Kinetic study of the reaction catalyzed by recombinant hGS . The insets represent the secondary plot of the slopes of Lineweaver - Burk plot obtained at the indicated concentrations of glufosinate ( C ) or MSO ( D ) used for determining the inhibitor constant Ki . GS inhibition ( % ) in presence of increasing concentrations of glufosinate ( ) or MSO ( ) results from the average of at least three independent experiments ( E ) . The control value for uninhibited hGS activity is 0 . 51 ± 0 . 08 μmol / ( min x mg protein ) .

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

Kinetic study of the reaction catalyzed by recombinant hGS. The insets represent the secondary plot of the slopes of Lineweaver-Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ±0.08 μmol/(min x mg protein).


Evaluation of M1 markers in macrophages by real - time PCR . Fold change of TNFA , CD80 , CXCL9 , and CXCL10 mRNA in IL10 , MSO - and glufosinate ( 10 and 20µM ) - stimulated IL10 macrophages ( n = 3 ) . Evaluation of M2 markers in macrophages by real - time PCR . Fold change of MRC1 , MSR1 , mRNA in IL10 , MSO - and glufosinate ( 10 and 20µM ) - stimulated IL10 macrophages ( n = 3 ) .

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

Evaluation of M1 markers in macrophages by real-time PCR. Fold change of TNFA, CD80, CXCL9, and CXCL10 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3). Evaluation of M2 markers in macrophages by real-time PCR. Fold change of MRC1, MSR1, mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).


Evaluation of M2 markers in macrophages by real - time PCR . Fold change of CCL17 and CCL18 mRNA in IL10 , MSO - and glufosinate ( 10 and 20µM ) - stimulated IL10 macrophages ( n = 3 ) .

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

Evaluation of M2 markers in macrophages by real-time PCR. Fold change of CCL17 and CCL18 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).


Evaluation of M1 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of TNFA , CXCL10 , CD86 mRNA in IL10 alone or glufosinate ( 10 and 20µM ) - and acriflavine / glufosinate ( 10 and 20µM ) - IL10 macrophages ( n = 3 ) .

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

Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of TNFA, CXCL10, CD86 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).


Evaluation of M1 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of CD80 and CXCL9 mRNA in IL10 alone or glufosinate ( 10 and 20µM ) - and acriflavine / glufosinate ( 10 and 20µM ) - IL10 macrophages ( n = 3 ) . Evaluation of M2 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of MRC1 , MSR1 , and CCL18 mRNA in IL10 , glufosinate ( 10 and 20µM ) - and acriflavine / glufosinate ( 10 and 20µM ) - IL10 macrophages ( n = 3 ) .

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

Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of CD80 and CXCL9 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3). Evaluation of M2 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of MRC1, MSR1, and CCL18 mRNA in IL10, glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).


( Q ) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10 , MSO / IL10 and glufosinate ( 10 and 20µM ) - IL10 treated macrophages after 24 h of incubation ( n = 6 ) .

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

(Q) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10, MSO/IL10 and glufosinate (10 and 20µM)- IL10 treated macrophages after 24 h of incubation (n=6).


( R ) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO / IL10 , and glufosinate ( 10 and 20µM ) / IL10 after 4 h of incubation with HUVEC cells ( n = 6 ) .

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

(R) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 after 4 h of incubation with HUVEC cells (n=6).


( S ) CD8 + T cell suppression by macrophages treated with IL10 or MSO / IL10 , and glufosinate ( 10 and 20µM ) / IL10 for 24 h ( n = 4 ) . Proliferation was evaluated by reading radioactivity as cpm ( counts per minute ) , after incubation with 1 μCi / well tritiated thymidine . The proliferation of T cells cultured without macrophages was used as control .

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

(S) CD8+ T cell suppression by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.


( T ) CD8 + T cell recruitment in a transwell system by macrophages treated with IL10 or MSO / IL10 , and glufosinate ( 10 and 20µM ) / IL10 for 24 h versus macrophages treated with LPS / IFNγ ; the migration of T cells cultured without macrophages ( Mφ - ) in presence of CXCL10 was used as positive control ( n = 4 ) .

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

(T) CD8+ T cell recruitment in a transwell system by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h versus macrophages treated with LPS/IFNγ; the migration of T cells cultured without macrophages (Mφ-) in presence of CXCL10 was used as positive control (n=4).


( U ) Representative image of Western blotting analysis of HIF1α , REDD1 , 4E - BP1 , S6 and P70S6K ( in their phosphorylated and unphosphorilated form ) to test mTOR activation in normoxic ( NRX ) and hypoxic ( HYP ) IL10 macrophages treated with glufosinate ( 20µM ) , rapamycin ( 20nM ) and a combination of both ( n = 3 ) .

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

(U) Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6 and P70S6K (in their phosphorylated and unphosphorilated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20µM), rapamycin (20nM) and a combination of both (n=3).


( V ) CD8 + T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate ( 20µM ) , rapamycin and a combination of both for 24 h ( n = 4 ) . Proliferation was evaluated by reading radioactivity as cpm ( counts per minute ) , after incubation with 1 μCi / well tritiated thymidine . The proliferation of T cells cultured without macrophages was used as control .

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

(V) CD8+ T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate (20µM), rapamycin and a combination of both for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.


Quantification of glufosinate ( B ) levels in wet tumor tissues of vehicle and glufosinate - treated mice ( n = 8 ) , by liquid chromatography - mass spectrometry ( LC - MS ) .

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

Quantification of glufosinate (B) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).


Quantification of glutamine ( GLN ) ( C ) levels in wet tumor tissues of vehicle and glufosinate - treated mice ( n = 8 ) , by liquid chromatography - mass spectrometry ( LC - MS ) .

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

Quantification of glutamine (GLN) (C) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).


Subcutaneous LLC tumor growth over the time ( D ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

Subcutaneous LLC tumor growth over the time (D) (pool of 2 independent experiments; 10 mice per condition in total).


number of lung metastatic nodules in vehicle and glufosinate ( 10 and 20mg / kg ) treated mice ( E ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

number of lung metastatic nodules in vehicle and glufosinate (10 and 20mg/kg) treated mice (E) (pool of 2 independent experiments; 10 mice per condition in total).


Quantification and representative images of F4 / 80 + CD11c + cells in the lung metastasis ( F ) , F4 / 80 + CD206 + ( G ) cells infiltration in tumors of vehicle and glufosinate - treated mice ( n = 6 ) .

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

Quantification and representative images of F4/80+ CD11c+ cells in the lung metastasis (F), F4/80+ CD206+ (G) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).


Quantification and representative images of F4 / 80 CD11c + ( H ) cells infiltration in tumors of vehicle and glufosinate - treated mice ( n = 6 ) .

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

Quantification and representative images of F4/80 CD11c+ (H) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).


RT - PCR quantification of M2 ( Ccl22 , Arg1 and Ccl17 ) ( I ) markers in vehicle and glufosinate treated mice ( n = 4 ) .

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

RT-PCR quantification of M2 (Ccl22, Arg1 and Ccl17) (I) markers in vehicle and glufosinate treated mice (n=4).


RT - PCR quantification of M1 ( Tnfa , Cxcl9 , Nos2 , Cd86 and Cd80 ) ( J ) markers in vehicle and glufosinate treated mice ( n = 4 ) .

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

RT-PCR quantification of M1 (Tnfa, Cxcl9, Nos2, Cd86 and Cd80) (J) markers in vehicle and glufosinate treated mice (n=4).


Quantification of total length of vessels ( A ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

Quantification of total length of vessels (A) in vehicle and glufosinate (20mg/kg) treated mice (n=6).


Quantification of CD31 + tumor vessel area ( B ) and of αSMA + pericyte - covered vessels over the total number of CD31 + vessels ( with representative images ) ( C ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

Quantification of CD31+ tumor vessel area (B) and of αSMA+ pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( D ) Quantification and representative images of Lectin + tumor vessel area over the total number of CD31 + vessels in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(D) Quantification and representative images of Lectin+ tumor vessel area over the total number of CD31+ vessels in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( E ) Quantification and representative images of PIMO + tumor hypoxic areas in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(E) Quantification and representative images of PIMO+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS quantification of TCRbeta + CD8 + cytotoxic T cells in tumors of vehicle and glufosinate - treated mice ( F ) .

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

FACS quantification of TCRbeta+ CD8+ cytotoxic T cells in tumors of vehicle and glufosinate-treated mice (F).


Evaluation of CD69 ( G ) and CD25 ( H ) proteins , expressed as Mean Fluorescence Intensity ( MFI ) , was determined by flow cytometry on the surface of responders CD8 + T cells ( n = 4 ) .

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

Evaluation of CD69 (G) and CD25 (H) proteins, expressed as Mean Fluorescence Intensity (MFI), was determined by flow cytometry on the surface of responders CD8+ T cells (n=4).


( I ) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate - treated mice ( n = 6 ) .

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

(I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).


( B ) Evaluation of body weight in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 8 ) . ( C ) Representative images of metastasis at the beginning and end of therapy . Evaluation of the metastatic burden following glufosinate treatment by H & E staining ( n = 8 ) .

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

(B) Evaluation of body weight in vehicle and glufosinate (20mg/kg) treated mice (n=8). (C) Representative images of metastasis at the beginning and end of therapy. Evaluation of the metastatic burden following glufosinate treatment by H&E staining (n=8).


( D ) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H & E staining in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 8 ) .

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

(D) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H&E staining in vehicle and glufosinate (20mg/kg) treated mice (n=8).


Quantification ( F ) of total metastatic area in LLC - tumor bearing mice preconditioned with IgG or anti - CSF1R antibody and treated with vehicle or glufosinate ( 20mg / Kg ) .

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

Quantification (F) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).


representative images ( G ) of total metastatic area in LLC - tumor bearing mice preconditioned with IgG or anti - CSF1R antibody and treated with vehicle or glufosinate ( 20mg / Kg ) .

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

representative images (G) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).


Intradermal YUMM 1 . 7 ( CD90 . 1 + ) tumor growth over the time ( B ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

Intradermal YUMM 1.7 (CD90.1+) tumor growth over the time (B) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


number of metastatic lesions per lung ( C ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

number of metastatic lesions per lung (C) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


( D ) Representative images of metastatic lesions checked by DAB - H stain for CD90 . 1 positivity in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per lung were analysed . Scale bar : 2mm . Zoomed area scale bar : 50µm .

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

(D) Representative images of metastatic lesions checked by DAB-H stain for CD90.1 positivity in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per lung were analysed. Scale bar: 2mm. Zoomed area scale bar: 50µm.


( E , F ) FACS quantification of M1 - like MHC class IIhigh ( E ) and MHC class IIlow ( F ) CD11c + CD206 - TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(E, F) FACS quantification of M1-like MHC class IIhigh (E) and MHC class IIlow (F) CD11c+ CD206- TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( G ) Quantification and representative images of CD11c + cells over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bars : 50 µm .

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

(G) Quantification and representative images of CD11c+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.


FACS quantification of M2 - like MHC class IIhigh CD11c - CD206 + TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

FACS quantification of M2-like MHC class IIhigh CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS quantification of M2 - like MHC class IIlow ( I ) CD11c - CD206 + TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

FACS quantification of M2-like MHC class IIlow (I) CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( J ) Quantification and representative images of CD206 + cells over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bars : 50 µm .

(J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.


FACS quantification of M1 - like MHC class IIhigh ( K ) TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

FACS quantification of M1-like MHC class IIhigh (K) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS quantification of M1 - like M2 - like MHC class IIlow ( L ) TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

FACS quantification of M1-like M2-like MHC class IIlow (L) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( M ) Quantification of MHC - II protein , expressed as Mean Fluorescence Intensity ( MFI ) , on F4 / 80 + cells , was obtained by flow cytometry ( n = 6 ) .

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

(M) Quantification of MHC-II protein, expressed as Mean Fluorescence Intensity (MFI), on F4/80+ cells, was obtained by flow cytometry (n=6).


( N ) RT - PCR quantification of M2 ( Arg1 and Cxcr4 ) and M1 ( Cxcl9 and Nos2 ) markers in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).


Quantification of total length of vessels ( A ) Six images per tumor were analysed . Scale bar : 50µm .

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

Quantification of total length of vessels (A) Six images per tumor were analysed. Scale bar: 50µm.


CD31 + tumor vessel area ( B ) and of αSMA + pericyte - covered vessels over the total number of CD31 + vessels ( with representative images ) ( C ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bar : 50µm .

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

CD31+ tumor vessel area (B) and of αSMA+pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.


( D ) Quantification and representative images of CA9 + tumor hypoxic areas in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bar : 50µm .

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

(D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.


( E , F ) FACS quantification of CD8 + cytotoxic T cells ( E ) and CD69 + CD8 + T cells ( F ) over the total number of CD45 + in vehicle or glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(E, F) FACS quantification of CD8+ cytotoxic T cells (E) and CD69+ CD8+ T cells (F) over the total number of CD45+ in vehicle or glufosinate (20mg/kg) treated mice (n=6).


Evaluation of 4T1 tumor growth over the time ( B ) , in vehicle and glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Evaluation of 4T1 tumor growth over the time (B) , in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


mice body weight ( C ) in vehicle and glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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mice body weight (C) in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


number of metastatic nodules ( D ) , in vehicle and glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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number of metastatic nodules (D), in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


Total metastatic area ( E ) in vehicle and glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . Six images per lung were analysed . Scale bar : 2mm .

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Total metastatic area (E) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.


number of lung metastatic lesions per size by H & E staining ( F ) with representative images ( G ) in vehicle and glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . Six images per lung were analysed . Scale bar : 2mm .

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number of lung metastatic lesions per size by H&E staining (F) with representative images (G) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.


( H , I ) Quantification of F4 / 80 + macrophage density ( H ) , of F4 / 80 + CD206 + cells infiltration in the lung metastasis and representative images ( I ) , in vehicle and glufosinate - treated mice ( n = 6 ) . Six images per lung were analysed . Scale bar : 20µm .

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(H, I) Quantification of F4/80+ macrophage density (H), of F4/80+ CD206+ cells infiltration in the lung metastasis and representative images (I), in vehicle and glufosinate-treated mice (n=6). Six images per lung were analysed. Scale bar: 20µm.


GFP + CTCs amount in blood , expressed as GFP expression levels , was determined by qPCR ( n = 8 ) ( K ) .

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GFP+ CTCs amount in blood, expressed as GFP expression levels, was determined by qPCR (n=8) (K).


Blood was drawn from treated and untreated tumor - bearing mice at the time of tumor resection and plated in culture dishes . The formation of tumor cell colonies was traced over time . Graph shows the number of GFP + tumor cell colonies ( n = 8 ) ( L ) .

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Blood was drawn from treated and untreated tumor-bearing mice at the time of tumor resection and plated in culture dishes. The formation of tumor cell colonies was traced over time. Graph shows the number of GFP+ tumor cell colonies (n=8) (L).


After treatment and tumor resection overall survival studies were performed until the natural death of the post - surgical mice ( n = 11 ) ( M ) .

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After treatment and tumor resection overall survival studies were performed until the natural death of the post-surgical mice (n=11) (M).


( O ) GFP + cancer cells amount in lungs , expressed as GFP expression levels , was determined by qPCR in vehicle and glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . The results were normalized to the vehicle group .

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(O) GFP+ cancer cells amount in lungs, expressed as GFP expression levels, was determined by qPCR in vehicle and glufosinate (10mg/kg) treated mice (n=8). The results were normalized to the vehicle group.


( A ) Time course evaluation of murine body weight during treatment in vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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(A) Time course evaluation of murine body weight during treatment in vehicle and glufosinate (10 and 20 mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


( B ) Blood counts of WBC and RBC in vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( n = 8 ) .

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(B) Blood counts of WBC and RBC in vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of liver toxicity following glufosinate treatment . Quantification of liver glufosinate ( C ) in serum of vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( n = 8 ) .

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Evaluation of liver toxicity following glufosinate treatment. Quantification of liver glufosinate (C) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of liver toxicity following glufosinate treatment . hepatotoxicity markers ALT ( Alanine Aminotransferase ) and AST ( Aspartate aminotransferase ) ( D ) in serum of vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( n = 8 ) .

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Evaluation of liver toxicity following glufosinate treatment. hepatotoxicity markers ALT (Alanine Aminotransferase) and AST (Aspartate aminotransferase) (D) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of neuronal toxicity following glufosinate treatment . Quantification of brain glufosinate ( E )

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Evaluation of neuronal toxicity following glufosinate treatment. Quantification of brain glufosinate (E)


Evaluation of neuronal toxicity following glufosinate treatment . grip strength for front paws ( triangular bar ) ( F ) , as an indicator of the development of tactile allodynia in vehicle and glufosinate - treated mice ( n = 4 ) , before the treatment ( baseline ) , after 1 day ( acute Toxicity ) and after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Evaluation of neuronal toxicity following glufosinate treatment. grip strength for front paws (triangular bar) (F), as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).


Evaluation of neuronal toxicity following glufosinate treatment . grip strength for all paws ( rectangle grid ) ( G ) , of the latency to fall off an accelerating rotarod ( H ) , and of the mean paw withdrawal force that caused animals ' response in the von Frey test ( left hindpaw , I ; right hindpaw , J ) as an indicator of the development of tactile allodynia in vehicle and glufosinate - treated mice ( n = 4 ) , before the treatment ( baseline ) , after 1 day ( acute Toxicity ) and after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Evaluation of neuronal toxicity following glufosinate treatment. grip strength for all paws (rectangle grid) (G), of the latency to fall off an accelerating rotarod (H), and of the mean paw withdrawal force that caused animals' response in the von Frey test (left hindpaw, I; right hindpaw, J) as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).


The C - terminal domains of IFT52 and IFT46 co - purify with the IFT81460 - C / 74460 - C / 27 / 251 - 136 protein complex ( C ) The SEC fractions indicated on the right with 1 - 3 were analysed by SDS - PAGE and stained with Coomassie to evaluate the protein composition .

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The C-terminal domains of IFT52 and IFT46 co-purify with the IFT81460-C/74460-C/27/251-136 protein complex (C) The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.


The C - terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623 - C of IFT81 and 615 - C of IFT74 ( D ) . The SEC fractions indicated on the right with 1 - 3 were analysed by SDS - PAGE and stained with Coomassie to evaluate the protein composition .

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The C-terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623-C of IFT81 and 615-C of IFT74 (D). The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.


( G ) Coomassie stained SDS PAGE gel of a GST - pulldown using the CrIFT88His - GST / 70 / 521 - 430 / 46 complex immobilized on GST beads as bait and the CrIFT811 - 387 - GFP / 74135 - 420 complex used as prey . The band highlighted by an * corresponds to pulled down CrIFT74135 - 420 . CrIFT811 - 387 - GFP runs on top of IFT70 and is not visible in the pull - down .

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(G) Coomassie stained SDS PAGE gel of a GST-pulldown using the CrIFT88His-GST/70/521-430/46 complex immobilized on GST beads as bait and the CrIFT811-387-GFP/74135-420 complex used as prey. The band highlighted by an * corresponds to pulled down CrIFT74135-420. CrIFT811-387-GFP runs on top of IFT70 and is not visible in the pull-down.


( B ) Pull - down experiments with purified IFT80Venus immobilized on GFP - binder beads and either IFT172 or IFT172968 - C . IFT80 pulls down full length IFT172 but not the truncated version lacking the N - terminal 967 residues .

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(B) Pull-down experiments with purified IFT80Venus immobilized on GFP-binder beads and either IFT172 or IFT172968-C. IFT80 pulls down full length IFT172 but not the truncated version lacking the N-terminal 967 residues.


( F ) GST tagged CrIFT1721 - 968 pulls down the CH - domain of CrIFT571 - 234 but not the mutated CH - domain of CrIFT57 where E77 and K84 residues were replaced by alanines .

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(F) GST tagged CrIFT1721-968 pulls down the CH-domain of CrIFT571-234 but not the mutated CH-domain of CrIFT57 where E77 and K84 residues were replaced by alanines.


( A ) Serum levels of miR - 483 - 3p / - 5p in IPAH patients ( n = 139 ) and HC ( n = 95 ) measured by qPCR . The data are fold change normalized to the averaged level of HC . Data information : Values are expressed as median ± interquartile range . Statistical test : Mann Whitney U test ( A )

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(A) Serum levels of miR-483-3p/-5p in IPAH patients (n = 139) and HC (n = 95) measured by qPCR. The data are fold change normalized to the averaged level of HC. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test (A)


( B ) ROC curve with sensitivity and specificity of serum levels of miR - 483 - 3p / - 5p for differentiating IPAH patients from HCs at diagnosis ( IPAH , n = 139 ; HC , n = 95 ) . Data information : Values are expressed as median ± interquartile range .

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(B) ROC curve with sensitivity and specificity of serum levels of miR-483-3p/-5p for differentiating IPAH patients from HCs at diagnosis (IPAH, n = 139; HC, n = 95). Data information: Values are expressed as median ± interquartile range.


( C ) Levels of miR - 483 - 3p / - 5p associated with PAH risk in 3 groups . IPAH patients were divided into a low risk group ( Low ) and an intermediate plus high risk group ( Inter & high ) according to the World Symposium on Pulmonary Hypertension 2018 [ 14 ] . Data information : Values are expressed as median ± interquartile range . Statistical test : Kruskal - Wallis U test ( C ) .

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(C) Levels of miR-483-3p/-5p associated with PAH risk in 3 groups. IPAH patients were divided into a low risk group (Low) and an intermediate plus high risk group (Inter&high) according to the World Symposium on Pulmonary Hypertension 2018 [14]. Data information: Values are expressed as median ± interquartile range. Statistical test: Kruskal-Wallis U test (C).


( D ) Levels of miR - 483 - 3p were inversely correlated with serum levels of ET - 1 ( IPAH , n = 118 ; HC , n = 95 ) and IL - 6 ( IPAH , n = 112 ; HC , n = 93 ) . Data information : Values are expressed as median ± interquartile range .

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(D) Levels of miR-483-3p were inversely correlated with serum levels of ET-1 (IPAH, n = 118; HC, n = 95) and IL-6 (IPAH, n = 112; HC, n = 93). Data information: Values are expressed as median ± interquartile range.


( A ) CD144 - enriched EVs were isolated from serum of IPAH patients ( n = 37 ) and HCs ( n = 34 ) . The levels of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as median ± interquartile range . Statistical test : Mann Whitney U test .

(A) CD144-enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.


PAECs were transfected with miR - 483 - 3p / - 5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA - seq . Data are results from two biological repeats . ( C ) Heat map comparison of log2 fold changes of the indicated genes . Data information : Values are expressed as median ± interquartile range . Statistical test : Mann Whitney U test .

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PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (C) Heat map comparison of log2 fold changes of the indicated genes. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression levels of miR - 483 - 3p / - 5p measured by qPCR Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of miR-483-3p/-5p measured by qPCR Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 mRNA were measured by qPCR Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were measured by qPCR Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 protein were measured by Western blot Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 protein were measured by Western blot Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


( E ) Bovine aortic ECs were transfected with a luciferase reporter fused with the 3 ' UTR of TGF - β ( Luc - TGF - β WT ) , TGFBR2 ( Luc - TGFBR2 WT ) , IL - 1β ( Luc - IL - 1β WT ) or ET - 1 ( Luc - ET - 1 WT ) or a binding site mutation ( Luc - TGF - β Mut , Luc - TGFBR2 Mut , Luc - IL - 1β Mut , ET - 1 - Mut ) , then infected with Lenti - pre - miR - 483 for additional 36 hr . Luciferase activity was measured . Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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(E) Bovine aortic ECs were transfected with a luciferase reporter fused with the 3'UTR of TGF-β (Luc-TGF-β WT), TGFBR2 (Luc-TGFBR2 WT), IL-1β (Luc-IL-1β WT) or ET-1 (Luc-ET-1 WT) or a binding site mutation (Luc-TGF-β Mut, Luc-TGFBR2 Mut, Luc-IL-1β Mut, ET-1-Mut), then infected with Lenti-pre-miR-483 for additional 36 hr. Luciferase activity was measured. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 36 hr . The Ago1 - or Ago2 - associated miRNAs and mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . Levels of miR - 483 - 3p / - 5p mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein . Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of miR-483-3p/-5p mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 36 hr . The Ago1 - or Ago2 - associated miRNAs and mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . Levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein . Data information : Values are expressed as mean ± SEM from 3 independent experiments . Statistical test : t - test ( * P < 0 . 05 between the indicated

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PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


( A ) Lung ECs were isolated from EC - miR - 483 transgenic rats ( Tg ) and their wild - type littermates ( WT ) , n = 3 in each group . MiR - 483 - 3p / - 5p levels were measured by qPCR . Data information : Values are expressed as mean ± SEM .

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(A) Lung ECs were isolated from EC- miR-483 transgenic rats (Tg) and their wild-type littermates (WT), n = 3 in each group. MiR-483-3p/-5p levels were measured by qPCR. Data information: Values are expressed as mean ± SEM.


( B ) Serum from WT and Tg rats ( n = 6 ) were collected . MiR - 483 - 3p and - 5p associated with CD144 were enriched by immunoprecipitation with anti - CD144 . Levels of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as mean ± SEM .

(B) Serum from WT and Tg rats (n = 6) were collected. MiR-483-3p and -5p associated with CD144 were enriched by immunoprecipitation with anti-CD144. Levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as mean ± SEM.


Lung ECs isolated from miR - 483 - Tg rat and WT littermates were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . Proliferation of ECs were measured by flow cytometry Data information : Values are expressed as mean ± SEM . Data are representative of three independent experiments Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation of ECs were measured by flow cytometry Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Lung ECs isolated from miR - 483 - Tg rat and WT littermates were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . migration of ECs were measured by wound healing assay Data information : Values are expressed as mean ± SEM . Data are representative of three independent experiments Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Tg and WT rats were injected with saline or MCT . Expression levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 mRNA and protein in lung tissues were measured by qPCR and Western blot , respectively ( E ) . Data information : Values are expressed as mean ± SEM .

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Tg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Data information: Values are expressed as mean ± SEM.


Tg and WT rats were injected with saline or MCT . Levels of miR - 483 - 3p / - 5p in serum and lung tissues were measured by qPCR ( F ) . Data information : Values are expressed as mean ± SEM .

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Tg and WT rats were injected with saline or MCT. Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Data information: Values are expressed as mean ± SEM.


Tg and WT rats were injected with saline or MCT . Ago1 - or Ago2 - associated miRNAs ( G ) were enriched from the isolated lung tissues by immunoprecipitation with anti - Ago1 or anti - Ago2 and quantified by qPCR ( n = 3 × 3 , samples were pooled from three animals for each assay , and 3 independent experiments [ a total of 9 animals ] were performed ) . Data information : Values are expressed as mean ± SEM .

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

Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated miRNAs (G) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.


Tg and WT rats were injected with saline or MCT . Ago1 - or Ago2 - associated mRNAs ( H ) were enriched from the isolated lung tissues by immunoprecipitation with anti - Ago1 or anti - Ago2 and quantified by qPCR ( n = 3 × 3 , samples were pooled from three animals for each assay , and 3 independent experiments [ a total of 9 animals ] were performed ) . Data information : Values are expressed as mean ± SEM .

Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.


EC - miR - 483 - Tg and WT rats were injected with saline or MCT . Analysis of mPAP for the indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) .

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

EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


EC - miR - 483 - Tg and WT rats were injected with saline or MCT . Analysis of RV hypertrophy [ RV / ( LV + S ) ] for the indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) .

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

EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


Vascularization evaluated by H & E staining of pulmonary arteries ( C ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . Scale bar : 20 µm ( C )

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

Vascularization evaluated by H&E staining of pulmonary arteries (C). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 µm (C)


Vascular integrity evaluated by intravenous injection of Evans blue dye ( D ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) . Scale bar : 0 . 5 cm ( D ) .

Vascular integrity evaluated by intravenous injection of Evans blue dye (D). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 0.5 cm (D).


EC - miR - 483 - Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks . Analysis of mPAP ( E ) for the indicated groups . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) .

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

EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


EC - miR - 483 - Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks . Analysis of RV hypertrophy [ RV / ( LV + S ) ] ( F ) for the indicated groups . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups ) .

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

EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of RV hypertrophy [RV/(LV+S)] (F) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


qPCR analysis of miR - 483 - 3p / - 5p in serum from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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

qPCR analysis of miR-483-3p/-5p in serum from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


qPCR analysis of miR - 483 - 3p / - 5p in lung tissue from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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

qPCR analysis of miR-483-3p/-5p in lung tissue from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


( D ) Protein and mRNA levels of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , and ET - 1 measured by Western blot and qPCR , respectively ( n = 3 × 3 , samples were pooled from three animals for each assay , and 3 independent experiments [ a total of 9 animals ] were performed ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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

(D) Protein and mRNA levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 measured by Western blot and qPCR, respectively (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Analysis of mPAP ( E ) for the indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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

Analysis of mPAP (E) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Analysis of RV hypertrophy [ RV / ( LV + S ) ] for the indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Analysis of systemic blood pressure ( SBP ) ( G ) for the indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

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

Analysis of systemic blood pressure (SBP) (G) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


( H ) RV hypertrophy shown by the H & E staining and RV / ( LV + S ) quantification . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups Scale bar : 0 . 5 cm ( H )

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

(H) RV hypertrophy shown by the H&E staining and RV/(LV+S) quantification. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 0.5 cm (H)


( I ) Vascularization revealed by H & E staining ( n = 7 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated groups Scale bar : 20 µm ( I ) .

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

(I) Vascularization revealed by H&E staining (n = 7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 20 µm (I).


( J ) Survival rate of 3 groups ( n = 15 rats in each group ) . Data information : Values are expressed as mean ± SEM . Statistical test : t - test ( * P < 0 . 05 vs . respective controls or between the indicated

(J) Survival rate of 3 groups (n = 15 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


A - C 10x106 ZsGreen - and luciferase - expressing U - 2932 , RC - K8 and RIVA cells were intravenously injected into six - week - old male ( M ) and female ( F ) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks . The color scales on the right indicate the radiance , i . e . the sum of the photons per second from each pixel inside the ROI / number of pixels ( photons / sec / cm2 / sr ) .

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

A-C 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. The color scales on the right indicate the radiance, i.e. the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr).


10x106 ZsGreen - and luciferase - expressing U - 2932 , RC - K8 and RIVA cells were intravenously injected into six - week - old male ( M ) and female ( F ) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks . D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line .

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

10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line.


E Gating strategy for the identification of U - 2932 cells residing in spleen and bone marrow , among all leukocytes in these organs . Cells were stained for human and mouse CD45 . Very few hCD45 + U - 2932 cells were detectable in the spleen , whereas their frequencies were much higher in the bone marrow ( BM ) . In BM , > 90 % of U - 2932 cells were positive for ZsGreen . The green curve shows U - 2932 cells recovered from BM , overlayed with unlabelled U - 2932 cells grown in vitro ( grey curve ) .

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

E Gating strategy for the identification of U-2932 cells residing in spleen and bone marrow, among all leukocytes in these organs. Cells were stained for human and mouse CD45. Very few hCD45+ U-2932 cells were detectable in the spleen, whereas their frequencies were much higher in the bone marrow (BM). In BM, >90% of U-2932 cells were positive for ZsGreen. The green curve shows U-2932 cells recovered from BM, overlayed with unlabelled U-2932 cells grown in vitro (grey curve).


F , G Frequencies of human lymphoma cells , as judged by hCD45 expression , among all ( human and mouse , h + m ) CD45 + leukocytes in the spleen ( S ) , femoral bone marrow ( BM ) and jaw bone marrow ( JB ) at the study endpoint of the mice shown in D ( four weeks post injection for U - 2932 , five weeks p . i . for RIVA and six weeks p . i . for RC - K8 ) .

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

F, G Frequencies of human lymphoma cells, as judged by hCD45 expression, among all (human and mouse, h+m) CD45+ leukocytes in the spleen (S), femoral bone marrow (BM) and jaw bone marrow (JB) at the study endpoint of the mice shown in D (four weeks post injection for U-2932, five weeks p.i. for RIVA and six weeks p.i. for RC-K8).


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . The reconstitution efficiency ( frequency of human among all human and mouse CD45 + leukocytes ) is shown in

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution efficiency (frequency of human among all human and mouse CD45+ leukocytes) is shown in A


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . the average human leukocyte composition in the bone marrow of the mice is shown in B .

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. the average human leukocyte composition in the bone marrow of the mice is shown in B.


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . Tumor volumes over time are presented in

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. Tumor volumes over time are presented in C


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . tumor weights and volumes at the study endpoint in D , where each data point represents one tumor .

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. tumor weights and volumes at the study endpoint in D, where each data point represents one tumor.


MISTRG mice were reconstituted or not at birth with ~ 200 . 000 human CD34 + cord blood HSPCs , and subcutaneously transplanted with 10x106 U - 2932 cells at six weeks of age . The reconstitution level directly correlates with the tumor weight at the endpoint ( E ) .

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

MISTRG mice were reconstituted or not at birth with ~200.000 human CD34+ cord blood HSPCs, and subcutaneously transplanted with 10x106 U-2932 cells at six weeks of age. The reconstitution level directly correlates with the tumor weight at the endpoint (E).


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age . The lymphoma burden at the study endpoint was quantified in the spleen ( F ) and bone marrow ( G ) by staining for hCD45 ( as well as hCD19 and hCD20 for U - 2932 and RC - K8 , respectively )

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

MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the spleen (F) and bone marrow (G) by staining for hCD45 (as well as hCD19 and hCD20 for U-2932 and RC-K8, respectively)


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with RC - K8 cells at six weeks of age . The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC - K8 cells , also over time by IVIS ( H ) .

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

MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with RC-K8 cells at six weeks of age. The lymphoma burden at the study endpoint was quantified by ZsGreen expression and in the case of RC-K8 cells, also over time by IVIS (H).


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age . The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation ( I ) ,

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

MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. The frequency of tissue involvement was assessed by ex vivo IVIS and macroscopic evaluation (I),


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPCs , and intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age . the occurrence of clinical symptoms ( weight loss , hind leg paralysis ) was recorded for each mouse ( J ) .

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

MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPCs, and intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age. the occurrence of clinical symptoms (weight loss, hind leg paralysis) was recorded for each mouse (J).


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint ( A ) .

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

MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A).


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint ( A ) . The lymphoma burden at the study endpoint ( six weeks p . i . for RC - K8 and four weeks p . i . for U - 2932 ) was quantified in the spleen ( B ) and bone marrow ( C ) by flow cytometric staining for hCD45 and by ZsGreen expression .

MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint (A). The lymphoma burden at the study endpoint (six weeks p.i. for RC-K8 and four weeks p.i. for U-2932) was quantified in the spleen (B) and bone marrow (C) by flow cytometric staining for hCD45 and by ZsGreen expression.


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D ( solid bars , MISTRG ; hatched bars , MISTRG6 ) ,

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

MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint The involvement of the indicated tissues as assessed by ex vivo IVIS is shown in D (solid bars, MISTRG; hatched bars, MISTRG6),


MISTRG and MISTRG6 mice were intravenously injected with 10x106 U - 2932 or RC - K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint mice that have developed clinical symptoms ( paralysis of hind legs ) are plotted in E .

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

MISTRG and MISTRG6 mice were intravenously injected with 10x106 U-2932 or RC-K8 cells at six weeks of age and monitored weekly by IVIS until the study endpoint mice that have developed clinical symptoms (paralysis of hind legs) are plotted in E.


F Surface expression of the IL - 6 receptor α - chain ( CD126 ) as assessed by flow cytometry of U - 2932 and RC - K8 cells . Data is representative of two stainings .

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

F Surface expression of the IL-6 receptor α-chain (CD126) as assessed by flow cytometry of U-2932 and RC-K8 cells. Data is representative of two stainings.


1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . The spleen weights ( B , representative pictures in A ) are

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

1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. The spleen weights (B, representative pictures in A) are shown


1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . with representative H & E - stained spleen ( MISTRG6 ) and lymph node ( patient ) sections in

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

1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. with representative H&E-stained spleen (MISTRG6) and lymph node (patient) sections in C


1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . the lymphoma burden at the study endpoint eight weeks p . i . , as quantified in the spleen and bone marrow in D Representative flow cytometry plots are shown in D for bone marrow and spleen ( MISTRG , MISTRG6 ) .

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

1x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow in D Representative flow cytometry plots are shown in D for bone marrow and spleen (MISTRG, MISTRG6).


x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients . the lymphoma burden at the study endpoint eight weeks p . i . , as quantified in the spleen and bone

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

x106 primary DLBCL cells obtained by passaging through a MISTRG6 mouse were intravenously injected into MISTRG or MISTRG6 recipients. the lymphoma burden at the study endpoint eight weeks p.i., as quantified in the spleen and bone marrow


MISTRG mice were reconstituted or not at birth with human CD34 + cord blood HSPC , and intravenously injected with 1x106 primary DLBCL cells at six weeks of age . The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un - reconstituted animals by flow cytometric staining for the indicated surface markers .

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MISTRG mice were reconstituted or not at birth with human CD34+ cord blood HSPC, and intravenously injected with 1x106 primary DLBCL cells at six weeks of age. The lymphoma burden at the study endpoint was quantified in the bone marrow of reconstituted and un-reconstituted animals by flow cytometric staining for the indicated surface markers.


Primary lymphoma cells harvested at the study endpoint express the IL - 6R α chain ( CD126 , as assessed by flow cytometry , G ) and are positive for phosphorylated STAT3 ( G ) .

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Primary lymphoma cells harvested at the study endpoint express the IL-6R α chain (CD126, as assessed by flow cytometry, G) and are positive for phosphorylated STAT3 (G).


Primary lymphoma cells harvested and the signaling chain gp130 ( as assessed by immunohistochemistry , H )

Primary lymphoma cells harvested and the signaling chain gp130 (as assessed by immunohistochemistry, H)


The expression of the IL - 6Rα ( CD126 ) and signaling chains ( gp130 ) was assessed by flow cytometry ( A ) on a panel of DLBCL cell lines . ABC - and GCB - DLBCL cell lines are color - coded in red and green ; RC - K8 are depicted in grey , as their subtype is a matter of

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The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by flow cytometry (A) on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate


The expression of the IL - 6Rα ( CD126 ) and signaling chains ( gp130 ) was assessed by SYBR or Taqman qRT - PCR ( B , C ) respectively , on a panel of DLBCL cell lines . ABC - and GCB - DLBCL cell lines are color - coded in red and green ; RC - K8 are depicted in grey , as their subtype is a matter of

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The expression of the IL-6Rα (CD126) and signaling chains (gp130) was assessed by SYBR or Taqman qRT-PCR (B, C) respectively, on a panel of DLBCL cell lines. ABC- and GCB-DLBCL cell lines are color-coded in red and green; RC-K8 are depicted in grey, as their subtype is a matter of debate


D and E . The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry , and correlated with the ABC / non - GCB vs . GCB subtype , as assessed via Hans classification . Representative micrographs are shown in D , alongside the quantification of gp130 + cases among all samples , and among the two subtypes , in E . Scale bar , 100 μm .

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D and E. The expression of gp130 in 114 tumor biopsies of DLBCL patients was assessed by immunohistochemistry, and correlated with the ABC/non-GCB vs. GCB subtype, as assessed via Hans classification. Representative micrographs are shown in D, alongside the quantification of gp130+ cases among all samples, and among the two subtypes, in E. Scale bar, 100 μm.


A IL - 6 secretion of the indicated cell lines ( ABC - DLBCL in red , GCB - DLBCL in green ) and primary patient material , as quantified by ELISA and normalized to cell number ( shown per 1000 cells ) . n . d . , not detectable .

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A IL-6 secretion of the indicated cell lines (ABC-DLBCL in red, GCB-DLBCL in green) and primary patient material, as quantified by ELISA and normalized to cell number (shown per 1000 cells). n.d., not detectable.


STAT3 phosphorylation as assessed by Western blotting ( B , D ) of the indicated ABC - DLBCL and GCB - DLBCL cell lines , with and without 1h of exposure to 50ng / ml hIL - 6 ( B , D ) . STAT3 expression levels are shown as Western blot control .

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STAT3 phosphorylation as assessed by Western blotting (B, D) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, with and without 1h of exposure to 50ng/ml hIL-6 (B,D). STAT3 expression levels are shown as Western blot control.


STAT3 phosphorylation as assessed by flow cytometry ( C ) of the indicated ABC - DLBCL and GCB - DLBCL cell lines , Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL - 6 .

STAT3 phosphorylation as assessed by flow cytometry (C) of the indicated ABC-DLBCL and GCB-DLBCL cell lines, Histograms in C represent STAT3 phosphorylation in the absence of exogenously added IL-6.


E STAT3 phosphorylation as assessed by Western blotting of CD126 - proficient ( WT ) or - deficient ( ko ) RC - K8 cells , with and without 1h of exposure to 50ng / ml hIL - 6 .

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E STAT3 phosphorylation as assessed by Western blotting of CD126-proficient (WT) or -deficient (ko) RC-K8 cells, with and without 1h of exposure to 50ng/ml hIL-6.


F Kinetics of STAT3 phosphorylation as assessed by Western blotting , of the IL - 6 - expressing SOCS1wt and SOCS1mut cell lines OCI - Ly3 and SU - DHL - 2 , under 1 , 6 and 24h exposure to an IL - 6 - neutralizing antibody ( α - IL - 6 , anti - IL - 6 neutralizing antibody ) .

F Kinetics of STAT3 phosphorylation as assessed by Western blotting, of the IL-6-expressing SOCS1wt and SOCS1mut cell lines OCI-Ly3 and SU-DHL-2, under 1, 6 and 24h exposure to an IL-6-neutralizing antibody (α-IL-6, anti-IL-6 neutralizing antibody).


STAT3 phosphorylation and gp130 expression of two representative cases ( G ) assessed by immunohistochemistry . The specific pSTAT3 and gp130 signal is seen as brown ( DAB staining ) , and counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130 expression ; the p - value was calculated by chi - square test . Scale bar , 100 μm .

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STAT3 phosphorylation and gp130 expression of two representative cases (G) assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression; the p-value was calculated by chi-square test. Scale bar, 100 μm.


STAT3 phosphorylation and gp130 expression of two representative cases ( G ) and summarized for the entire cohort of 114 patients ( H ) , as assessed by immunohistochemistry . The specific pSTAT3 and gp130 signal is seen as brown ( DAB staining ) , and counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130

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STAT3 phosphorylation and gp130 expression of two representative cases (G) and summarized for the entire cohort of 114 patients (H), as assessed by immunohistochemistry. The specific pSTAT3 and gp130 signal is seen as brown (DAB staining), and counterstaining was performed using hematoxylin (blue nuclei). STAT3 phosphorylation is more common in biopsies with gp130 expression


Disease - specific mortality ( A ) of cohorts of 9 - 12 mice each ( n indicated in figure ) of the indicated genotypes .

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Disease-specific mortality (A) of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.


lymphoma burden as determined by lymph node weight ( B ) , of cohorts of 9 - 12 mice each ( n indicated in figure ) of the indicated genotypes .

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lymphoma burden as determined by lymph node weight (B), of cohorts of 9-12 mice each (n indicated in figure) of the indicated genotypes.


Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B - cells ( C ) , centroblasts ( D ) and centrocytes ( E ) .

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Mice of the indicated genotypes were immunized once with sheep red blood cells and assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B-cells (C), centroblasts (D) and centrocytes (E).


Spleen sections were further stained for Ki67 to reveal GCs ( F ) , which were quantified both in number ( G ) and area ( H ) . Arrows in F point to GCs ; scale bar , 1000 μm .

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Spleen sections were further stained for Ki67 to reveal GCs (F), which were quantified both in number (G) and area (H). Arrows in F point to GCs; scale bar, 1000 μm.


Frequency of STAT3 phosphorylation ( A ) , of DLBCL patients stratified according to subtype and disease stage Phospho - STAT3 positivity was defined as expression in ≥ 17 % of tumor cells and more common found in ABC - DLBCL tumors , and in more advanced ( stage 3 and 4 , relative to stage 1 and 2 ) disease ( A ) .

Frequency of STAT3 phosphorylation (A), of DLBCL patients stratified according to subtype and disease stage Phospho-STAT3 positivity was defined as expression in ≥17% of tumor cells and more common found in ABC-DLBCL tumors, and in more advanced (stage 3 and 4, relative to stage 1 and 2) disease (A).


overall as well as disease - specific survival probability ( B ) of DLBCL patients pSTAT3 positivity was a strong negative prognostic factor in the entire cohort ( B )

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overall as well as disease-specific survival probability (B) of DLBCL patients pSTAT3 positivity was a strong negative prognostic factor in the entire cohort (B)


overall mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - and GCB - DLBCL

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overall mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes


disease - specific mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - and GCB - DLBCL

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disease-specific mortality of DLBCL patients stratified according to subtype and disease stage pSTAT3 positivity was associated with higher overall- as well as disease-specific mortality in both ABC- and GCB-DLBCL subtypes


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection , on ( E and F ) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells ( expanded by one serial passage through MISTRG6 mice ) ,

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, on (E and F) the spleen weights of MISTRG6 mice transplanted with 1x106 primary DLBCL cells (expanded by one serial passage through MISTRG6 mice),


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection , Representative microscopy images in F show STAT3 phosphorylation in spleen - colonizing primary cells exposed to either tocilizumab or isotype control antibody ( in brown , counterstained in blue with hematoxylin ) , and an H & E - stained section from the same mouse . TCZ , tocilizumab . Scale bars , 100 μm .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection, Representative microscopy images in F show STAT3 phosphorylation in spleen-colonizing primary cells exposed to either tocilizumab or isotype control antibody (in brown, counterstained in blue with hematoxylin), and an H&E-stained section from the same mouse. TCZ, tocilizumab. Scale bars, 100 μm.


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI - Ly3 - transplanted MISTRG mice ( G ) assessed at around four weeks post tumor cell injection .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the bone marrow lymphoma burden of OCI-Ly3-transplanted MISTRG mice (G) assessed at around four weeks post tumor cell injection.


Effect of IL - 6R neutralization by twice - weekly injection of 250 μg / dose of tocilizumab or an isotype control antibody , starting two weeks post tumor cell injection on the splenic lymphoma burden of RC - K8 - transplanted MISTRG6 mice ( H ) , all assessed at around four weeks post tumor cell injection .

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Effect of IL-6R neutralization by twice-weekly injection of 250 μg/dose of tocilizumab or an isotype control antibody, starting two weeks post tumor cell injection on the splenic lymphoma burden of RC-K8-transplanted MISTRG6 mice (H), all assessed at around four weeks post tumor cell injection.


Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 and 10 U / mL IFN - α2 , respectively ) before infection with Sendai virus ( SeV ) and influenza A virus ( IAV , strain PR8 ) for 6 hours as indicated . Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 . ISG response measured by ISG15 and RSAD2 . Each colour corresponds to a donor ; 9 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for the statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. ISG response measured by ISG15 and RSAD2. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 and 10 U / mL IFN - α2 , respectively ) before infection with Sendai virus ( SeV ) and influenza A virus ( IAV , strain PR8 ) for 6 hours as indicated . The column labelled c shows control samples collected directly after purification . Quantification of the presence of SeV or IAV . Each colour corresponds to a donor ; 9 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for the statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. Quantification of the presence of SeV or IAV. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL fluid and pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( A ) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 ( Donors marked in orange and blue were not quantifiable in all test conditions ) . ( B ) ISG response measured by ISG15 , RSAD2 , and IFIT1 .

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Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1.


Alveolar macrophages from different donors were purified from BAL fluid and pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( C ) Quantification of the presence of SARS - CoV - 2 .

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Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2.