ) in 122 patients in the internal validation data set . Kaplan - Meier curves of DFS ( E ) and OS ( F ) in 180 patients in the external independent validation data set . Hazard ratios ( HRs ) were calculated with a univariate Cox regression analysis , P - values were calculated with the log - rank test .

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A-F. Kaplan-Meier curves of DFS (A) and OS (B) in 249 patients in the training set. Kaplan-Meier curves of DFS (C) and OS (D) in 122 patients in the internal validation data set. Kaplan-Meier curves of DFS (E) and OS (F) in 180 patients in the external independent validation data set. Hazard ratios (HRs) were calculated with a univariate Cox regression analysis, P-values were calculated with the log-rank test.


A . RT - PCR products with divergent and convergent primers showing circularization of has _ circ _ 0079480 and has _ circ _ 0087391 . cDNA , complementary DNA ; gDNA , genomic DNA .

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A. RT-PCR products with divergent and convergent primers showing circularization of has_circ_0079480 and has_circ_0087391. cDNA, complementary DNA; gDNA, genomic DNA.


B . qRT - PCR evaluated the knockdown efficiency of has _ circ _ 0079480 and and has _ circ _ 0087319 in SW620 and HCT116 cells transfected with two unique shRNAs ( # 1 , # 2 ) . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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B. qRT-PCR evaluated the knockdown efficiency of has_circ_0079480 and and has_circ_0087319 in SW620 and HCT116 cells transfected with two unique shRNAs (#1, #2). **P < 0.01, Student's t-test, mean ± SD.


C . Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs .

C. Representative images of the migration phenotype in HCT116 cells with knockdown of candidate circRNAs.


D . The relative fold - change of the transwell migration for indicated knockdown cells over those of control cells . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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D. The relative fold-change of the transwell migration for indicated knockdown cells over those of control cells. **P < 0.01, Student's t-test, mean ± SD.


E - F . Representative hematoxylin and eosin ( H & E ) staining and statistical results of the micro - metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days . N = 8 per group . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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E-F. Representative hematoxylin and eosin (H&E) staining and statistical results of the micro-metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days. N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


G - H . Representative H & E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days . ( Five sections evaluated per lung ) . N = 8 per group . * * P < 0 . 01 , Student ' s t - test , mean ± SD .

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G-H. Representative H&E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days. (Five sections evaluated per lung). N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


C DNA breaks were measured using alkaline comet assay in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with 2 µM pyridostatin ( PDS ) for 16 hours and released into fresh medium without pyridostatin . Representative images are shown . Scale bar represents 100 µm . D Quantification of DNA breaks shown in ( C ) . Graph and error bars represent the mean and SEM of n = 3 independent experiments . A minimum of 50 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; NS , P > 0 . 5 . E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in ( C ) . A minimum of 200 cells were analysed per condition per experiment . Graph and error bars represent the mean and SEM of n = 3 independent experiments . P values were calculated using an unpaired two - tailed t - test . * * * , P ≤ 0 . 001 ; NS , P > 0 . 5 .

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C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm. D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5. E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.


F BRCA2 - deficient ( - BRCA2 ) human DLD1 cells were treated with 2 µM pyridostatin ( PDS ) for 24 hours . Next , pyridostatin was removed and cells were released in a medium containing 250 nM NU - 7441 ( NU ) . Whole - cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown . SMC1 was used as a loading control . KAP1 phosphorylation site is indicated in red .

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F BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.


G Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with pyridostatin ( PDS ) and NU - 7441 at the indicated concentrations for six days . Graphs represent average values obtained from of n = 3 independent experiments , each performed in technical triplicates .

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G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.


Quantitative RT - PCR of cells grown as in ( A ) and treated with 10 µM pyridostatin ( PDS ) for 3 days was performed using primers specific for the indicated genes . mRNA levels are expressed relative to GAPDH and to - DOX ( + BRCA2 ) cells . Error bars represent the SEM of n = 3 independent experiments , each performed in technical triplicate . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 .

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Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001.


BRCA2 + / + and BRCA2 - / - RPE - 1 cells were treated with 10 μM pyridostatin ( PDS ) for 2 days . Whole - cell extracts were immunoblotted as indicated . KAP1 , IRF3 and STAT1 phosphorylation sites are shown in red . Tubulin and GAPDH were used as loading controls .

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BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls.


H1299 + shBRCA2DOX cells treated as in ( B ) were fixed and prepared for immunofluorescence with antibody against cGAS . DNA was counterstained with DAPI . Scale bar represents 20 µm . Quantification of cGAS - positive micronuclei per cells shown in ( E ) . Graph and error bars represent the mean and SEM of of n = 3 independent experiments . A minimum of 250 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; NS , P > 0 . 5 .

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H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm. Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.


FVB female mice were injected intramuscularly with ( A ) BRCA1 - proficient KP3 . 33 ( Brca1 + / + ) or ( B ) BRCA1 / 53BP1 - deficient KB1PM5 ( Brca1 - / - , Tp53bp1 - / - ) mouse mammary tumour cells . Pyridostatin ( PDS ) was administered intravenously ( i . v . ; 7 . 5 mg / kg / day ) and talazoparib was administered orally ( p . o . ; 0 . 33 mg / kg / day ) , over the indicated periods of time . Vertical dotted line indicates end of treatment . Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment . Each experimental group included n = 5 mice . Error bars represent SEM . P values were calculated between treated and untreated tumours at day 16 , using an unpaired two - tailed t - test . * * * * , P ≤ 0 . 0001 ; NS , P > 0 . 5 .

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FVB female mice were injected intramuscularly with (A) BRCA1-proficient KP3.33 (Brca1+/+) or (B) BRCA1/53BP1-deficient KB1PM5 (Brca1-/-, Tp53bp1-/-) mouse mammary tumour cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 16, using an unpaired two-tailed t-test. ****, P ≤ 0.0001; NS, P > 0.5.


BRCA1 + / + , BRCA1 - / - and BRCA1 - / - / TP53BP1 - / - RPE - 1 cells were treated with 10 μM pyridostatin ( PDS ) or 2 μM olaparib for 2 days . Whole - cell extracts were immunoblotted as indicated . KAP1 , IRF3 and STAT1 phosphorylation sites are shown in red . SMC1 and GAPDH were used as loading controls .

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BRCA1+/+, BRCA1-/- and BRCA1-/-/TP53BP1-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) or 2 μM olaparib for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. SMC1 and GAPDH were used as loading controls.


B VHIO179 PDTXs were grafted into CB17 - SCID female mice . Pyridostatin was administered intravenously ( 7 . 5 mg / kg / day ) over the indicated periods of time . Vertical dotted line indicates end of treatment . Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment . Each experimental group included n = 7 mice . Error bars represent SEM . P values were calculated between treated and untreated tumours at day 24 , using an unpaired two - tailed t - test . * * * * , P ≤ 0 . 0001 .

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B VHIO179 PDTXs were grafted into CB17-SCID female mice. Pyridostatin was administered intravenously (7.5 mg/kg/day) over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 7 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 24, using an unpaired two-tailed t-test. ****, P ≤ 0.0001.


Kaplan - Meier survival curve for mice treated as in ( C ) , with each treatment group containing n = 5 mice . Statistical significance was assessed by Log - rank test ( * * * * P < 0 . 0001 ) .

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Kaplan-Meier survival curve for mice treated as in (C), with each treatment group containing n = 5 mice. Statistical significance was assessed by Log-rank test (****P <0.0001).


Kaplan - Meier survival curve for mice treated as in ( C ) , with each treatment group containing n = 6 mice . Statistical significance was assessed by Log - rank test ( * * * * P < 0 . 0001 ) .

Kaplan-Meier survival curve for mice treated as in (C), with each treatment group containing n = 6 mice. Statistical significance was assessed by Log-rank test (****P <0.0001).


( C ) Acoustic startle reflex test to compare hearing ability between Wt ( N = 7 ) and Hpn - / - ( N = 4 ) mice .

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(C) Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn-/- (N = 4) mice.


( F ) Immunoblot analysis of phospho - Smad2 / 3 ( TGFβ pathway signaling marker ) and total Smad2 / 3 in lysates from indicated tissues isolated from Wt , Hpn + / - and Hpn - / - mice . GAPDH was used as the loading control . The histogram depicts quantification of pSmad2 / 3 compared to Wt normalized to total Smad2 / 3 .

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(F) Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt normalized to total Smad2/3.


( B ) Immunoblot analysis of Wap - Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin ( T # denotes individual tumors ) . GAPDH was used as the loading control . Lysates were derived from Wap - Myc mammary tumors from 6 mice with and 6 mice without DOX - induced hepsin

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(B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression


( D ) Immunoblot analysis of phospho - Smad2 / 3 , total Smad2 / 3 , and GAPDH ( loading control ) expression in Wt tumors transplanted into either Wt or Hpn - / - recipients ( n = 10 tumors each ) .

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(D) Immunoblot analysis of phospho-Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn-/- recipients (n = 10 tumors each).


( E ) Western blot analysis of phospho - Smad2 / 3 , total Smad2 / 3 , hepsin , and β - tubulin ( loading control ) in MCF10A - pIND20 - HPN cells treated with doxycycline ( DOX ; overexpression of hepsin ) and the TGFβR1 inhibitor Galunisertib ( TGFβR1i , 10µM ) as indicated .

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(E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated.


( G ) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A - pIND20 - HPN pL6 - TGFβ1 cells with ( DOX + ) or without ( DOX - ) hepsin overexpressing . The TGFβ ELISA assay detects active TGFβ levels ( first 2 columns ) , but heat treatment of the conditioned medium activates all TGFβ , thus effectively measuring total TGFβ ( right two columns ) .

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(G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns).


( H ) Silver - stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin . SLC was incubated with increasing concentrations of recombinant hepsin ( numbers indicate the concentration of hepsin in nM ) .

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(H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).


( A ) Coomassie - stained protein gels with cell lysates and concentrated culture supernatants of MCF10A - pIND20 - HPN cells , with ( + DOX ) or without ( - DOX ) hepsin overexpression . M indicates the media only control . R and NR above the gels indicate reducing and non - reducing conditions , respectively . Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry ; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left . The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions , highlighting the part from which fibronectin was identified . Red boxes indicate the lanes that were analyzed by mass

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(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry


( C ) Immunoblot analysis of fibronectin , hepsin , and β - tubulin ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells with ( DOX + ) or without ( DOX - ) hepsin overexpression . The graph shows the quantification of fibronectin levels .

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(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.


( D ) Silver - stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin ( upper panel ) or the 70 kDa most N - terminal fragment of fibronectin ( lower panel ) . Full - length fibronectin ( 1μg ) or the 70 kDa fibronectin fragment ( 1μg ) were incubated with increasing concentrations of recombinant hepsin . Arrowheads indicate full - length fibronectin , the 70kDa fragment , the 50 kDa cleavage fragment generated by hepsin , and hepsin . The schematic figure shows the full - length fibronectin protein and the 70KDa N - terminal fragment . The red arrow indicates the location of the putative hepsin cleavage site . RGD indicated the RGD - binding domain in fibronectin .

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(D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.


( A ) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt , Hpn + / - and Hpn - / - mouse ( representative of at least three repeats ) . GAPDH blot is used here as the loading control .

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(A) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mouse (representative of at least three repeats). GAPDH blot is used here as the loading control.


( C ) Immunoblot analysis of fibronectin , hepsin , and β - tubulin ( loading control ) in prostate lysates of control mice or mice with prostate - specific hepsin overexpression N = 2 mice each , the two prostate lobes were run separately ( a and b ) .

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(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in prostate lysates of control mice or mice with prostate-specific hepsin overexpression N=2 mice each, the two prostate lobes were run separately (a and b).


( A ) Immunoblot analysis of fibronectin , pSmad2 / 3 , total Smad2 / 3 , and GAPDH ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells without hepsin overexpression . The graph depicts quantification of pSmad2 / 3 normalized to total Smad2 / 3 levels , compared to siCtrl ( N = 3 biological repeats ) . ( B ) Immunoblot analysis of cell lysates ( pSmad2 / 3 , total Smad2 / 3 and vinculin as loading control ) and concentrated cell culture supernatant ( anti - V5 for TGFβ ) from MCF10A - pIND20 - HPN pL6 - TGFβ1 - V5 cells without hepsin overexpression . Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant . The graph depicts quantification of pSmad2 / 3 normalized to total Smad2 / 3 levels , compared to siCtrl ( N = 3 biological repeats ) .

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(A) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (B) Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3 and vinculin as loading control) and concentrated cell culture supernatant (anti-V5 for TGFβ) from MCF10A-pIND20-HPN pL6-TGFβ1-V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats).


( E ) Immunoblot analysis of fibronectin , pSmad2 / 3 , total Smad2 / 3 , hepsin , and GAPDH ( loading control ) in cell lysates from MCF10A - pIND20 - HPN cells with ( DOX + ) and without ( DOX - ) hepsin overexpression , and with or without fibronectin silencing ( siFN1 ) . The graph depicts quantification of pSmad2 / 3 normalized to total Smad2 / 3 levels ( N = 3 biological repeats ) .

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(E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats).


Luciferase - based imaging of drug response in Vegfr3Luc ; Tyr : CreERT2 ; BrafV600E ; Ptenflox / flox mice . Panels labeled as " basal " and " induced " correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH - tamoxifen ( 5 mM , topical administration , 3 consecutive days ) for the induction of melanomas . ( Right panels ) Treatment with anti - PD - L1 antibody ( αPD - L1 ; clone 10F . 9G2 , 3 weeks ) or the corresponding control IgG ( 200 ug / dose , twice per week , 3 weeks ) ; vemurafenib ( Vem , 50 mg / Kg , oral once per day , 3 weeks ) or BO - 110 ( BO , 0 . 8 mg / kg , twice per week , 3 weeks ) . Scale : p / s / cm2 / sr ( x106 ) .

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Luciferase-based imaging of drug response in Vegfr3Luc;Tyr:CreERT2;BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) Treatment with anti-PD-L1 antibody (αPD-L1; clone 10F.9G2, 3 weeks) or the corresponding control IgG (200 ug/dose, twice per week, 3 weeks); vemurafenib (Vem, 50 mg/Kg, oral once per day, 3 weeks) or BO-110 (BO, 0.8 mg/kg, twice per week, 3 weeks). Scale: p/s/cm2/sr (x106).


Treatment with BO - 110 of human patient - derived xenografts ( PDX ) implanted in Vegfr3Luc nu / nu . 42 days after implantation ( when systemic luciferase was detected ) , animals were randomized for treatment with vehicle ( V ) or with 0 . 8 mg / kg BO - 110 ( BO , twice per week ) , and luciferase emission was acquired at the indicated times . Scale , p / s / cm2 / sr ( x106 ) .

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Treatment with BO-110 of human patient-derived xenografts (PDX) implanted in Vegfr3Luc nu/nu. 42 days after implantation (when systemic luciferase was detected), animals were randomized for treatment with vehicle (V) or with 0.8 mg/kg BO-110 (BO, twice per week), and luciferase emission was acquired at the indicated times. Scale, p/s/cm2/sr (x106).


qRT - PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0 . 5 ug / ml BO - 110 ( BO ) , 10 µM vemurafenib ( Vem ) , or vehicle control ( V ) . Data correspond to the mean ± SD of 3 biological replicates . Statistical significance was determined by t - test . qRT - PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0 . 5 or 1 ug / ml BO - 110 ( VO ) , 10 µM vemurafenib ( Vem ) , or the corresponding vehicle control ( V ) . Data correspond to the mean ± SD of 3 biological replicates . Statistical significance was determined by ANOVA .

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qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml BO-110 (BO), 10 µM vemurafenib (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.   qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.


Luciferase signal driven by FLT4 ( VEGFR3 ) - promoter transduced into HLEC treated with vehicle ( v ) or BO - 110 ( BO ) at the indicated doses ( ug / ml ) as indicated in methods . Results were normalized to vehicle control . N = 4 biological replicates . Error bars correspond to mean ± SD . Statistical significance was determined by ANOVA .

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Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA.


Analysis of apoptotic cells at the indicated time points . HLEC cells were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for the indicated time points . Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) , as determined by qRT - PCR . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Inhibitory effect of the indicated doses of BO - 110 ( in µg / ml ) or vehicle ( V ) on MDK mRNA expression determined by qRT - PCR in SK - Mel - 147 ( 16 h after treatment ) . Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD . Statistical significance was determined by ANOVA . qRT - PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK - Mel - 147 with the indicated doses of BO - 110 ( in µg / ml ) ( BO ) . Data correspond to the mean ± SD of three experiments with three technical replicates . Statistical significance was determined by ANOVA .

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Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA expression determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.   qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.


Analysis of apoptotic cells at the indicated time points . SK - Mel - 147 cells were treated with vehicle ( V ) or 0 . 5 µg / ml BO - 110 ( BO ) for the indicated time points . Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods . Data correspond to the mean ± SD of 3 experiments . Statistical significance was determined by t - test .

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Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.


Immunohistochemical analysis of MDK repression ( pink staining ) in SK - Mel - 147 xenografts and PDX lesions after treatment with BO - 110 ( BO , 0 . 8 mg / kg , 2 doses / week ) . Histological staining in tumors extracted from animals treated with vehicle control ( V ) are included as a reference . Nuclei were counterstained in with hematoxylin .

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Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained in with hematoxylin.


Type I IFN mRNA induction ( IFNA2 and IFNB1 ) in SK - Mel - 147 melanoma cells treated for 16 h with the indicated amounts of BO - 110 ( in mg / ml ) . Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control . Statistical significance was determined by ANOVA .

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Type I IFN mRNA induction (IFNA2 and IFNB1) in SK-Mel-147 melanoma cells treated for 16 h with the indicated amounts of BO-110 (in mg/ml). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Statistical significance was determined by ANOVA.


Heatmap depicting expression changes in interferon - related genes ( GO : 0034340 ) in SK - Mel - 147 melanoma cells ( left panel ) and HLEC ( right panel ) treated with vehicle or 0 . 5 g / ml of BO - 110 for 10 hours . CD274 , LAG3 and PDCD1 genes were also included as a reference .

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Heatmap depicting expression changes in interferon-related genes (GO:0034340) in SK-Mel-147 melanoma cells (left panel) and HLEC (right panel) treated with vehicle or 0.5 g/ml of BO-110 for 10 hours. CD274, LAG3 and PDCD1 genes were also included as a reference.


IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO - 110 treatment ( 0 . 5 µg / ml ) of SK - Mel - 147 melanoma cells or HLEC ( left and right graphs , respectively ) . Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control .

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IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control.


Quantification of the impact of BO - 110 as single agent or in the presence of the indicated blocking antibodies for type I interferon ( IFNB1 or IFNAR1 ) . Upper graphs show the effect of these agents on MDK mRNA levels in SK - Mel - 147 melanoma cells . Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA ( middle graphs ) and tube formation capacity ( lower graphs ) . Data correspond to the mean ± SD of 3 biological replicates in triplicate .

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Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.


Growth of B16 melanoma xenografts in siblings of Ifnar1 + / + , Ifnar1 + / - or Ifnar1 - / - mice . Treatment started 10 days after tumor cell implantation . BO - 110 was administered at 0 . 8 mg / kg , every third day for 2 weeks . N = 6 mice per condition . Graphs show the mean tumor size ± SD at each time point . Statistical significance was determined by Two - Way ANOVA .

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Growth of B16 melanoma xenografts in siblings of Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA.


Impact of BO - 110 on tumor - bearing mice implanted with syngeneic B16R2L ( 5 x 105 cells ) . When tumors have an average size on 100 mm3 , mice were randomized into two groups and treated with BO - 110 ( BO ) or vehicle ( V ) , every second day for 2 weeks . The arrow indicates the start of the treatment . Tumor size was measured with a caliper at the indicated time points . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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Impact of BO-110 on tumor-bearing mice implanted with syngeneic B16R2L (5 x 105 cells). When tumors have an average size on 100 mm3, mice were randomized into two groups and treated with BO-110 (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points. , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment ( A ) . N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment (A). N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


G . ELISA analysis of Mdk blood levels in mice in experiment ( A ) . H . ELISA analysis of mouse Ifn - β blood levels in mice in experiment ( A ) . , N = 4 mice per condition . Statistical significance was determined by Two - Way - ANOVA .

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G. ELISA analysis of Mdk blood levels in mice in experiment (A). H. ELISA analysis of mouse Ifn-β blood levels in mice in experiment (A). , N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.


Efficacy of BO - 110 as an adjuvant ( preventing relapse after surgical removal of the primary lesion ) . Shown are representative examples of Vegfr3Luc mice implanted with mCherry - SK - Mel - 147 and imaged for luciferase emission ( prior to and after tumor removal . Animals were left to recover from surgery ( 4 days ) and then treated for 2 weeks ( 4 dosis ) with 0 . 8 mg / Kg BO - 110 or vehicle control ( n = 8 for control and n = 10 for treatment arm ) . Scale , p / s / cm2 / sr x106 . mCherry emission from tumor cells of the animals in ( A ) . Scale , p / s / cm2 / sr x109 .

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Efficacy of BO-110 as an adjuvant (preventing relapse after surgical removal of the primary lesion). Shown are representative examples of Vegfr3Luc mice implanted with mCherry-SK-Mel-147 and imaged for luciferase emission (prior to and after tumor removal. Animals were left to recover from surgery (4 days) and then treated for 2 weeks (4 dosis) with 0.8 mg/Kg BO-110 or vehicle control (n= 8 for control and n=10 for treatment arm). Scale, p/s/cm2/sr x106.   mCherry emission from tumor cells of the animals in (A). Scale, p/s/cm2/sr x109.


Kaplan - Meier survival curves of animals treated as in ( A ) . 8 / 8 animals treated with vehicle ( V ) control had to be sacrificed for humane reasons 110 days after surgery . 9 / 10 animals in the BO - 110 arm ( BO ) remained tumor - free 8 months after stopping treatment . The grey box marks the period of treatment with BO - 110 . Statistical significance was determined by logrank test .

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Kaplan-Meier survival curves of animals treated as in (A). 8/8 animals treated with vehicle (V) control had to be sacrificed for humane reasons 110 days after surgery. 9/10 animals in the BO-110 arm (BO) remained tumor-free 8 months after stopping treatment. The grey box marks the period of treatment with BO-110. Statistical significance was determined by logrank test.


( B ) Fluorescence microscopy images of typical mouse small intestinal organoids under different culture conditions . Lgr5 + intestinal stem cells are labeled with GFP , which are ubiquitously present , restricted to the bottom of the crypts and absent in CV , ENR and EN , respectively . * is auto - fluorescence

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(B) Fluorescence microscopy images of typical mouse small intestinal organoids under different culture conditions. Lgr5+ intestinal stem cells are labeled with GFP, which are ubiquitously present, restricted to the bottom of the crypts and absent in CV, ENR and EN, respectively. * is auto-fluorescence


( A ) Two row - matched heatmaps showing the relative change in mRNA expression ( left ) and protein expression ( right ) of significantly changing proteins . Known markers of intestinal homeostasis are highlighted . Relative expression is shown as log2 fold change over the row mean

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(A) Two row-matched heatmaps showing the relative change in mRNA expression (left) and protein expression (right) of significantly changing proteins. Known markers of intestinal homeostasis are highlighted. Relative expression is shown as log2 fold change over the row mean


( B ) Hematoxylin and ( Periodic acid - Shiff ) PAS staining of WT ( left ) and Hnf4g KO ( right ) intestine . Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS

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(B) Hematoxylin and (Periodic acid-Shiff) PAS staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS


( C ) Alcian Blue and Nuclear Fast Red staining of WT ( left ) and Hnf4g KO ( right ) small intestinal organoids . Cells are visualized using Nuclear Fast Red . Intra - and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue

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(C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue


( D ) Strand specific RNA - seq data , Hnf4g ChIP - seq in EN , and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown . Data from different organoid cultures are color coded . RNA - seq reads mapping to the positive strand ( sense strand ) are plotted in the top window of every sample , while RNA - seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample . The two isoforms of Hnf4a that are expressed are shown in the bottom , together with the antisense transcript Hnf4aos . The " phyloP30way " track from the UCSC genome browser is plotted at the bottom to show sequence conservation

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(D) Strand specific RNA-seq data, Hnf4g ChIP-seq in EN, and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown. Data from different organoid cultures are color coded. RNA-seq reads mapping to the positive strand (sense strand) are plotted in the top window of every sample, while RNA-seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample. The two isoforms of Hnf4a that are expressed are shown in the bottom, together with the antisense transcript Hnf4aos. The "phyloP30way" track from the UCSC genome browser is plotted at the bottom to show sequence conservation


( E ) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine . Dots represent the amount of goblet cells counted per villus . ( n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice ) . P value is from two - tailed Mann - Whitney U test

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(E) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine. Dots represent the amount of goblet cells counted per villus. (n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice). P value is from two-tailed Mann-Whitney U test


A . HEK293 cells were treated for 8 and 24 h with 100 μM H2O2 . GSNOR was evaluated by Western blot . Densitometry of each lane is normalized to GADPH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n ≥ 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 calculated with regard to H2O2 - untreated cells .

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A. HEK293 cells were treated for 8 and 24 h with 100 μM H2O2. GSNOR was evaluated by Western blot. Densitometry of each lane is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01 calculated with regard to H2O2-untreated cells.


B . HEK293 cells were treated for 8 h with 100 or 200 μM H2O2 . Nrf2 ( left panel ) and GSNOR ( right panel ) were evaluated by Western blot performed in nuclear and cytosol - enriched fractions . GAPDH and Lamin A / C were used as loading and purity controls of cytosol and nuclei , respectively . Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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B. HEK293 cells were treated for 8 h with 100 or 200 μM H2O2. Nrf2 (left panel) and GSNOR (right panel) were evaluated by Western blot performed in nuclear and cytosol-enriched fractions. GAPDH and Lamin A/C were used as loading and purity controls of cytosol and nuclei, respectively. Densitometry of GSNOR immunoreactive bands is normalized to GADPH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


C . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline ( Trig ) . Heme oxygenase 1 ( HMOX - 1 ) , glutamate : cysteine ligase ( GCL ) and GSNOR expressions were evaluated RT - qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate . * p < 0 . 05 ; n . s . , not significant .

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C. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline (Trig). Heme oxygenase 1 (HMOX-1), glutamate:cysteine ligase (GCL) and GSNOR expressions were evaluated RT-qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate. *p<0.05; n.s., not significant.


D . RT - qPCR analyses of GSNOR mRNA after 2 - to - 24 h incubation with 100 μM H2O2 . Results shown are the means ± SEM of n = 3 experiments run in triplicate , analyzed using ANOVA with Dunnett multiple comparisons test . n . s . , not significant .

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D. RT-qPCR analyses of GSNOR mRNA after 2-to-24 h incubation with 100 μM H2O2. Results shown are the means ± SEM of n = 3 experiments run in triplicate, analyzed using ANOVA with Dunnett multiple comparisons test. n.s., not significant.


E . Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline ( Trig ) . Densitometry of GSNOR immunoreactive bands is normalized to GADPH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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E. Western blot analysis of GSNOR in HEK293 cells treated for 8 h with 100 μM H2O2 in the presence or absence of trigonelline (Trig). Densitometry of GSNOR immunoreactive bands is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.


HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide ( CHX , F ) , GSNOR was evaluated by Western blot . Vertical dotted lines represent a virtual division of the nitrocellulose filter , as immunoreactive bands reported in figure - although part of the same experiment / gel - were not contiguous . Vehicle : PBS . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 4 independent experiments . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . , not significant .

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HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of cycloheximide (CHX, F), GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant.


G . HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 ( G ) . GSNOR was evaluated by Western blot . Vertical dotted lines represent a virtual division of the nitrocellulose filter , as immunoreactive bands reported in figure - although part of the same experiment / gel - were not contiguous . Vehicle : PBS . Densitometry of GSNOR immunoreactive bands is normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 4 independent experiments . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . , not significant .

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

G. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of in the presence or absence of the proteasome inhibitor MG132 (G). GSNOR was evaluated by Western blot. Vertical dotted lines represent a virtual division of the nitrocellulose filter, as immunoreactive bands reported in figure - although part of the same experiment/gel - were not contiguous. Vehicle: PBS. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 4 independent experiments. *, p<0.05; ***, p<0.001; n.s., not significant.


H . HEK293 cells were treated for 8 h ( blue ) or 24 h ( red ) with 100 μM H2O2 . Polysome profile showing monosomes and polysomes was obtained from control ( Vehicle ) and H2O2 treated lysates ( for either 8 or 24 h ) by separation on 5 - 50 % sucrose linear density gradient and collection using a gradient fractionation system .

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H. HEK293 cells were treated for 8 h (blue) or 24 h (red) with 100 μM H2O2. Polysome profile showing monosomes and polysomes was obtained from control (Vehicle) and H2O2 treated lysates (for either 8 or 24 h) by separation on 5-50% sucrose linear density gradient and collection using a gradient fractionation system.


I . RT - qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 - treated samples ( for either 8 or 24 h ) . H3A mRNA expression level was used as housekeeping gene control . Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A . * * * , p < 0 . 001 Vehicle : PBS .

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I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p<0.001 Vehicle: PBS.


J . HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin ( tyrosyl - tRNA mimic used to label nascent proteins ) , or cycloheximide ( CHX , used to block protein translation ) . Nascent polypeptides were assessed by Western blot performed with an anti - puromycin ( anti - PMC ) antibody . Vehicle : PBS . β - actin was used as loading control .

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J. HEK293 cells treated for 8 h or 24 h with 100 μM H2O2 in the presence or absence of puromycin (tyrosyl-tRNA mimic used to label nascent proteins), or cycloheximide (CHX, used to block protein translation). Nascent polypeptides were assessed by Western blot performed with an anti-puromycin (anti-PMC) antibody. Vehicle: PBS. β-actin was used as loading control.


A . HEK293 cells treated for 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM and CHK2 were assessed by Western blot . Phospho : basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n ≥ 4 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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A. HEK293 cells treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2 were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 immunoreactive bands are normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 4 independent experiments. *, p<0.05; **, p<0.01.


B . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 ( KU ) . Basal and phospho - active forms of ATM , CHK2 , and GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Phospho : basal level ratios of ATM and CHK2 ( normalized to Vinculin ) , along with densitometry of GSNOR immunoreactive bands ( normalized to GAPDH ) are expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 .

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B. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the ATM inhibitor KU55933 (KU). Basal and phospho-active forms of ATM, CHK2, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05.


C . HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 ( AZD ) . Basal and phospho - active forms of CHK2 , together with GSNOR were assesses by Western blot . Phospho : basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH , selected as loading control , and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . , not significant .

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C. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of the CHK2 inhibitor AZD7762 (AZD). Basal and phospho-active forms of CHK2, together with GSNOR were assesses by Western blot. Phospho:basal level ratios of CHK2 and densitometry of GSNOR immunoreactive bands are normalized to GAPDH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.


D . HEK293 cells were transfected with a pool of siRNA against ATM ( siATM ) or with control siRNA ( siScr ) for 18 h and treated for additional 24 h with 100 μM H2O2 . Basal and phospho - active forms of ATM , together with GSNOR were assessed by Western blot . Vinculin and GAPDH were used as loading controls . Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units . Values shown represent the means ± SD of n = 3 independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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D. HEK293 cells were transfected with a pool of siRNA against ATM (siATM) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM, together with GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01.