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

O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O

(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 during treatment in vehicle glufosinate ( 10 20 mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O

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

O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O

(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 . of liver glufosinate ( C ) in serum of vehicle glufosinate ( 10 20 mg / kg ) treated mice ( n = 8 ) .

O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-TISSUE B-SMALL_MOLECULE O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O

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 ) AST ( Aspartate aminotransferase ) ( D ) in serum of vehicle glufosinate ( 10 20 mg / kg ) treated mice ( n = 8 ) .

O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O

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 . of brain glufosinate ( E )

O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-TISSUE B-SMALL_MOLECULE O O O

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 development of tactile allodynia in vehicle glufosinate - treated mice ( n = 4 ) , before treatment ( baseline ) , after 1 day ( acute Toxicity ) after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O

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

O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE O O 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-DISEASE I-DISEASE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O

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).


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

O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O

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.


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

O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O

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 CrIFT88His - GST / 70 / 521 - 430 / 46 immobilized on GST beads as bait CrIFT811 - 387 - GFP / 74135 - 420 used as prey . band highlighted by an * corresponds to pulled down CrIFT74135 - 420 . CrIFT811 - 387 - GFP runs on top of IFT70 is not visible in pull - down .

O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY B-ORGANISM O O O B-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(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 either IFT172 or IFT172968 - C . IFT80 pulls down full IFT172 but not truncated version lacking N - terminal 967 residues .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O

(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 CH - domain of CrIFT571 - 234 but not mutated CH - domain of CrIFT57 where E77 K84 residues were replaced by alanines .

O O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O

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

O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-DISEASE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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

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

O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-DISEASE O O O O O B-DISEASE O O 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-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE 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) 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 ( n = 37 ) HCs ( n = 34 ) . of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as median ± interquartile range . Statistical : Mann Whitney U .

O O O B-GENEPROD O O B-SUBCELLULAR O O O B-TISSUE O B-DISEASE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(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 for 36 hr before isolation then analyzed by - seq . Data are results from two biological repeats . ( C ) Heat map comparison of log2 fold changes of indicated genes . Data information : Values are expressed as median ± interquartile range . Statistical : Mann Whitney U .

B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of miR - 483 - 3p / - 5p measured by qPCR Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 mRNA were measured by qPCR Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 were measured by Western blot Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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) 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 . Ago1 - or Ago2 - associated miRNAs mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . of miR - 483 - 3p / - 5p mRNA were detected by qPCR normalized to those of Ago1 or Ago2 . Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 . Ago1 - or Ago2 - associated miRNAs mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 mRNA were detected by qPCR normalized to those of Ago1 or Ago2 . Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) their wild - type ( WT ) , n = 3 in each group . MiR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as mean ± .

O O O B-TISSUE B-CELL_TYPE O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O

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

O O O B-TISSUE O O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O

(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 WT 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 ± . Data are representative of three independent experiments Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

B-TISSUE B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 WT were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . migration of ECs were measured by wound healing Data information : Values are expressed as mean ± . Data are representative of three independent experiments Statistical : t - ( * P < 0 . 05 vs . respective controls or between 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 WT rats were injected with saline or MCT . Expression of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 mRNA in lung tissues were measured by qPCR Western blot , respectively ( E ) . Data information : Values are expressed as mean ± .

O O B-ORGANISM O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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. 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 WT rats were injected with saline or MCT . of miR - 483 - 3p / - 5p in serum lung tissues were measured by qPCR ( F ) . Data information : Values are expressed as mean ± .

O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

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

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

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

B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O 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 WT rats were injected with saline or MCT . of RV hypertrophy [ RV / ( LV + S ) ] for indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O B-TISSUE B-EXP_ASSAY O B-TISSUE O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O 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 of pulmonary arteries ( C ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) . Scale bar : 20 µm ( C )

B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O 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 ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) . Scale bar : 0 . 5 cm ( D ) .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 WT were injected with SU5416 then exposed to hypoxia for 3 had reoxygenation for 2 or injected with DMSO exposed to normoxia for 5 . of mPAP ( E ) for indicated groups . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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 WT were injected with SU5416 then exposed to hypoxia for 3 had reoxygenation for 2 or injected with DMSO exposed to normoxia for 5 . of RV hypertrophy [ RV / ( LV + S ) ] ( F ) for indicated groups . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE B-EXP_ASSAY O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O 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 of miR - 483 - 3p / - 5p in serum from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE O B-DISEASE O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O 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 of miR - 483 - 3p / - 5p in lung tissue from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-TISSUE O O B-DISEASE O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O 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 ) mRNA of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 measured by Western blot qPCR , respectively ( n = 3 × 3 , samples were pooled from three for each , 3 independent experiments [ a total of 9 ] were performed ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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


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

O B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O O O O 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


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

O B-TISSUE B-EXP_ASSAY O B-TISSUE O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O 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 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


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

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O 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 H & E RV / ( LV + S ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups Scale bar : 0 . 5 cm ( H )

O O O B-TISSUE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O 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 ( n = 7 rats in each group ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups Scale bar : 20 µm ( I ) .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O 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 ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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 - luciferase - expressing U - 2932 , RC - K8 RIVA were intravenously injected into six - week - old ( M ) ( F ) MISTRG mice monitored weekly using IVIS for at least four up to six . D frequency of involvement of indicated tissues is shown for one cohort of mice is representative of two independently analyzed cohorts per .

O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-ORGANISM O 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-ORGANISM 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 identification of U - 2932 residing in spleen bone marrow , among leukocytes in these organs . were stained for human mouse CD45 . Very few hCD45 + U - 2932 were detectable in spleen , whereas their frequencies were much higher in bone marrow ( BM ) . In BM , > 90 % of U - 2932 were positive for ZsGreen . green curve shows U - 2932 recovered from BM , overlayed with unlabelled U - 2932 grown in vitro ( grey curve ) .

O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-TISSUE I-TISSUE I-TISSUE O O B-CELL_TYPE O O O O O O O B-ORGANISM I-ORGANISM B-GENEPROD O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-TISSUE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 , as judged by hCD45 expression , among ( human mouse , h + m ) CD45 + leukocytes in spleen ( S ) , femoral bone marrow ( BM ) jaw bone marrow ( JB ) at study endpoint of mice shown in D ( four post injection for U - 2932 , five p . i . for RIVA six p . i . for RC - K8 ) .

O O O O O B-ORGANISM B-DISEASE O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-ORGANISM O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-CELL_LINE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 , subcutaneously transplanted with 10x106 U - 2932 at six of . reconstitution efficiency ( frequency of human among human mouse CD45 + leukocytes ) is shown in

O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O B-ORGANISM I-ORGANISM B-GENEPROD O B-CELL_TYPE 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 , subcutaneously transplanted with 10x106 U - 2932 at six of . average human leukocyte composition in bone marrow of mice is shown in B .

O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-ORGANISM B-CELL_TYPE O O B-TISSUE I-TISSUE O B-ORGANISM 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 , subcutaneously transplanted with 10x106 U - 2932 at six of . volumes over time are presented in

O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY 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 , subcutaneously transplanted with 10x106 U - 2932 at six of . weights volumes at study endpoint in D , where each data point represents one .

O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 , subcutaneously transplanted with 10x106 U - 2932 at six of . reconstitution level directly correlates with at endpoint ( E ) .

O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 , intravenously injected with 10x106 U - 2932 or RC - K8 at six of . lymphoma burden at study endpoint was quantified in spleen ( F ) bone marrow ( G ) by for hCD45 ( as well as hCD19 hCD20 for U - 2932 RC - K8 , respectively )

O B-ORGANISM O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-DISEASE O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE 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 , intravenously injected with RC - K8 at six of . lymphoma burden at study endpoint was quantified by ZsGreen expression in case of RC - K8 , also over time by IVIS ( H ) .

O B-ORGANISM O O O O O O O B-ORGANISM B-GENEPROD O B-TISSUE I-TISSUE B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-DISEASE O O O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 , intravenously injected with 10x106 U - 2932 or RC - K8 at six of . frequency of tissue involvement was assessed by ex vivo IVIS macroscopic evaluation ( I ) ,

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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 , intravenously injected with 10x106 U - 2932 or RC - K8 at six of . occurrence of clinical symptoms ( loss , hind leg paralysis ) was recorded for each mouse ( J ) .

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

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

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

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

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

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

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

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

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

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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 , intravenously injected with 1x106 DLBCL at six of . lymphoma burden at study endpoint was quantified in bone marrow of reconstituted un - reconstituted by flow cytometric for 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.


lymphoma harvested at study endpoint express IL - 6R α chain ( CD126 , as assessed by flow cytometry , G ) 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).


lymphoma harvested signaling chain gp130 ( as assessed by immunohistochemistry , H )

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


expression of IL - 6Rα ( CD126 ) signaling chains ( gp130 ) was assessed by flow cytometry ( A ) on a panel of DLBCL lines . ABC - GCB - DLBCL lines are color - coded in red 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


expression of IL - 6Rα ( CD126 ) signaling chains ( gp130 ) was assessed by SYBR or Taqman qRT - PCR ( B , C ) respectively , on a panel of DLBCL lines . ABC - GCB - DLBCL lines are color - coded in red 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 E . expression of gp130 in 114 biopsies of DLBCL was assessed by immunohistochemistry , correlated with ABC / non - GCB vs . GCB subtype , as assessed via Hans classification . Representative are shown in D , alongside of gp130 + cases among samples , among 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 indicated lines ( ABC - DLBCL in red , GCB - DLBCL in green ) material , as quantified by ELISA normalized to number ( shown per 1000 ) . 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 indicated ABC - DLBCL GCB - DLBCL lines , with without 1h of exposure to 50ng / ml hIL - 6 ( B , D ) . STAT3 expression 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 indicated ABC - DLBCL GCB - DLBCL lines , Histograms in C represent STAT3 phosphorylation in absence of exogenously added IL - 6 .

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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 , with 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 IL - 6 - expressing SOCS1wt SOCS1mut lines OCI - Ly3 SU - DHL - 2 , under 1 , 6 24h exposure to an IL - 6 - neutralizing antibody ( α - IL - 6 , anti - IL - 6 neutralizing antibody ) .

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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 gp130 expression of two representative cases ( G ) assessed by immunohistochemistry . specific pSTAT3 gp130 signal is seen as brown ( DAB ) , counterstaining was performed using hematoxylin ( blue nuclei ) . STAT3 phosphorylation is more common in biopsies with gp130 expression ; p - value was calculated by chi - square . 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 gp130 expression of two representative cases ( G ) summarized for entire cohort of 114 ( H ) , as assessed by immunohistochemistry . specific pSTAT3 gp130 signal is seen as brown ( DAB ) , 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 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 ( B ) , of cohorts of 9 - 12 mice each ( n indicated in figure ) of 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 indicated genotypes were immunized once with sheep red blood assessed 10 days later by flow cytometry of splenocytes for their frequencies of GC B - ( C ) , centroblasts ( D ) 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 ) ( 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 stratified according to subtype disease stage Phospho - STAT3 positivity was defined as expression in ≥ 17 % of more common found in ABC - DLBCL tumors , in more advanced ( stage 3 4 , relative to stage 1 2 ) disease ( A ) .

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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 pSTAT3 positivity was a strong negative prognostic factor in 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 stratified according to subtype disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - 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 stratified according to subtype disease stage pSTAT3 positivity was associated with higher overall - as well as disease - specific mortality in both ABC - 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 post injection , on ( E F ) spleen weights of MISTRG6 mice transplanted with 1x106 DLBCL ( 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 post injection , Representative microscopy in F show STAT3 phosphorylation in spleen - colonizing exposed to either tocilizumab or isotype control antibody ( in brown , counterstained in blue with hematoxylin ) , an H & E - stained section from 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 post injection on bone marrow lymphoma burden of OCI - Ly3 - transplanted MISTRG mice ( G ) assessed at around four post 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 post injection on splenic lymphoma burden of RC - K8 - transplanted MISTRG6 mice ( H ) , assessed at around four post 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 culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 10 U / mL IFN - α2 , respectively ) before infection with Sendai ( SeV ) influenza A ( IAV , PR8 ) for 6 hours as indicated . Total was harvested subjected to qPCR for of IFN response measured by IFNA2 , IFNB1 , IFNL1 . ISG response measured by ISG15 RSAD2 . Each colour corresponds to a donor ; 9 different donors were analysed . dashed represents detection limit . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 was harvested subjected to qPCR for of IFN response measured by IFNA2 , IFNB1 , IFNL1 ( Donors marked in orange blue were not quantifiable in conditions ) . ( B ) ISG response measured by ISG15 , RSAD2 , IFIT1 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O

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 pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( C ) of presence of SARS - CoV - 2 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O

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.


Alveolar macrophages from different donors were purified from BAL fluid infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( B ) Total was harvested subjected to qPCR for of IFN response measured by IFNA2 , IFNB1 , IFNL1 . ( C ) ISG response measured by ISG15 , RSAD2 , IFIT1 ( Donors marked in green red were not quantifiable in conditions ) .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

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


Alveolar macrophages from different donors were purified from BAL fluid infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( D ) of presence of SARS - CoV - 2 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O

Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.


Alveolar macrophages from different donors were purified from BAL fluid infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( E ) Western blots were performed using samples from two donors . ( A - D ) Each colour corresponds to a donor ; 5 different donors were analysed . dashed represents detection limit . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for 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 treated with SARS - CoV - 2 for either 2 or 24 hours . ( A ) amount of SARS - CoV - 2 nucleocapsid phosphoprotein was quantified using LC - MS / MS . ( B ) After subtraction of background ( unchallenged AMs ) , between amount of SARS - CoV - 2 nucleocapsid phosphoprotein at 24 2 hours was calculated . Each colour corresponds to a donor ; 3 different donors were analyzed . An ordinary one - way - ANOVA was used for statistical : ns , not significant ; * , P ≤ 0 . 05 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Alveolar macrophages from different donors were purified from BAL and treated with SARS-CoV-2 for either 2 or 24 hours. (A) The amount of SARS-CoV-2 nucleocapsid phosphoprotein was quantified using LC-MS/MS. (B) After subtraction of the background (unchallenged AMs), the ratio between the amount of SARS-CoV-2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated. Each colour corresponds to a donor; 3 different donors were analyzed. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05.


( A ) Total was harvested from AMs human airway epithelial ( HAE ) subjected to qPCR for of ACE2 receptor . Each colour corresponds to a donor . dashed represents detection limit .

O O O O O O O B-CELL_TYPE B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O

(A) Total RNA was harvested from AMs and primary human airway epithelial cells (HAE) and subjected to qPCR for quantification of the ACE2 receptor. Each colour corresponds to a donor. The dashed line represents the detection limit.


( B ) presence of nucleoprotein mRNA of SARS - CoV - 2 in AMs HAE was also quantified after SARS - CoV - 2 treatment . Each colour corresponds to a donor . An unpaired t - was used for statistical : * * * * , P ≤ 0 . 0001 .

O O O O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O 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) The presence of nucleoprotein mRNA of SARS-CoV-2 in AMs and primary HAE was also quantified after SARS-CoV-2 treatment. Each colour corresponds to a donor. An unpaired t-test test was used for statistical analysis: ****, P≤0.0001.


( C ) Primer efficiency was tested using serial dilutions of positive samples using qPCR . Ct values were plotted on a logarithmic scale along with corresponding dilutions before performing a linear regression in order to calculate slope of trend through data points . This revealed an amplification factor of 1 . 92 , 1 . 91 , 1 . 85 for SARS - CoV - 2 , IAV , SeV primer set , respectively . different colours correspond to different primer sets .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O

(C) Primer efficiency was tested using serial dilutions of positive samples using qPCR. The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points. This analysis revealed an amplification factor of 1.92, 1.91, and 1.85 for the SARS-CoV-2, IAV, and SeV primer set, respectively. The different colours correspond to different primer sets.


( A ) Proportion change of Chinese SL isolates from different phylogenetic clades . Chinese isolates were classified into two groups on their sampling time ( 1993 - 2010 vs 2011 - 2020 ) . of isolates from each clade in each period was compared .

O O O O O O O B-ORGANISM O O O O O O O O O O O O O O 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) Proportion change of Chinese SL isolates from different phylogenetic clades. The Chinese isolates were classified into two groups based on their sampling time (1993-2010 vs 2011-2020). The percentage of isolates from each clade in each period was compared.


( A ) resistance to desiccation . Representative isolates that are phylogenetically distinct within Clade - 1 ( n = 2 ) , Clade - 2 ( n = 1 ) , Clade - 4 ( n = 2 ) Clade - 5 ( n = 6 ) were used for desiccation Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( A ) were calculated using unpaired t - ( two - ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(A) Bacterial resistance to desiccation. Representative isolates that are phylogenetically distinct within Clade-1 (n=2), Clade-2 (n=1), Clade-4 (n=2) and Clade-5 (n=6) were used for desiccation assay Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (A) were calculated using unpaired t-test (two-tailed) (*:P<0.05, **:P<0.01, ***:P<0.001).


( B ) survivability under oxidative stress . Representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) C5 ( n = 6 ) were treated with hydrogen peroxide . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( B ) , were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial survivability under oxidative stress. Representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were treated with hydrogen peroxide. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (B), were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) Biofilm formation ability . biofilm of representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) C5 ( n = 6 ) were formed at 22 ° C for 48 h . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( C ) were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Biofilm formation ability. The biofilm of the representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were formed at 22°C for 48 h. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (C) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( D ) Comparison of survival rates of chick after challenging SL C5 C1 isolates . Comparison was on 6 day - post - infection ( dpi ) . Ten were used for each group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( D ) were calculated using Log - rank ( Mantel - Cox , chi - square ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(D) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates. Comparison was on 6 day-post-infection (dpi). Ten embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (D) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( E ) Survival rates of zebrafish after challenging SL C5 C1 isolates . comparison was on 14 dpi . Ten zebrafish were used for each group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( E ) were calculated using Log - rank ( Mantel - Cox , chi - square ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates. The comparison was on 14 dpi. Ten zebrafish embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (E) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( F ) loads in distinct organs of mice after challenging SL C5 ( L42 ) C1 ( L1 ) isolates . in liver , spleen , cecum of mice were measured on 3 5 dpi . Ten mice were used per group . Data information : experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( F ) were calculated using unpaired t - ( two - ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial loads in distinct organs of mice after challenging SL C5 (L42) and C1 (L1) isolates. The bacterial counts in the liver, spleen, and cecum of mice were measured on 3 and 5 dpi. Ten mice were used per group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (F) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) day 20 ± 2 cerebral organoids derived from controls , EML1 - heKOs stained for neuronal marker MAPT adherens junction marker NCAD , counterstained with DAPI . Ectopic neural rosettes neuronal heterotopia are highlighted with dotted yellow lines . ( D ) of VZ areas with ectopic neural rosettes ( 3 batches , 3 organoids analyzed per batch , significance on Kruskal - Wallis - , P = 0 . 0001 , Dunn ' s post hoc for multiple comparisons performed to define statistical differences between genotypes , single data points are presented colored by batch ) . ( E ) of heterotopic , disorganized or organized cortical areas ( 3 batches , 3 organoids per batch , significance on Kruskal - Wallis - , P = 0 . 0001 for " organized " " heterotopic " ; no significant difference for " disorganized " , Dunn ' s post hoc performed for multiple comparisons to define statistical differences between genotypes ) . Data information : * marks statistical significance in relation to Control 1 , # in relation to Control 2 . P - values : * * * / # # # < 0 . 001 . VZ : ventricular zone ; Data in graphs are represented as means ± SD . Scale bars : 50µm .

O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O

(C) day 20±2 cerebral organoids derived from controls, patients and EML1-heKOs stained for the neuronal marker MAPT and the adherens junction marker NCAD, counterstained with DAPI. Ectopic neural rosettes and neuronal heterotopia are highlighted with dotted yellow lines. (D) Quantification of VZ areas with ectopic neural rosettes (3 batches, 3 organoids analyzed per batch, significance based on Kruskal-Wallis-test, P=0.0001, Dunn's post hoc test for multiple comparisons performed to define statistical differences between genotypes, single data points are presented colored by batch). (E) Quantification of heterotopic, disorganized or organized cortical areas (3 batches, 3 organoids per batch, significance based on Kruskal-Wallis-test, P=0.0001 for "organized" and "heterotopic"; no significant difference for "disorganized", Dunn's post hoc test performed for multiple comparisons to define statistical differences between genotypes). Data information: * marks statistical significance in relation to Control 1, # in relation to Control 2. P-values: ***/###<0.001. VZ: ventricular zone; Data in graphs are represented as means ± SD. Scale bars: 50µm.


( A ) Hybrid organoids generated from controls mixed with control - EGFP + or EML1 - heKO - EGFP + ( day 20 ± 2 ) . Yellow dotted lines indicate VZ areas . ( B ) of control - EGFP + or EML1 - heKO - EGFP + within or at basal side of VZ areas ( 3 batches , 3 organoids each , significance on Mann Whitney , * * * P = 0 . 0001 for " EGFP + at basal side of VZ areas " ) . Data information : Data in graphs are represented as means ± SD . Scale bars : ( A )

O O O O B-TISSUE O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(A) Hybrid organoids generated from controls mixed with control-EGFP+ or EML1-heKO-EGFP+ cells (day 20±2). Yellow dotted lines indicate VZ areas. (B) Quantification of control-EGFP+ or EML1-heKO-EGFP+ cells within or at the basal side of VZ areas (3 batches, 3 organoids each, significance based on Mann Whitney test, ***P= 0.0001 for "EGFP+ cells at the basal side of VZ areas"). Data information: Data in graphs are represented as means ± SD. Scale bars: (A) 50µm


( C ) Representative scheme of cleavage plan orientation . Upper panel : immunostained for pVim ( red ) TPX2 ( green ) , counterstained with DAPI , dotted indicates cleavage plane orientation , yellow dotted VZ - surface . ( D ) of horizontal , vertical oblique plane of divisions in control EML1 - deficient organoids ( day 20 ± 2 ; 3 batches , 3 organoids each ) . Data information : Scale bars :

O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O

(C) Representative images and scheme of cleavage plan orientation. Upper panel: cells immunostained for pVim (red) and TPX2 (green), counterstained with DAPI, white dotted line indicates cleavage plane orientation, yellow dotted line VZ-surface. (D) Quantification of horizontal, vertical and oblique plane of cell divisions in control and EML1-deficient organoids (day 20±2; 3 batches, 3 organoids each). Data information: Scale bars: 5µm


( F ) Electron microscopy of cilia derived from control or EML1 - deficient cortical progenitors in DMSO - control or following EpothiloneD ( EpoD ) exposure ( blue arrow : basal , yellow arrow : appendages , red arrow : ciliary pockets ) . Data information : Scale bars : 0 , 5µm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O B-TISSUE O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O

(F) Electron microscopy of primary cilia derived from control or EML1-deficient cortical progenitors in DMSO-control or following EpothiloneD (EpoD) exposure (blue arrow: basal body, yellow arrow: appendages, red arrow: ciliary pockets). Data information: Scale bars: 0,5µm.