Developmental rate of embryos treated with TSA at different time points ( 0 - 20 hpi and 8 - 28 hpi ) versus control .

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

Developmental rate of embryos treated with TSA at different time points (0-20 hpi and 8-28 hpi) versus control.


( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assays . * P < 0 . 05 ( one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA).


( B ) HT29 cells were infected with Shigella WT or ∆ ospD3 and incubated for 8 h . Infected cells were fixed and subjected to TUNEL and PI staining . Percentages of positive cells ( TUNEL , green ; PI , red ) are shown in graph at right . The nuclei were stained with DAPI ( blue ) . Scale bar : 100 μm . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


( C ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospD3 and incubated for 8 h . Infected cells were subjected to Giemsa staining . Arrows indicate cells in which the cytoplasm disappeared . Scale bar : 20 μm .

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

(C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.


( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Cell lysates were subjected to immunoblotting .

O O O O O O O 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) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting.


( B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to

O O O 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

(B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting


C ) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

O O O 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

C) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay


( D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


E ) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h , and then cell lysates were subjected to immunoblotting .

O O O 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) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting.


( B ) 293T cells were transfected with Myc - tagged ospD expression plasmids . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 .

O O O O O O O O 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) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.


D ) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 .

O O O O O O O O O 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) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.


( E HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting


F ) HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays


( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 8 activation . Caspase - 8 activity is reported as relative light units ( RLU ) of infected samples , minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-8 activation. Caspase-8 activity is reported as relative light units (RLU) of infected samples, minus the value in uninfected samples. *P<0.05 (one-way ANOVA).


( B ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . Arrows indicate cleaved forms of caspase - 8 , PARP , GSDMD , and IL - 18 , respectively .

(B) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. Arrows indicate cleaved forms of caspase-8, PARP, GSDMD, and IL-18, respectively.


( C ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assay . * P < 0 . 05 ; n . s . , not significant ( one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assay. *P<0.05; n.s., not significant (one-way ANOVA).


( D ) HT29 cells were infected with Shigella WT or ∆ ospC1 mutant and incubated for 8 h . Infected cells were then fixed and stained with cleaved caspase - 8 ( green ) , rhodamine - phalloidin ( red ) , and DAPI ( blue ) . Percentages of positive cells are shown in the graph at right ( * P < 0 . 05 ; unpaired two - tailed Student ' s t - test ) . Scale bar : 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.


( E ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 3 / 7 activation . Caspase - 3 / 7 activity is reported as relative light units ( RLU ) of infected samples minus uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

(E) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-3/7 activation. Caspase-3/7 activity is reported as relative light units (RLU) of infected samples minus uninfected samples. *P<0.05 (one-way ANOVA).


( F ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospC1 strains , and incubated for up to 8 h . After incubation for the indicated times , cells were subjected to real - time Annexin V apoptosis assay . Annexin V binding is reported as relative light units ( RLU ) in infected samples minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with Shigella WT, S325, or ∆ospC1 strains, and incubated for up to 8 h. After incubation for the indicated times, cells were subjected to real-time Annexin V apoptosis assay. Annexin V binding is reported as relative light units (RLU) in infected samples minus the value in uninfected samples. *P<0.05 (one-way ANOVA).


( A HT29 cells were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting


B ) HT29 cells were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay


( C ) HT29 cells were infected with Shigella WT , ∆ ospD3 , or ∆ ospC1 . Cell lysates obtained at the indicated time points were subjected to immunoblotting .

(C) HT29 cells were infected with Shigella WT, ∆ospD3, or ∆ospC1. Cell lysates obtained at the indicated time points were subjected to immunoblotting.


( D HT29 cells treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to

O O O O O O O O O O O O O O O O O O O O O O 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 HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting


E ) HT29 cells treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay


( F ) HT29 cells treated with the control or caspase - 8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting .

(F) HT29 cells treated with the control or caspase-8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting.


A Peroxisomes were induced by growing the WT strain expressing Pot1 ‐ GFP in oleate medium to mid ‐ log ‐ phase , then transferred to SD ‐ N starvation medium with or without GABA to trigger pexophagy for 6 h . GFP cleavage was analyzed at the indicated time points by immunoblotting .

O O O O O O O O O O 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

A Peroxisomes were induced by growing the WT strain expressing Pot1‐GFP in oleate medium to mid‐log‐phase, then transferred to SD‐N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.


B Mitochondria were induced by growing the WT strain expressing OM45 ‐ GFP in YPL medium to mid ‐ log ‐ phase and subsequently transferring cells to either SD ‐ N with or without GABA to trigger mitophagy for 12 h . GFP cleavage was analyzed at the indicated time points by immunoblotting .

B Mitochondria were induced by growing the WT strain expressing OM45‐GFP in YPL medium to mid‐log‐phase and subsequently transferring cells to either SD‐N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.


C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45 ‐ GFP grown in YPL medium for 12 h to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA for 24 h . Bar , 5 μm .

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

C Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45‐GFP grown in YPL medium for 12 h to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA for 24 h. Bar, 5 μm.


D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA , grown to mid ‐ log ‐ phase , after which samples were analyzed for Ape1 maturation .

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

D The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid‐log‐phase, after which samples were analyzed for Ape1 maturation.


E Ribophagy was monitored by growing the WT strain expressing Rpl25 ‐ GFP in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 24 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 B-CONTROLLED_VAR O O O O

E Ribophagy was monitored by growing the WT strain expressing Rpl25‐GFP in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 24 h.


F Autophagy was monitored by growing the WT strain expressing GFP ‐ Atg8 in SD medium to mid ‐ log ‐ phase and transferring cells to SD ‐ N either with or without GABA for 6 h .

F Autophagy was monitored by growing the WT strain expressing GFP‐Atg8 in SD medium to mid‐log‐phase and transferring cells to SD‐N either with or without GABA for 6 h.


APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A .

APeroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig 1A.


B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1 ‐ GFP grown in oleate medium to mid ‐ log ‐ phase in the presence of FM4 ‐ 64 , and transferred to either SD ‐ N medium with or without GABA or to SD ‐ N with GABA and rapamycin for 6 h . Bar , 5 μm .

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

B Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1‐GFP grown in oleate medium to mid‐log‐phase in the presence of FM4‐64, and transferred to either SD‐N medium with or without GABA or to SD‐N with GABA and rapamycin for 6 h. Bar, 5 μm.


C Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .


A WT cells expressing OM45 ‐ GFP , along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing OM45 ‐ GFP were grown in YPL medium to mid ‐ log ‐ phase . To monitor mitophagy , strains were transferred to SD ‐ N starvation medium ( with or without rapamycin ) .

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

A WT cells expressing OM45‐GFP, along with the uga2∆ strain over‐expressing the GAD1 gene and expressing OM45‐GFP were grown in YPL medium to mid‐log‐phase. To monitor mitophagy, strains were transferred to SD‐N starvation medium (with or without rapamycin).


B WT strain along with the uga2 ∆ strain over ‐ expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin . Samples were taken at the indicated time points , and Pot1 degradation was analyzed by immunoblotting ( 45 kD ) .

O O O O O O O O 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

B WT strain along with the uga2∆ strain over‐expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).


C To monitor autophagy , WT cells expressing GFP ‐ Atg8 along with the uga2 ∆ strain over ‐ expressing the GAD1 gene and expressing GFP ‐ Atg8 were grown in SD medium and transferred to SD ‐ N .

O O O O O O O O O O O O O O O O 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 To monitor autophagy, WT cells expressing GFP‐Atg8 along with the uga2∆ strain over‐expressing the GAD1 gene and expressing GFP‐Atg8 were grown in SD medium and transferred to SD‐N.


A , B WT cells were cultured under pexophagy ( A ) or mitophagy ( B ) conditions with or without GABA and rapamycin . S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control .

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

A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control.


C Samples were analyzed for Pot1 degradation by immunoblotting ( 45 kD ) .

C Samples were analyzed for Pot1 degradation by immunoblotting (45 kD).


D GFP production during mitophagy was analyzed by immunoblotting .

D GFP production during mitophagy was analyzed by immunoblotting.


A S6 phosphorylation after 6 h in SD ‐ N was analyzed by immunoblotting with a loading control .

A S6 phosphorylation after 6 h in SD‐N was analyzed by immunoblotting with a loading control.


B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting .

B GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.


A , B WT , WT with GABA , WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under ( A ) pexophagy and ( B ) mitophagy conditions . After 24 h incubation , cells were stained with DHR − 123 and propidium iodide for 1 h . Living cells were analyzed for DHR − 123 fluorescence by flow cytometry . Data represent mean + s . d . ( n = 3 ) . * P < 0 . 005 , * * P < 0 .

O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O 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 O O 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 WT, WT with GABA, WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under (A) pexophagy and (B) mitophagy conditions. After 24 h incubation, cells were stained with DHR−123 and propidium iodide for 1 h. Living cells were analyzed for DHR−123 fluorescence by flow cytometry. Data represent mean + s.d. (n = 3). *P < 0.005, **P < 0.01


C , D Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under ( C ) pexophagy or ( D ) mitophagy conditions . Significant differences between the treatments and strains were determined using an unpaired two ‐ tailed t ‐ test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Yeast cells stained with 5 μM propidium iodide were used to differentiate between living and dead cells under (C) pexophagy or (D) mitophagy conditions. Significant differences between the treatments and strains were determined using an unpaired two‐tailed t‐test. **P < 0.01.


E Pexophagy assay was monitored by the degradation of Pot1 ‐ GFP and analyzed for GFP cleavage by immunoblotting .

E Pexophagy assay was monitored by the degradation of Pot1‐GFP and analyzed for GFP cleavage by immunoblotting.


F Mitophagy assay was monitored by the degradation of Om45 ‐ GFP and analyzed for GFP cleavage by immunoblotting .

F Mitophagy assay was monitored by the degradation of Om45‐GFP and analyzed for GFP cleavage by immunoblotting.


A , B Example images of Parkin ‐ expressing HeLa cells analyzed using a tandem fluorochrome protein ( mito ‐ RFP ‐ GFP ) mitophagy assay under ( A ) control conditions or ( B ) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes . Bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Example images of Parkin‐expressing HeLa cells analyzed using a tandem fluorochrome protein (mito‐RFP‐GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 μm.


C Percentage of cells displaying mitophagy + s . d . , * * P < 0 . 01 using an unpaired two ‐ tailed t ‐ test , n > 80 .

O O O O O O O O O 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 Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two‐tailed t‐test, n > 80.


A Electron microscopy images of mitochondria from WT ( n = 44 ) and SSADH ‐ deficient mice ( Aldh5a1 − / − ) ( n = 80 ) were calculated for area size .

O O O O O O O O O O O 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 Electron microscopy images of mitochondria from WT (n = 44) and SSADH‐deficient mice (Aldh5a1−/−) (n = 80) were calculated for area size.


B Electron microscopy images showing typical sizes of WT and Aldh5a1 − / − mice liver mitochondria . Bar , 0 . 5 μm .

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

B Electron microscopy images showing typical sizes of WT and Aldh5a1−/− mice liver mitochondria. Bar, 0.5 μm.


C Quantification of mitochondrial numbers from electron microscopy images of liver from WT ( n = 31 ) and Aldh5a1 − / − mice treated with vehicle ( n = 39 ) or rapamycin ( n = 34 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life .

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C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.


D Quantification of mitochondrial numbers from electron microscopy images of brain from WT ( n = 23 ) and Aldh5a1 − / − mice treated with vehicle ( n = 30 ) or rapamycin ( n = 41 ) ( 5 mg / kg body weight per day ) via intraperitoneal injections for 3 successive days starting at day 7 of life .

D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.


E Aldh5a1 − / − mice were treated with vehicle or rapamycin ( 10 mg / kg body weight per day ) via intraperitoneal injections for 10 successive days starting at day 10 of life . WT mice served as non ‐ disease controls ( set to 1 ) . After sacrifice , liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay .

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E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.


F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis ( WT set to 1 ) .

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F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1).


G Immunofluorescence images showing typical nuclear staining ( DAPI , blue ) and SOD2 staining ( red ) from WT , Aldh5a1 − / − mice treated with vehicle and Aldh5a1 − / − mice treated with rapamycin . Bar , 10 μm .

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G Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1−/− mice treated with vehicle and Aldh5a1−/− mice treated with rapamycin. Bar, 10 μm.


A Quantification of S6 phosphorylation of liver lysates from WT ( n = 5 ) and Aldh5a1 − / − mice treated with vehicle ( n = 4 ) or rapamycin ( n = 5 ) after normalization ( WT set to 1 ) .

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A Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1−/− mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1).


B S6 phosphorylation of liver lysates analyzed by immunoblotting .

B S6 phosphorylation of liver lysates analyzed by immunoblotting.


C Quantification of S6 phosphorylation of brain lysates from WT ( n = 2 ) and Aldh5a1 − / − mice treated with vehicle ( n = 3 ) or rapamycin ( n = 3 ) after normalization ( WT set to 1 ) .

C Quantification of S6 phosphorylation of brain lysates from WT (n = 2) and Aldh5a1−/− mice treated with vehicle (n = 3) or rapamycin (n = 3) after normalization (WT set to 1).


D S6 phosphorylation of brain lysates analyzed by immunoblotting .

D S6 phosphorylation of brain lysates analyzed by immunoblotting.


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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


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

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


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

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


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

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


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

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


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

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


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

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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 ) .

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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 ) .

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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 )

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


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

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


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

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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 ) .

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


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

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

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


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

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

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


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

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(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

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O 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 ) .

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 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

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 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

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 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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O 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 ] .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).