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 .

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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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

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

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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


B The expression of mature miR - 200b and miR - 200a in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 ; Student ' s t - test ) .

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

B The expression of mature miR-200b and miR-200a in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01; Student's t-test).


C The expression of Ttll10 ( the host gene of miR - 200b / a ) mRNA in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; p = 0 . 3540 ; Student ' s t - test ) .

O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O 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 The expression of Ttll10 (the host gene of miR-200b/a) mRNA in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; p=0.3540; Student's t-test).


D The locomotor activity of the miR - 200b / a KD mice was similar to that of the NC mice ( NC : n = 13 mice , miR - 200b / a knockdown : n = 15 mice ; data represent the mean ± SEM ; p = 0 . 5030 ; Student ' s t - test ) .

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

D The locomotor activity of the miR-200b/a KD mice was similar to that of the NC mice (NC: n=13 mice, miR-200b/a knockdown: n=15 mice; data represent the mean±SEM; p=0.5030; Student's t-test).


E The odorant habituation results shown as the ratio of odor sniffs / water of the miR - 200b / a KD mice were significantly reduced compared to those of the NC mice . These odorants included male urine ( 1 : 50 ) , female urine ( 1 : 50 ) , isopentyl acetate ( 50 μM ) , citral ( 50 μM ) , 2 - heptanone ( 50 μM ) and propyl propionate ( 50 μM ) ( n = 6 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The odorant habituation results shown as the ratio of odor sniffs/water of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice. These odorants included male urine (1:50), female urine (1:50), isopentyl acetate (50 μM), citral (50 μM), 2-heptanone (50 μM) and propyl propionate (50 μM) (n=6 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test).


The resident / intruder experiment showed that the attack frequency ( F ) of the miR - 200b / a KD mice were impaired compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) .

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

The resident/intruder experiment showed that the attack frequency (F) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).


G The resident / intruder experiment showed that the attack duration ( G ) of the miR - 200b / a KD mice were impaired compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) .

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

G The resident/intruder experiment showed that the attack duration (G) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).


H , I The analysis of male - female mating behaviors showed that the mating frequency ( H ) and duration ( I ) of the miR - 200b / a KD mice were significantly reduced compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) .

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

H, I The analysis of male-female mating behaviors showed that the mating frequency (H) and duration (I) of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; **p<0.01, ***p<0.001; Student's t-test).


B Representative IF staining with Ki67 antibody in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . C Quantification of the numbers of Ki67 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 , * * * * p < 0 . 0001 ; Student ' s t - test ) .

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

B Representative IF staining with Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. C Quantification of the numbers of Ki67+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01, ****p<0.0001; Student's t-test).


D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the EdU + cells and the yellow arrows indicate the cells positive for both EdU and Ki67 . D ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . D : Scale bars , 20 μm ; D ' : Scale bars , 2 μm . E Quantification of the numbers of costained Ki67 + and EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 3 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR - 200b / a KD mice compared with their NC controls [ the cell cycle exit index was calculated using the following formula : ( EdU + Ki67 − cells / total EdU + cells ) × 100 ; n = 3 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' s t - test ] .

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

D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the EdU+ cells and the yellow arrows indicate the cells positive for both EdU and Ki67. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the numbers of costained Ki67+ and EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR-200b/a KD mice compared with their NC controls [the cell cycle exit index was calculated using the following formula: (EdU+ Ki67− cells/total EdU+ cells)×100; n=3 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test].


G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the BrdU + cells , and the yellow arrows indicate the cells positive for both BrdU and EdU . G ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . G : Scale bars , 20 μm ; G ' : Scale bars , 2 μm .

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

G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the BrdU+ cells, and the yellow arrows indicate the cells positive for both BrdU and EdU. G' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). G: Scale bars, 20 μm; G': Scale bars, 2 μm.


H The S phase length of the GBCs in the MOE of the NC and miR - 200b / a KD mice ( the S phase length was calculated using the following formula : 3 h × BrdU + / BrdU + EdU - ; n = 3 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The S phase length of the GBCs in the MOE of the NC and miR-200b/a KD mice (the S phase length was calculated using the following formula: 3 h×BrdU+/BrdU+EdU-; n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).


I Total cell cycle length of the GBCs in the MOE of the NC and miR - 200b / a KD mice ( total cell cycle length was calculated using the following formula : 20 h + ( Ts × EdU + BrdU - / Edu + ) ; n = 3 mice each group ; p = 0 . 9785 ; data represent the mean ± SEM ; Student ' s t - test ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Total cell cycle length of the GBCs in the MOE of the NC and miR-200b/a KD mice (total cell cycle length was calculated using the following formula: 20 h+(Ts×EdU+BrdU-/Edu+); n=3 mice each group; p=0.9785; data represent the mean±SEM; Student's t-test).


B The mRNA levels of CK14 , Trp63 , Sox2 and Mash1 in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; CK14 : p = 0 . 2135 , Trp63 : p = 0 . 1930 , Sox2 : p = 0 . 5190 , * p < 0 . 05 ; Student ' s t - test ) .

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

B The mRNA levels of CK14, Trp63, Sox2 and Mash1 in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; CK14: p=0.2135, Trp63: p=0.1930, Sox2: p=0.5190, *p<0.05; Student's t-test).


C Representative IF staining for CK14 , Sox2 , and Mash1 in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . D Quantification of the number of CK14 + , basal Sox2 + , apical Sox2 + , and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; CK14 : p = 0 . 8998 , basal Sox2 : p = 0 . 7056 , apical Sox2 : p = 0 . 4455 , * * p < 0 . 01 ; Student ' s t - test ) . E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR - 200b / a KD mice ( n = 2 mice each group ) . GAPDH and Tubulin served as loading controls . The molecular weight of each band is indicated at the right .

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

C Representative IF staining for CK14, Sox2, and Mash1 in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. D Quantification of the number of CK14+, basal Sox2+, apical Sox2+, and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; CK14: p=0.8998, basal Sox2: p=0.7056, apical Sox2: p=0.4455, **p<0.01; Student's t-test). E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR-200b/a KD mice (n=2 mice each group). GAPDH and Tubulin served as loading controls. The molecular weight of each band is indicated at the right.


F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both Sox2 and EdU . F ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( right images ) . F : Scale bars , 20 μm ; F ' : Scale bars , 2 μm . G Quantification of the number of Sox2 + EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; p = 0 . 7988 ; Student ' s t - test ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O 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 Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Sox2 and EdU. F' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). F: Scale bars, 20 μm; F': Scale bars, 2 μm. G Quantification of the number of Sox2+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; p=0.7988; Student's t-test).


H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both Mash1 and EdU . H ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( right images ) . H : Scale bars , 20 μm ; H ' : Scale bars , 2 μm . I Quantification of the number of Mash1 + EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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 Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Mash1 and EdU. H' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). H: Scale bars, 20 μm; H': Scale bars, 2 μm. I Quantification of the number of Mash1+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).


A Representative IF staining for Tuj1 , GAP43 , and OMP in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . B Quantification of the number of Tuj1 + , GAP43 + , and OMP + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) . C The mRNA levels of Ngn1 , GAP43 and OMP in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) .

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

A Representative IF staining for Tuj1, GAP43, and OMP in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. B Quantification of the number of Tuj1+, GAP43+, and OMP+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test). C The mRNA levels of Ngn1, GAP43 and OMP in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).


D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR - 200b / a KD mice injected with BrdU at 3 d . The white arrows indicate the cells positive for both GAP43 and BrdU . D ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . D : Scale bars , 20 μm ; D ' : Scale bars , 2 μm . E Quantification of the ratio of the number of BrdU + GAP43 + cells and the number of total GAP43 + cells in the MOE of the NC and miR - 200b / a KD mice injected with BrdU at 3 d ( n = 3 mice each group ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) .

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

D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d. The white arrows indicate the cells positive for both GAP43 and BrdU. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the ratio of the number of BrdU+ GAP43+ cells and the number of total GAP43+ cells in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d (n=3 mice each group; data represent the mean±SEM; ****p<0.0001; Student's t-test).


F Representative IF staining with Cas3 antibody in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . G Quantification of the number of Cas3 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) .

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

F Representative IF staining with Cas3 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. G Quantification of the number of Cas3+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).


A The number of differentially expressed olfrs from RNA sequencing in the MOE of the NC and miR - 200b / a KD mice .

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

A The number of differentially expressed olfrs from RNA sequencing in the MOE of the NC and miR-200b/a KD mice.


C TET 3 ' UTR - dependent expression of a luciferase reporter gene was suppressed by miR - 200a overexpression . Luciferase reporter gene under the control of the wild - type ( WT ) or mutant ( Mut ) TET3 3 ' UTR was transfected along with the negative control ( nc ) RNA or miR - 200a mimic duplexes into HeLa cells ( n = 6 from six independent experiments ; data represent the mean ± SEM ; Mut - nc vs . Mut - mimic : p = 0 . 3697 , * p < 0 . 05 ; Student ' s t - test ) .

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

C TET 3' UTR-dependent expression of a luciferase reporter gene was suppressed by miR-200a overexpression. Luciferase reporter gene under the control of the wild-type (WT) or mutant (Mut) TET3 3' UTR was transfected along with the negative control (nc) RNA or miR-200a mimic duplexes into HeLa cells (n=6 from six independent experiments; data represent the mean±SEM; Mut-nc vs. Mut-mimic: p=0.3697, *p<0.05; Student's t-test).


D , E The expression of mature miR - 200a ( D ) and TET3 ( E ) mRNA in the 3T3 - L1 cells treated with miR - 200a inhibitor , miR - 200a mimic or control , as revealed by qPCR analysis ( n = 4 from four independent experiments ; data represent the mean ± SEM ; D : F = 66 . 37 , E : F = 16 . 39 , NC vs . mimic : p = 0 . 2597 , * p < 0 . 05 , * * * p < 0 . 001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E The expression of mature miR-200a (D) and TET3 (E) mRNA in the 3T3-L1 cells treated with miR-200a inhibitor, miR-200a mimic or control, as revealed by qPCR analysis (n=4 from four independent experiments; data represent the mean±SEM; D: F=66.37, E: F=16.39, NC vs. mimic: p=0.2597, *p<0.05, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons).


F Western Blot analysis of the TET3 protein level in the 3T3 - L1 cells treated with miR - 200a inhibitor , miR - 200a mimic or control ( n = 3 from three independent experiments ) . Actin served as a loading control . The molecular weight of each band is indicated at the right .

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

F Western Blot analysis of the TET3 protein level in the 3T3-L1 cells treated with miR-200a inhibitor, miR-200a mimic or control (n=3 from three independent experiments). Actin served as a loading control. The molecular weight of each band is indicated at the right.


Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR - 200a ( G ) NC mimic ( n = 3 mice each group ) . Actin served as a loading control . The molecular weight of each band is indicated at the right .

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

Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR-200a (G) NC mimic (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right.


H Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR - 200b ( H ) or NC mimic ( n = 3 mice each group ) . Actin served as a loading control . The molecular weight of each band is indicated at the right .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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 Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR-200b (H) or NC mimic (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right.


I Western Blot analysis of the TET3 protein level in the MOE of the NC and miR - 200b / a KD mice ( n = 3 mice each group ) . Actin served as a loading control . The molecular weight of each band is indicated at the right . The blue number shows the ratio of the average intensity of TET3 / Actin .

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

I Western Blot analysis of the TET3 protein level in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right. The blue number shows the ratio of the average intensity of TET3/Actin.


J Representative IF staining of TET3 in the MOE of the NC and miR - 200b / a KD mice . Scale bars : 20 μm .

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

J Representative IF staining of TET3 in the MOE of the NC and miR-200b/a KD mice. Scale bars: 20 μm.


K Quantification of the number of TET3 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) .

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

K Quantification of the number of TET3+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).


L Representative IF staining of 5mC in the MOE of the NC and miR - 200b / a knockdown mice . L ' is shown at higher magnification of the boxed region . L : Scale bars , 20 μm ; L ' : Scale bars , 2 μm .

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

L Representative IF staining of 5mC in the MOE of the NC and miR-200b/a knockdown mice. L' is shown at higher magnification of the boxed region. L: Scale bars, 20 μm; L': Scale bars, 2 μm.


M Quantification of the number of 5mC + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) .

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

M Quantification of the number of 5mC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).


N Representative IF staining of 5hmC in the MOE of the NC and miR - 200b / a knockdown mice . N ' is shown at higher magnification of the boxed region . N : Scale bars , 20 μm ; N ' : Scale bars , 2 μm .

N Representative IF staining of 5hmC in the MOE of the NC and miR-200b/a knockdown mice. N' is shown at higher magnification of the boxed region. N: Scale bars, 20 μm; N': Scale bars, 2 μm.


Quantification of the number of 5hmC + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' s t - test ) .

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

Quantification of the number of 5hmC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).


A Representative IF staining for costaining with TET3 and Mash1 antibodies in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both TET3 and Mash1 . A ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . A : Scale bars , 20 μm ; A ' : Scale bars , 2 μm . B Quantification of the number of costained TET3 + and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) .

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

A Representative IF staining for costaining with TET3 and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both TET3 and Mash1. A' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). A: Scale bars, 20 μm; A': Scale bars, 2 μm. B Quantification of the number of costained TET3+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).


C , D The resident / intruder experiment showed that the attack frequency ( C ) and duration ( D ) of the miR - 200b / a + TET3 DKD mice were significantly improved compared with those of the miR - 200b / a KD controls ; [ 200 KD : miR - 200b / a KD ; ( 200 + T ) DKD : ( miR - 200b / a + TET3 ) DKD ; ( NC : n = 18 mice , miR - 200b / a KD : n = 14 mice , miR - 200b / a + TET3 DKD : n = 16 mice ; data represent the mean ± SEM ; C : F = 17 . 84 , D : F = 12 . 06 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; One - way ANOVA and Bonferroni pairwise comparisons ) ] .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D The resident/intruder experiment showed that the attack frequency (C) and duration (D) of the miR-200b/a+TET3 DKD mice were significantly improved compared with those of the miR-200b/a KD controls; [200 KD: miR-200b/a KD; (200+T) DKD: (miR-200b/a+TET3) DKD; (NC: n=18 mice, miR-200b/a KD: n=14 mice, miR-200b/a+TET3 DKD: n=16 mice; data represent the mean±SEM; C: F=17.84, D: F=12.06, *p<0.05, ***p<0.001, ****p<0.0001; One-way ANOVA and Bonferroni pairwise comparisons)].


E , F The analysis of male - female mating behaviors showed that the frequency ( E ) and duration ( F ) of mating to females of the miR - 200b / a + TET3 DKD mice were significantly reduced compared with those of their NC controls and were not dramatically different compared with those of the miR - 200b / a KD mice ( NC : n = 18 mice , miR - 200b / a KD : n = 14 mice , miR - 200b / a + TET3 DKD : n = 16 mice ; data represent the mean ± SEM ; E : F = 15 . 62 , F : F = 15 . 56 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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

E, F The analysis of male-female mating behaviors showed that the frequency (E) and duration (F) of mating to females of the miR-200b/a+TET3 DKD mice were significantly reduced compared with those of their NC controls and were not dramatically different compared with those of the miR-200b/a KD mice (NC: n=18 mice, miR-200b/a KD: n=14 mice, miR-200b/a+TET3 DKD: n=16 mice; data represent the mean ±SEM; E: F=15.62, F: F=15.56, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).


G Representative IF staining for Mash1 , Tuj1 , Cas3 and Ki67 in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + TET3 DKD mice . Scale bars : 20 μm . H - K Quantification of the numbers of Mash1 + ( H ) , Tuj1 + ( I ) , Cas3 + ( J ) and Ki67 + ( K ) cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + TET3 DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; Mash1 : F = 41 . 45 , Tuj1 : F = 113 . 9 , Cas3 : F = 92 . 51 , Ki67 : F = 61 . 35 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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

G Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice. Scale bars: 20 μm. H-K Quantification of the numbers of Mash1+(H), Tuj1+(I), Cas3+(J) and Ki67+(K) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=41.45, Tuj1: F=113.9, Cas3: F=92.51, Ki67: F=61.35, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).


A The REST mRNA expression in the MOE of the NC , miR - 200b / a KD and miR - 200b / a + TET3 DKD mice , as revealed by qPCR analysis [ 200 KD : miR - 200b / a KD ; ( 200 + T ) DKD : ( miR - 200b / a + TET3 ) DKD ; n = 4 mice each group ; data represent the mean ± SEM ; F = 9 . 409 , NC vs . 200 : p = 0 . 1367 , 200 vs . 200 + T : p = 0 . 1896 , * * p < 0 . 01 ; one - way ANOVA and Bonferroni pairwise

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

A The REST mRNA expression in the MOE of the NC, miR-200b/a KD and miR-200b/a+TET3 DKD mice, as revealed by qPCR analysis [200 KD: miR-200b/a KD; (200+T) DKD: (miR-200b/a+TET3) DKD; n=4 mice each group; data represent the mean±SEM; F=9.409, NC vs. 200: p=0.1367, 200 vs. 200+T: p=0.1896, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisons


B Western Blot analysis of the REST protein level in the MOE of the NC , miR - 200b / a KD and miR - 200b / a + TET3 DKD mice ( n = 3 mice each group ) . Actin served as a loading control .

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

B Western Blot analysis of the REST protein level in the MOE of the NC, miR-200b/a KD and miR-200b/a+TET3 DKD mice (n=3 mice each group). Actin served as a loading control.


C Representative IF staining for REST in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a and TET3 DKD mice . Scale bars : 20 μm .

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

C Representative IF staining for REST in the MOE of the NC, miR-200b/a KD, and miR-200b/a and TET3 DKD mice. Scale bars: 20 μm.


D Quantification of the number of REST + cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + TET3 DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; F = 12 . 11 , * p < 0 . 05 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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

D Quantification of the number of REST+ cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice (n=4 mice each group; data represent the mean±SEM; F=12.11, *p<0.05; one-way ANOVA and Bonferroni pairwise comparisons).


Representative IF costaining with REST and Mash1 antibodies in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both REST and Mash1 . E ' are shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . E : Scale bars , 20 μm ; E ' : Scale bars , 2 μm .

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

Representative IF costaining with REST and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both REST and Mash1. E' are shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). E: Scale bars, 20 μm; E': Scale bars, 2 μm.


F Quantification of the number of costained REST + and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' s t - test ) .

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

F Quantification of the number of costained REST+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).


G Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against TET3 . Input protein ( 100 ug ) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies . IgG antibodies were used as the negative control .

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

G Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against TET3. Input protein (100 ug) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. IgG antibodies were used as the negative control.


H Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against REST . Input protein ( 100 ug ) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies . IgG antibodies were used as the negative control .

H Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against REST. Input protein (100 ug) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. IgG antibodies were used as the negative control.


A , B The resident / intruder experiment showed that the attack frequency ( A ) and duration ( B ) of the miR - 200b / a + REST DKD mice were dramatically improved compared with those of the miR - 200b / a KD mice [ 200 KD : miR - 200b / a KD ; ( 200 + R ) DKD : ( miR - 200b / a + REST ) DKD ; NC : n = 10 mice , miR - 200b / a KD : n = 9 mice , miR - 200b / a + REST DKD : n = 13 mice ; data represent the mean ± SEM ; A : F = 10 . 62 , B : F = 10 . 54 , * * * p < 0 . 001 ; one - way ANOVA and Bonferroni pairwise comparisons ] .

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

A, B The resident/intruder experiment showed that the attack frequency (A) and duration (B) of the miR-200b/a+REST DKD mice were dramatically improved compared with those of the miR-200b/a KD mice [200 KD: miR-200b/a KD; (200+R) DKD: (miR-200b/a+REST) DKD; NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; A: F=10.62, B: F=10.54, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons].


C , D The analysis of male - female mating behaviors showed that the frequency ( C ) and duration ( D ) of mating to females of the miR - 200b / a + REST DKD mice were significantly improved compared with those of the miR - 200b / a KD mice ( NC : n = 10 mice , miR - 200b / a KD : n = 9 mice , miR - 200b / a + REST DKD : n = 13 mice ; data represent the mean ± SEM ; C : F = 6 . 162 , D : F = 6 . 360 , * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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

C, D The analysis of male-female mating behaviors showed that the frequency (C) and duration (D) of mating to females of the miR-200b/a+REST DKD mice were significantly improved compared with those of the miR-200b/a KD mice (NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; C: F=6.162, D: F=6.360, *p<0.05, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisons).


Representative IF staining for Mash1 , Tuj1 , Cas3 and Ki67 in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + REST DKD mice . Scale bars : 20 μm . F - I Quantification of the number of Mash1 + ( F ) , Tuj1 + ( G ) , Cas3 + ( H ) and Ki67 + ( I ) cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + REST DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; Mash1 : F = 35 . 53 , Tuj1 : F = 62 . 28 , Cas3 : F = 115 . 7 , Ki67 : F = 77 . 43 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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

Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice. Scale bars: 20 μm. F-I Quantification of the number of Mash1+(F), Tuj1+(G), Cas3+(H) and Ki67+(I) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=35.53, Tuj1: F=62.28, Cas3: F=115.7, Ki67: F=77.43, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).


The rate of glucose uptake ( a , e ) , lactate released after 4 h of incubation ( b , f ) , glycolytic flux ( c , g ) and pentose - phosphate flux ( d , h ) were assessed in MEF and mouse cortical neurons in primary culture , as indicated , obtained from either WT or Pink1 KO ( Pink1 − / − ) mice .

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

The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.


Blood concentrations of glucose ( i ) and lactate ( j ) , and the rate of 14C - lactate appearance in the blood from acutely injected [ U - 14C ] glucose in the tail vein ( k ) of WT and Pink1 KO mice were analysed . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO miceMEF ( a ) ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT .

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

Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO miceMEF (a) ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null mice MEF ( b ) ß - Actin was used as loading control .

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

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) ß-Actin was used as loading control.


Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO neurons ( c ) . ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT .

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

Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null neurons ( d ) ; ß - Actin was used as loading control .

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null neurons (d); ß-Actin was used as loading control.


( e ) Western blot against PINK1 , GLUT1 , GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples ; α - Tubulin was used as loading control . Only representative western blots are shown per condition ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . The mean ± s . e . m . values of the mRNA data were calculated from the fold change of each ß - Actin - normalized transcript abundance in the Pink1 KO samples versus that in the WT . Thus , in all cases , the WT values were considered = 1 . 00 . * P0 . 05 versus the corresponding WT ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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

(e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student's t-test; n=3-4 independent experiments).


( a ) RT - qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α , or siControl ( 100 nM ) , after 72 h .

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

(a) RT-qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h.


( b ) siHif1α effectively knockdowns expressed Hif1α cDNA WT , but not a Hif1α cDNA silent mutant form refractory to siHif1α ( Hif1α cDNA REFR . ) in mice MEF .

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

(b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF.


( c ) RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α . ß - Actin mRNA was used for normalization , and all data are expressed as the change relative to WT - siControl .

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

(c) RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl.


( d ) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α ; ß - actin was used as loading control . As shown , siHif1α partially prevented the increase in GLUT1 , HK2 and GAPDH protein abundances .

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

(d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances.


The increased rate of lactate released after 4 h of incubation ( e ) and glycolytic flux ( f ) observed in the Pink1 KO MEF was prevented by siHif1α .

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

The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.


siHif1α was also effective at preventing the endogenous increased HIF1α protein ( g ) , as well as the increased glycolytic flux ( h ) observed in Pink1 KO primary neurons . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information .

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

siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.


( a ) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase ( CS ) , and ( b ) mitochondrial inner membrane potential ( Δψm ) in WT and Pink1 KO mice MEF .

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

(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.


( c ) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α ( or its siControl ) ; ß - actin was used as loading control .

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

(c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control.


d ) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl .

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

d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl.


Rate of whole - cell H2O2 ( e ) and mitochondrial O2 · − ( f ) detection in WT and Pink1 KO

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

Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF


( g ) Detection of mitochondrial O2 · − using the mitochondrial - specific probe MitoSox in MEF incubated in the absence ( − ) or in the presence ( + ) of the plasma membrane permeable GSH - EE , or by expressing mitochondrial - tagged forms of glutamate - cysteine ligase , ( + mitoGCL ; empty vector , pIRES2 - EGFP , denoted as − ) or catalase ( + mitoCAT ; empty vector denoted as − ) , shows effective mitochondrial ROS removal .

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

(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.


( h ) Western blot against HIF1α in Pink1 KO MEF incubated with ( or without ) GSH - EE , or transfected with empty vector , mitoGCL or mitoCAT , showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS ; α - TUBULIN was used as loading control . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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

(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


( a ) Proportion of BrdU incorporation into DNA and cell cycle phases , and ( b ) cell growth in WT and Pink1 KO MEF after 72 h in the absence ( denoted as - 2DG ) or in the presence ( + 2DG ) of the glucose - metabolizing inhibitor , 2 - deoxyglucose ( 2DG , 1 mM ) .

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

(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).


( c ) Proportions of BrdU incorporation into DNA and cell cycle phases , and ( d ) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α ( + siHif1α ) or siControl ( denoted as - siHif1α ) .

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

(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).


( e ) Ki67 , Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain , and the corresponding quantification . * P0 . 05 ; # P0 . 05 versus all other conditions ( analysis of variance followed by Bonferroni test ; n = 3 - 4 independent experiments ) . All data are expressed as mean ± s . e . m . Scale bar , 100 μm .

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

(e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.


a ) Rate of mitochondrial O2 · - detection in WT and Pink1 KO mice primary neurons .

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

a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.


( b ) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons .

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

(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.


( c ) Proportion of apoptosis in WT and Pink1 KO neurons , as assessed by the proportion of annexin V - positive / 7 - AAD - negative cells , determined by flow cytometry . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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(c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


( A , B ) Airyscan confocal analysis of PC1 ( 568 , red ) co - localization with GFP - LC3 ( green ) in ( A ) MEF ( B ) Saos2 . Scale bars = 10 µm . ( C ) Airyscan confocal analysis of PC2 ( 647 , red ) co - localization with LC3 ( 488 , green ) in RCS cells . Scale bars = 10 µm . ( A - C ) The insets show higher magnification ( A = x4 . 68 ; B = x6 . 76 ; C = x7 . 33 ) and single colour channels of the boxed area . ( D ) Quantification of GFP ( A , B ) or LC3 ( C ) vesicles positive for PC1 or PC2 , expressed as % of total LC3 ( mean + / - SEM ) , n = 18 cells ( MEFs and Saos2 ) ; n = 12 ( RCS ) from 3 independent experiments .

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

(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments.


( E - G ) Scanning confocal microscopy analysis of MEFs , Saos2 and RCS cells treated with BafA1 , immunolabeled for PC1 or PC2 and LAMP1 . Nuclei were stained with Hoechst . ( E , F ) Scale bars = 10 µm , ( G ) Scale bars = 5 µm . The insets show higher magnification ( E = x4 . 99 ; F = x6 . 49 ; G = x2 . 01 ) and single colour channels of the boxed area .

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(E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.


( H ) Transmission EM analysis in Saos2 cells , treated with BafA1 , showing in detail a lysosome which contains immunolabeled PC1 ( with nanogold particles ) , as indicated by arrows .

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(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.


( I ) Scanning confocal microscopy analysis of Saos2 WT and CRISPR - Cas9 IDUA Saos2 at steady state , immunolabeled for PC1 and LAMP1 . Nuclei were stained with Hoechst . Scale bar = 10 µm . The insets show higher magnification ( left = x3 . 09 ; right = x3 . 12 ) and single colour channels of the boxed area . Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell ( mean + / - SEM ) . n = 31 WT cells , n = 33 CRISPR cells counted ; 3 independent experiments . Student ' s unpaired , two - tailed T - Test * * * P < 0 . 0001 .

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(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.


( J ) WT and CRISPR IDUA Saos2 lysed and analysed by western blot . Data are representative of 3 independent experiments .

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(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.


U2OS expressing ( A ) GFP - LC3 mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification ( A = x3 . 93 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm.


U2OS expressing ( B ) GFP - 2 - FYVE ( green ) , mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification B = x3 . 42 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm.


( C ) Airyscan analysis of Saos2 cells expressing GFP - LC3 ( green ) and immunolabeled for PC1 ( 405 , blue ) and CANX ( 647 , red ) . The insets show higher magnification ( x5 . 26 ) and single colour channels of the boxed area . Scale bar = 10 µm .

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(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.


( D ) Correlative Light Electron Microscopy ( CLEM ) and Electron Tomography of Saos2 cells transfected with GFP - LC3 ( green ) and labelled for PC1 ( 568 , red and nanogold particles ) and CANX ( 647 , blue ) . Cells were first imaged by confocal microscopy ( top left panel ) and then the same region was retraced in EM ( upper middle panel ) and overlay is shown ( upper right panel ) . Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules .

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(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.


( E ) Single tomography slice ( left panel , taken from boxed are in D at a magnification of x2 . 84 ) , overlay with immunofluorescence ( IF ) ( central panel ) and IF 3D rendering of AV ( green ) and the CANX positive vesicle containing gold particles of labelled collagen ( blue and white respectively ) inside an AV ( right panel ) .

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(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).