A . Immunofluorescence of SATB2 ( green ) GSC marker SOX2 or OLIG2 ( red ) on frozen tissue sections of human GBM surgical specimens . SATB2 is preferentially expressed by GSCs in human GBMs . Scale bar , 25 μm . B . of fraction of SATB2 + in SOX2 + or OLIG2 + in human GBMs . More than 90 % SOX2 + or OLIG2 + showed SATB2 . n = 3 GBMs .

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A. Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm. B. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n=3 GBMs.


C . qPCR of SATB2 mRNA expression in GSCs matched non - ( NSTCs ) ( n = 5 ) .

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C. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n=5).


D . Immunoblot of SATB2 , SOX2 OLIG2 expression in lysates of GSCs matched NSTCs .

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D. Immunoblot analysis of SATB2, SOX2 and OLIG2 expression in cell lysates of GSCs and matched NSTCs.


E . Immunofluorescenc of SATB2 ( green ) SOX2 ( red ) in T3359 GSCs matched NSTCs . Scale bar , 50 μm .

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E. Immunofluorescenc of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm.


F . qPCR of SATB2 mRNA expression in GSCs neural progenitor ( NPCs ) ( n = 3 ) .

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F. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n=3).


G . Immunoblot of SATB2 SOX2 expression in lysates of GSCs NPCs .

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G. Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs.


H . Immunoblot of SATB2 , GSC marker SOX2 differentiation marker GFAP expression during serum - induced GSC differentiation .

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H. Immunoblot analysis of SATB2, GSC marker SOX2 and differentiation marker GFAP expression during serum-induced GSC differentiation.


A . Immunoblot of SATB2 expression in GSCs transduced with lentiviral - mediated non - targeting shRNA ( shNT ) or SATB2 shRNA ( shSATB2 ) .

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A. Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral-mediated non-targeting shRNA (shNT) or SATB2 shRNA (shSATB2).


B . viability of GSCs transduced with shNT or shSATB2 ( n = 5 ) .

B. Cell viability of GSCs transduced with shNT or shSATB2 (n=5).


C . EdU incorporation of GSCs transduced with shNT or shSATB2 . Scale bar : 50 μm . D . of ( C ) showing of EdU + ( n = 5 ) .

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C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm. D. Quantification of (C) showing the percentage of EdU+ cells (n=5).


E . Tumorsphere of GSCs transduced with shNTor shSATB2 . Scale bar : 100 μm . of diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) .

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E. Tumorsphere images of GSCs transduced with shNTor shSATB2. Scale bar: 100 μm. Quantification of the diameter of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).


G . of number of tumorspheres formed by GSCs expressing shNT or shSATB2 ( F : n = 9 ; G : n = 5 ) .

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G. Quantification of the number of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n=9; G: n=5).


H . In vitro limiting dilution of tumorsphere formations of GSCs expressing shNT or shSATB2 . Silencing SATB2 attenuated self - renewal capacity of GSCs .

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H. In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2. Silencing SATB2 attenuated the self-renewal capacity of GSCs.


A . Bioluminescent of GBM xenografts derived from luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM growth .

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A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth .


A . Bioluminescent of GBM xenografts derived from luciferase - labeled T3359 GSCs expressing NT or SATB2 shRNA . Representative on day 21 posttransplantation are shown ( n = 5 mice per group ) . Silencing SATB2 significantly delayed GBM

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A. Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n=5 mice per group). Silencing SATB2 significantly delayed GBM growth


C . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 ( shNT : n = 7 mice ; shSATB2 - 1 or shSATB2 - 2 : n = 5 mice ) . Median survival : shNT , 28 days ; shSATB2 - 1 , 48 days ; shSATB2 - 2 , 45 Days . bearing GSC - derived xenografts expressing SATB2 shRNA survived longer than control .

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C. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n=7 mice; shSATB2-1 or shSATB2-2: n=5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 Days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals.


D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . E . of SATB2 in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) .

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D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. E. Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).


D . Immunofluorescence of SATB2 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm .

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D. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.


F . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm . G . of Ki67 positive in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) .

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F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm. G. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group).


F . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 5 tumors per group ) . Scale bar : 40 μm .

F. Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=5 tumors per group). Scale bar: 40 μm.


H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm . I . of SOX2 positive in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) .

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm. I. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group).


H . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 ( n = 4 tumors per group ) . Scale bar : 40 μm .

H. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n=4 tumors per group). Scale bar: 40 μm.


A . of differentially expressed genes between SATB2 silencing H2S GSCs ( shSATB2 ) control H2S GSCs ( shNT ) . Differentially expressed genes had a 1 . 8 - fold or greater expression difference . Among differentially expressed genes , 160 are upregulated 185 are downregulated .

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A. Heatmap analysis of differentially expressed genes between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT). Differentially expressed genes had a 1.8-fold or greater expression difference. Among differentially expressed genes, 160 are upregulated and 185 are downregulated.


C . qPCR of FOXM1 SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 ( n = 3 ) .

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C. qPCR analysis of FOXM1 and SATB2 mRNA expression in GSCs transduced with shNT or shSATB2 (n=3).


D . Immunoblot of FOXM1 SATB2 expression in GSCs transduced with shNT or shSATB2 .

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D. Immunoblot analysis of FOXM1 and SATB2 expression in GSCs transduced with shNT or shSATB2.


E . Immunoblot of FOXM1 SATB2 expression in xenografts - derived from T3359 GSCs expressing shNT or shSATB2 .

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E. Immunoblot analysis of FOXM1 and SATB2 expression in tumor xenografts-derived from T3359 GSCs expressing shNT or shSATB2.


F . of FOXM1 downstream targets between SATB2 silencing H2S GSCs ( shSATB2 ) control H2S GSCs ( shNT ) from microarray . Differentially expressed genes had a 1 . 5 - fold or greater expression difference .

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F. Heatmap analysis of FOXM1 downstream targets between SATB2 silencing H2S GSCs (shSATB2) and control H2S GSCs (shNT) from microarray analysis. Differentially expressed genes had a 1.5-fold or greater expression difference.


G . qPCR of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 ( n = 3 ) .

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G. qPCR analysis of FOXM1 downstream targets in H2S GSCs transduced with shNT or shSATB2 (n=3).


A . Immunoblot of FOXM1 SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 .

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A. Immunoblot analysis of FOXM1 and SATB2 expression in T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2.


B . viability of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored proliferation impaired by SATB2 silencing .

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B. Cell viability assay of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the cell proliferation impaired by SATB2 silencing.


C . Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( n = 5 ) . Ectopic expression of FOXM1 restored tumorsphere formation of GSCs impaired by SATB2 silencing .

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C. Tumorsphere number of T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (n=5). Ectopic expression of FOXM1 restored the tumorsphere formation of GSCs impaired by SATB2 silencing.


In vivo bioluminescent of xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM growth .

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In vivo bioluminescent images of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.


E . In vivo bioluminescent or of xenografts derived from luciferase - labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 5 mice ; shNT + FOXM1 : n = 4 mice ; shSATB2 : n = 5 mice ; shSATB2 + FOXM1 : n = 5 mice ) . Representative on day 21 posttransplantation are shown . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM growth .

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E. In vivo bioluminescent images or quantification of the tumor xenografts derived from luciferase-labeled T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=5 mice; shNT+FOXM1: n=4 mice; shSATB2: n=5 mice; shSATB2+ FOXM1: n=5 mice). Representative images on day 21 posttransplantation are shown. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly restored GBM tumor growth.


F . Kaplan - Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 8 mice ; shNT + FOXM1 : n = 7 mice ; shSATB2 : n = 6 mice ; shSATB2 + FOXM1 : n = 8 mice ) . Median survival : shNT , 30 days ; shNT + FOXM1 , 28 days ; shSATB2 , 50 . 5 Days ; shSATB2 + FOXM1 , 34 days . Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated increased survival of mice bearing GSC - derived GBMs .

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F. Kaplan-Meier survival curves of mice intracranially implanted with T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=8 mice; shNT+FOXM1: n=7 mice; shSATB2: n=6 mice; shSATB2+ FOXM1: n=8 mice). Median survival: shNT, 30 days; shNT+FOXM1, 28 days; shSATB2, 50.5 Days; shSATB2+FOXM1, 34 days. Ectopic expression of FOXM1 in GSCs expressing shSATB2 markedly attenuated the increased survival of mice bearing the GSC-derived GBMs.


G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . H . of Ki67 positive in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

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G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. H. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


G . Immunofluorescence of Ki67 ( Green ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm .

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G. Immunofluorescence of Ki67 (Green) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.


I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm . J . of SOX2 positive in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) .

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm. J. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors).


I . Immunofluorescence of SOX2 ( Red ) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 ( shNT : n = 6 tumors ; shNT + FOXM1 : n = 5 tumors ; shSATB2 : n = 6 tumors ; shSATB2 + FOXM1 : n = 6 tumors ) . Scale bar : 40 μm .

I. Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs transduced with FOXM1 or vector control in combination with shNT or shSATB2 (shNT: n=6 tumors; shNT+FOXM1: n=5 tumors; shSATB2: n=6 tumors; shSATB2+ FOXM1: n=6 tumors). Scale bar: 40 μm.


B . ChIP assays with SATB2 antibody or IgG using GSCs , NSTCs NPCs . PCR primers amplified a fragment flanking MAR of FOXM1 gene locus . Note that abundant SATB2 binds to MAR of FOXM1 gene locus in GSCs .

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B. ChIP assays with the SATB2 antibody or IgG using GSCs, NSTCs and NPCs. PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Note that abundant SATB2 binds to the MAR of FOXM1 gene locus in GSCs.


C . qPCR of ChIP assays with SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking MAR of FOXM1 gene locus . Silencing SATB2 decreased its binding amount to MAR of FOXM1 gene locus .

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C. qPCR analysis of ChIP assays with the SATB2 antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 decreased its binding amount to the MAR of FOXM1 gene locus.


D . CoIP assays of endogenous interaction in GSCs . Immunoblots of precipitated or total lysates were performed using indicated antibodies . Note that SATB2 associates with endogenous CBP while not P300 .

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D. CoIP assays of endogenous protein interaction in GSCs. Immunoblots of precipitated proteins or total lysates were performed using indicated antibodies. Note that SATB2 associates with endogenous CBP while not P300.


E . qPCR of ChIP assays with CBP antibody or IgG using GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking MAR of FOXM1 gene locus . Silencing SATB2 reduced binding of CBP to MAR of FOXM1 gene locus .

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E. qPCR analysis of ChIP assays with the CBP antibody or IgG using GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced the binding of CBP to the MAR of FOXM1 gene locus.


F . qPCR of ChIP assays with indicated antibody ( AcH3K18 , AcH3K27 , AcH4 ) using T3359 GSCs transduced with shNT or shSATB2 ( n = 3 ) . PCR primers amplified a fragment flanking MAR of FOXM1 gene locus . Silencing SATB2 reduced acetylation of H3K18 , H3K27 H4 on MAR of FOXM1 locus .

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F. qPCR analysis of ChIP assays with the indicated antibody (AcH3K18, AcH3K27, AcH4) using T3359 GSCs transduced with shNT or shSATB2 (n=3). PCR primers amplified a fragment flanking the MAR of FOXM1 gene locus. Silencing SATB2 reduced acetylation of H3K18, H3K27 and H4 levels on the MAR of FOXM1 locus.


G . qPCR of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both ( n = 3 ) .

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G. qPCR analysis of FOXM1 mRNA expression in T3359 GSCs transduced with shSATB2 or shCBP or both (n=3).


H . Immunoblot of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both .

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H. Immunoblot analysis of FOXM1 expression in T3359 GSCs transduced with shSATB2 or shCBP or both.


A . viability of T3359 GSCs treated with indicated doses of C646 or vehicle control ( n = 5 ) .

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A. Cell viability of T3359 GSCs treated with indicated doses of C646 or the vehicle control (n=5).


B . Tumorsphere of T3359 GSCs treated with indicated doses of C646 or vehicle control for 6 days . Scale bar : 100 μm .

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B. Tumorsphere images of T3359 GSCs treated with indicated doses of C646 or the vehicle control for 6 days. Scale bar: 100 μm.


C . qPCR of SATB2 , FOXM1 FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or vehicle control for 24 hours ( n = 3 ) .

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C. qPCR analysis of SATB2, FOXM1 and FOXM1 downstream targets in T3359 GSCs treated with indicated doses of C646 or the vehicle control for 24 hours (n=3).


E . Bioluminescent imaging of growth in mice bearing xenografts derived from luciferase - labeled T3359 GSCs treated with C646 or vehicle control at indicated days after GSC transplantation ( n = 5 mice per group ) . F . of growth from ( E ) ( n = 5 mice per group ) .

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E. Bioluminescent imaging of tumor growth in mice bearing xenografts derived from the luciferase-labeled T3359 GSCs treated with C646 or the vehicle control at indicated days after GSC transplantation (n=5 mice per group). F. Quantification of tumor growth from (E) (n=5 mice per group).


G . Kaplan - Meier survival curves of mice bearing T3359 GSC - derived xenografts treated with C646 or vehicle control ( Vehicle control : n = 9 mice ; C646 : n = 8 mice ) . Median survival : Vehicle control , 27 days ; C646 , 33 . 5 days .

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G. Kaplan-Meier survival curves of mice bearing T3359 GSC-derived xenografts treated with C646 or the vehicle control (Vehicle control: n=9 mice; C646: n= 8 mice). Median survival: Vehicle control, 27 days; C646, 33.5 days.


H . Immunofluorescence of Ki67 ( Green ) in T3359 GSC - derived xenografts from mice treated with with C646 or vehicle control ( n = 6 tumors per group ) . Scale bar : 40 μm . I . of Ki67 positive in T3359 GSC - derived xenografts from mice treated with with C646 or vehicle control ( n = 6 tumors per group ) .

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H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm. I. Quantification of Ki67 positive cells in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group).


H . Immunofluorescence of Ki67 ( Green ) in T3359 GSC - derived xenografts from mice treated with with C646 or vehicle control ( n = 6 tumors per group ) . Scale bar : 40 μm .

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H. Immunofluorescence of Ki67 (Green) in T3359 GSC-derived xenografts from mice treated with with C646 or the vehicle control (n=6 tumors per group). Scale bar: 40 μm.


A Left , time lapse confocal of PHcrac ( red ) LimE ( green ) in waves at basal surface of a giant . Right , plot across blue dotted arrow in image " 30 sec " .

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A Left, time lapse confocal images of PHcrac (red) and LimE (green) in waves at the basal surface of a giant cell. Right, intensity plot across the blue dotted arrow in image "30 sec".


Various biosensors in excitable waves . snapshots of biosensors ( top ) plots ( bottom ) across blue dotted arrows , with black plots showing corresponding biosensors in above , red ( B ) showing PHcrac in same , Scatter plots of biosensor peak locations relative to peak of PHcrac ( C ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that peak precedes that of PHcrac along direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 for RalGDS , RBD , PKBA , , respectively . Scale bars in above represent 20 μm . Grey arrows in plots represent direction of wave propagation .

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Various biosensors in excitable waves. snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, red (B) showing PHcrac in the same cell, Scatter plots of biosensor peak locations relative to the peak of PHcrac (C) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for RalGDS, RBD, PKBA, , respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.


Various biosensors in excitable waves . D , snapshots of biosensors ( top ) plots ( bottom ) across blue dotted arrows , with black plots showing corresponding biosensors in above , green ( D ) showing LimE . E , Scatter plots of biosensor peak locations relative to peak of or first peak of LimE ( E ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that peak precedes that of LimE ) along direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 for , RacGEF1 , PAK1 - GBD , Coronin , respectively . Scale bars in above represent 20 μm . Grey arrows in plots represent direction of wave propagation .

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Various biosensors in excitable waves. D, snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, green (D) showing LimE. E, Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE (E) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of LimE) along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for , RacGEF1, PAK1-GBD, and Coronin, respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.


F Left , confocal of PHcrac ( red ) PAK1 - GBD ( green ) in same treated with LatA . Scale bars represent 10 μm . Right , temporal profiles of normalized mean intensities of PHcrac ( red ) PAK1 - GBD ( green ) ( mean ± s . e . m . , n = 18 ) in response to cAMP .

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F Left, confocal images of PHcrac (red) and PAK1-GBD (green) in the same cells treated with LatA. Scale bars represent 10 μm. Right, temporal profiles of normalized mean cytosolic intensities of PHcrac (red) and PAK1-GBD (green) (mean±s.e.m., n=18 cells) in response to cAMP.


A Confocal image of LimE in waves ( scale bar , 20 μm ) .

A Confocal image of LimE in waves (scale bar, 20 μm).


B Top , kymograph of LimE in a fixed region across which a piece of wave propagates , with arrows pointing to same . Bottom , box plot of ' s lifetime ( n = 100 ) .

B Top, kymograph of LimE in a fixed region across which a piece of wave propagates, with white arrows pointing to the same puncta. Bottom, box plot of puncta's lifetime (n=100 puncta).


H A two - dimensional snapshot of a propagating wave ( direction of propagation indicated with dashed arrow ) . A scan corresponding to STEN - CEN profile is shown on right .

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H A two-dimensional snapshot of a propagating wave (direction of propagation indicated with the dashed arrow). A line scan corresponding to the STEN-CEN profile is shown on the right.


A Time lapse confocal of PHcrac ( top ) LimE ( bottom ) in same giant ( scale bar , 20 μm ) . arrows point to wave breaking events .

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A Time lapse confocal images of PHcrac (top) and LimE (bottom) in the same giant cell (scale bar, 20 μm). White arrows point to wave breaking events.


E Time lapse confocal of LimE at basal surface of a giant , which recruits Inp54p to induced by rapamycin at time 0 . arrow ( - 45 s ) points to a nascent wave before Inp54p recruitment , while red ( 60 s ) arrow after .

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E Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits Inp54p to membrane induced by rapamycin at time 0. The white arrow (-45 s) points to a nascent wave before Inp54p recruitment, while the red (60 s) arrow after.


F Color coded overlays of LimE before after Inp54p recruitment . arrows point to nascent waves at time 0 . G Kymographs of LimE in regions outlined by dotted boxes in f .

F Color coded overlays of LimE before and after Inp54p recruitment. White arrows point to nascent waves at time 0. G Kymographs of LimE in regions outlined by dotted line boxes in f.


J Time lapse confocal of PHcrac at bottom of giant under LatA treatment , where rapamycin is added at time 0 to induce Inp54p recruitment . arrows point to wave patches right before rapamycin addition .

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J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment, where rapamycin is added at time 0 to induce Inp54p recruitment. White arrows point to wave patches right before rapamycin addition.


K Confocal image of PI5K ( left ) LimE ( right ) at bottom of same . scale bars in represent 20 μm .

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K Confocal image of PI5K (left) and LimE (right) at the bottom of the same cell. All scale bars in images represent 20 μm.


A Time lapse confocal of LimE at basal surface of a giant , which recruits PKBA to induced by rapamycin at time 0 .

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A Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.


B T - stack of LimE in region outlined by colored box in A .

B T-stack of LimE in the region outlined by the colored line box in A.


C Box plots of mean wave speed ( left ) , mean wave patch ( middle ) , average number of wave patches per frame ( right ) before after PKBA recruitment ( red bars indicate median , n = 24 ) . Error bars are from student ' s t - .

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C Box plots of mean wave speed (left), mean wave patch area (middle), and average number of wave patches per frame (right) before and after PKBA recruitment (red bars indicate median, n=24 cells). Error bars are from the student's t-test.


D Time lapse confocal of PHcrac at basal surface of a giant , which recruits PKBA to induced by rapamycin at time 0 .

D Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits PKBA to membrane induced by rapamycin at time 0.


E Confocal of PHcrac ( left ) PHcrac merged with LimE ( middle , PHcrac as red , LimE as green ) in a giant after steady PKBA recruitment . Right , plots of PHcrac ( red ) LimE ( green ) across blue box .

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E Confocal images of PHcrac (left) and PHcrac merged with LimE (middle, PHcrac as red, LimE as green) in a giant cell after steady PKBA recruitment. Right, intensity plots of PHcrac (red) and LimE (green) across the blue box.


F Kymograph of PHcrac ( red ) merged with LimE ( green ) along dashed from E .

F Kymograph of PHcrac (red) merged with LimE (green) along the white dashed line from E.


G Time lapse confocal of PHcrac at bottom of a giant treated with LatA , where rapamycin is added at time 0 to induce PKBA recruitment . scale bars above represent 20 μm .

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G Time lapse confocal images of PHcrac at the bottom of a giant cell treated with LatA, where rapamycin is added at time 0 to induce PKBA recruitment. All scale bars above represent 20 μm.


A Left , time lapse confocal of LimE in single following RacGEF1ΔN recruitment , induced by rapamycin addition at time 0 . Right , temporal profile of normalized mean LimE at cortex ( mean ± s . e . m . , n = 20 ) .

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A Left, time lapse confocal images of LimE in single cells following RacGEF1ΔN recruitment, induced by rapamycin addition at time 0. Right, temporal profile of normalized mean LimE intensity at cell cortex (mean±s.e.m., n=20 cells).


B Time lapse confocal of LimE at basal surface of a giant , which recruits RacGEF1ΔN to induced by rapamycin at time 0 .

B Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane induced by rapamycin at time 0.


C T - stack of LimE in region outlined by colored box in B .

C T-stack of LimE in the region outlined by the colored line box in B.


D Right , kymographs of LimE along a across giant after RacGEF1ΔN recruitment . Top left , confocal image with a dotted as an example used to make kymographs . arrows in image kymographs point to examples of actin . Bottom left , box plot of lifetime of ( red bar indicates median , n = 100 from 10 ) . scale bars above represent 20 μm .

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D Right, kymographs of LimE along a line across giant cells after RacGEF1ΔN recruitment. Top left, confocal image with a dotted line as an example used to make kymographs. White arrows in image and kymographs point to examples of actin puncta. Bottom left, box plot of lifetime of puncta (red bar indicates median, n=100 puncta from 10 cells). All scale bars above represent 20 μm.


A Time lapse confocal of PHcrac at basal surface of a giant , which recruits RacGEF1ΔN to at time 0 induced by rapamycin .

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A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin.


B Confocal of PHcrac ( left ) PHcrac merged with LimE ( right , PHcrac as red , LimE as green ) in a giant after RacGEF1ΔN recruitment .

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B Confocal images of PHcrac (left) and PHcrac merged with LimE (right, PHcrac as red, LimE as green) in a giant cell after RacGEF1ΔN recruitment.


C Kymograph of LimE ( top , green ) PHcrac ( bottom , red ) across dotted in B .

C Kymograph of LimE (top, green) and PHcrac (bottom, red) across the dotted line in B.


D Confocal image of RalGDS ( left ) in a giant after RacGEF1ΔN recruitment corresponding kymograph ( right ) .

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D Confocal image of RalGDS (left) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph (right).


E Time lapse confocal of PHcrac in single , which recruit RacGEF1ΔN to induced by rapamycin at time 0 . arrow points to PHcrac patches at cortex .

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E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex.


F Kymographs of PHcrac at cortex before after RacGEF1ΔN recruitment . Red dashed lines in F indicate rapamycin addition at time 0 .

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F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment. Red dashed lines in F indicate rapamycin addition at time 0.


G Temporal profiles of normalized PHcrac intensities at cortex before after RacGEF1ΔN recruitment ( mean ± s . e . m . , n = 20 ) . Red dashed lines in F G indicate rapamycin addition at time 0 .

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G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0.


H Distribution of different sizes of PHcrac patches at cortex under LatA treatment , with ( solid ) or without ( hollow ) RacGEF1ΔN recruitment ( ~ 6000 from 3 independent experiments ) .

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H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments).


I Temporal profiles of normalized intensities of PHcrac in response to 1 nM cAMP stimulation at time 0 , with ( red ) or without ( blue ) RacGEF1ΔN recruitment ( mean ± s . e . m . , n = 25 each ) .

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I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each).


J Confocal of GflB ( top ) RapGAP1 ( bottom ) in waves at basal surface of a giant .

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J Confocal images of GflB (top) and RapGAP1 (bottom) in waves at the basal surface of a giant cell.


K plots of GflB ( blue ) RapGAP1 ( orange ) across green dotted arrow in J .

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K Intensity plots of GflB (blue) and RapGAP1 (orange) across the green dotted arrow in J.


L Scatter plots of first peak distances , of GflB relative to LimE , RapGAP1 relative to LimE , RapGAP1 relative to GflB in same giant ( red lines indicate median , n = 19 each ) .

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L Scatter plots of first peak distances, of GflB relative to LimE, RapGAP1 relative to LimE, and RapGAP1 relative to GflB in the same giant cells (red lines indicate median, n=19 cells each).


M Confocal of GflB ( top ) RapGAP1 ( bottom ) at basal surface of same giant after RacGEF1ΔN recruitment .

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M Confocal images of GflB (top) and RapGAP1 (bottom) at the basal surface of the same giant cell after RacGEF1ΔN recruitment.


N kymographs of GflB ( top ) RapGAP1 ( bottom ) across green dotted lines in m . scale bars represent 20 μm .

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N kymographs of GflB (top) and RapGAP1 (bottom) across the green dotted lines in m. All scale bars represent 20 μm.


A , B Left , confocal of LimE ( top ) kymographs of cortical LimE ( bottom ) in single with PKBA ( A ) or RacGEF1ΔN ( B ) steadily recruited ( scale bars , 10 μm ) . Right , color coded overlays of LimE in single .

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A, B Left, confocal images of LimE (top) and kymographs of cortical LimE (bottom) in single cells with PKBA (A) or RacGEF1ΔN (B) steadily recruited (scale bars, 10 μm). Right, color coded overlays of LimE in single cells.


C Box plots of roundness ( left ) ( middle ) comparing wt , with RacGEF1ΔN recruited , spreading phases of oscillators after Inp54p recruitment ( n = 100 each ) . Right , box plots of coefficient of variance of with above conditions in a 10 - minute time window ( n = 30 each ) . Red bars indicate median .

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C Box plots of roundness (left) and cell area (middle) comparing wt, cells with RacGEF1ΔN recruited, and spreading phases of oscillators after Inp54p recruitment (n=100 cells each). Right, box plots of coefficient of variance of cell area with the above conditions in a 10-minute time window (n=30 cells each). Red bars indicate median.


D Phalloidin of after perturbations . Maximal projections are shown .

D Phalloidin staining of cells after perturbations. Maximal intensity projections are shown.


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

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

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

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

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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).


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

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

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

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

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

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

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

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

O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O 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 BrdU antibody in MOE of NC miR - 200b / a KD mice . arrows indicate BrdU + , yellow arrows indicate positive for both BrdU EdU . G ' is shown at higher magnification of boxed region , as three channel merged ( left image ) separated channel ( middle right ) . G : Scale bars , 20 μm ; G ' : Scale bars , 2 μm .

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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 with Cas3 antibody in MOE of NC miR - 200b / a KD mice . Scale bars , 20 μm . G of number of Cas3 + in MOE of NC miR - 200b / a KD mice ( n = 4 mice each group ; data represent mean ± ; * * * p < 0 . 001 ; Student ' s t - ) .

O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O 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 number of differentially expressed olfrs from sequencing in MOE of NC miR - 200b / a KD mice .

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

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


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

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

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

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of TET3 level in 3T3 - L1 treated with miR - 200a inhibitor , miR - 200a mimic or control ( n = 3 from three independent experiments ) . Actin served as a loading control . molecular of each band is indicated at right .

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

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

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

O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 of TET3 level in MOE of NC miR - 200b / a KD mice ( n = 3 mice each group ) . Actin served as a loading control . molecular of each band is indicated at right . blue number shows of average of TET3 / Actin .

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

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


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

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

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


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

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

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


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

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

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

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 of 5hmC in MOE of NC miR - 200b / a knockdown mice . N ' is shown at higher magnification of 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.


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

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

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O 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 resident / intruder experiment showed that attack frequency ( C ) duration ( D ) of miR - 200b / a + TET3 DKD mice were significantly improved compared with those of 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 mean ± ; C : F = 17 . 84 , D : F = 12 . 06 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; One - way ANOVA Bonferroni pairwise comparisons ) ] .

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

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 of - mating behaviors showed that frequency ( E ) duration ( F ) of mating to of miR - 200b / a + TET3 DKD mice were significantly reduced compared with those of their NC controls were not dramatically different compared with those of 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 mean ± ; E : F = 15 . 62 , F : F = 15 . 56 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA Bonferroni pairwise comparisons ) .

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

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 for Mash1 , Tuj1 , Cas3 Ki67 in MOE of NC , miR - 200b / a KD , miR - 200b / a + TET3 DKD mice . Scale bars : 20 μm . H - K of numbers of Mash1 + ( H ) , Tuj1 + ( I ) , Cas3 + ( J ) Ki67 + ( K ) in MOE of NC , miR - 200b / a KD , miR - 200b / a + TET3 DKD mice ( n = 4 mice each group ; data represent mean ± ; 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 Bonferroni pairwise comparisons ) .

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

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

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

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 of level in MOE of NC , miR - 200b / a KD 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O 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 for in MOE of NC , miR - 200b / a KD , miR - 200b / a TET3 DKD mice . Scale bars : 20 μm .

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

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


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

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

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

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

Representative IF 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 of number of costained + Mash1 + in MOE of NC miR - 200b / a KD mice ( n = 4 mice each group ; data represent mean ± ; * * p < 0 . 01 ; Student ' s t - ) .

O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O 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 were immunoprecipitated with polyclonal antibodies against TET3 . Input ( 100 ug ) immunoprecipitates were subjected to immunoblot with indicated antibodies . IgG antibodies were used as negative control .

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

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 were immunoprecipitated with polyclonal antibodies against . Input ( 100 ug ) immunoprecipitates were subjected to immunoblot with indicated antibodies . IgG antibodies were used as 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

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

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

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


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

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 for Mash1 , Tuj1 , Cas3 Ki67 in MOE of NC , miR - 200b / a KD , miR - 200b / a + DKD mice . Scale bars : 20 μm . F - I of number of Mash1 + ( F ) , Tuj1 + ( G ) , Cas3 + ( H ) Ki67 + ( I ) in MOE of NC , miR - 200b / a KD , miR - 200b / a + DKD mice ( n = 4 mice each group ; data represent mean ± ; 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 Bonferroni pairwise comparisons ) .

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

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).


A ) oxLDL plasma in ACM HC ( n = 36 ; Mann - Whitney ) . B ) oxLDL plasma in mutated ACM ( n = 7 ) NON ACM relatives , carriers of same causative mutation ( n = 9 ; Mann - Whitney ) . C ) oxLDL plasma in ACM carriers of a PKP2 mutation ACM carriers of other desmosomal or non desmosomal mutations , or gene elusive ( n = 10 vs . n = 26 ; One - Way ANOVA ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

A) oxLDL plasma concentration in ACM patients and HC (n=36; Mann-Whitney test). B) oxLDL plasma concentration in mutated ACM (n=7) and NON ACM relatives, carriers of the same causative mutation (n=9; Mann-Whitney test). C) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n=10 vs. n=26; One-Way ANOVA). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


D ) Representative of MDA immunostaining ( green ) on ACM HC ventricular tissue sections relative ( n = 4 biological replicates ; Two - Student ' s t - ) . Nuclei are counterstained with Hoechst33342 ( blue ) . E ) Representative of CD36 immunostaining on ACM HC ventricular tissue sections relative ( n = 4 biological replicates ; Two - Student ' s t - ) . Nuclei are counterstained with Hoechst33342 ( blue ) . Data information : mean ± . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

D) Representative images of MDA immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). E) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.


B ) Left panels : representative of three cases of ACM in Steady State Free Procession sequences at cardiac MRI . On left a case with RV dilation , regional bulging of RV wall without fat infiltration , oxLDL below cut - off ; in center on right , two cases with fat infiltration in right or left ventricle wall ( arrow ) , respectively , oxLDL above cut - off . Right panel : of myocardial fat mass of two ACM subpopulations ( above or below cut - off ) whose MRI was available for re - ( n = 14 oxLDL < 86ng / ml , n = 25 oxLDL > 86ng / ml ; Two - Student ' s t - ) .

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

B) Left panels: representative images of three cases of ACM in Steady State Free Procession sequences at cardiac MRI. On the left a case with RV dilation, regional bulging of the RV wall without fat infiltration, and oxLDL levels below the cut-off; in the center and on the right, two cases with fat infiltration in the right or left ventricle wall (white arrow), respectively, and oxLDL levels above the cut-off. Right panel: quantification of the myocardial fat mass of the two ACM subpopulations (above or below the cut-off) whose MRI was available for re-analysis (n=14 oxLDL<86ng/ml, n=25 oxLDL>86ng/ml; Two-tailed Student's t-test).


G ) Kaplan - Meier of actual MAE free survival of belonging to two ACM cohort subgroups in first 5 - year follow - up ( n = 26 oxLDL < 86ng / ml vs . n = 41oxLDL > 86ng / ml ; Log - Rank p < 0 . 0001 ; HR = 0 . 223 [ 0 . 116 - 0 . 428 ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier analysis of actual MAE free survival of patient belonging to the two ACM cohort subgroups in the first 5-year follow-up (n=26 oxLDL<86ng/ml vs. n=41oxLDL>86ng/ml; Log-Rank p<0.0001; HR=0.223[0.116-0.428]).


B ) Left panels : representative of MDA immunostaining ( green ) on ACM HC C - MSC in GM . Nuclei are counterstained with Hoechst33342 ( blue ) . Right panel : image ( n = 4 biological replicates ACM , n = 5 biological replicates HC ; Two - Student ' s t - ) . Data information : mean ± . * p < 0 . 05 , * * p < 0 . 01 .

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

B) Left panels: representative images of MDA immunostaining (green) on ACM and HC C-MSC in GM. Nuclei are counterstained with Hoechst33342 (blue). Right panel: image quantification (n=4 biological replicates ACM, n=5 biological replicates HC; Two-tailed Student's t-test). Data information: mean ± SEM. * p<0.05, ** p<0.01.


D ) Top panel : representative of Western Blot of extracted from ACM HC C - MSC cultured in GM , hybridized with anti - CD36 anti - PPARγ antibodies . Immunostaining of housekeeping GAPDH is shown for normalization . Bottom panel : d . a . of PPARγ ( n = 18 biological replicates ) CD36 ( n = 17 vs . n = 16 biological replicates ) , normalized on GAPDH ( Mann - Whitney ) . Data information : mean ± . * p < 0 . 05 , * * 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 B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Top panel: representative images of Western Blot analysis of proteins extracted from ACM and HC C-MSC cultured in GM, hybridized with anti-CD36 and anti-PPARγ antibodies. Immunostaining of the housekeeping GAPDH is shown for normalization. Bottom panel: d.a. of PPARγ (n=18 biological replicates) and CD36 (n=17 vs. n=16 biological replicates) levels, normalized on GAPDH (Mann-Whitney test). Data information: mean ± SEM. * p<0.05, ** p<0.01.


F ) Top panels : representative of internalization of oxLDL ( red ) in HC ACM , cultured either in GM or in AM , subjected to 10μg / ml DiI - oxLDL treatment . Nuclei are counterstained with Hoechst33342 ( blue ) . Bottom panel : of relative mean DiI for each sample , measured by FACS ( n = 3 biological replicates ; Two - Way Anova ) . Data information : mean ± . * p < 0 . 05 , * * p < 0 . 01 .

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

F) Top panels: representative images of internalization of oxLDL (red) in HC and ACM cells, cultured either in GM or in AM, and subjected to 10μg/ml DiI-oxLDL treatment. Nuclei are counterstained with Hoechst33342 (blue). Bottom panel: quantification of the relative mean DiI fluorescence for each sample, measured by FACS analysis (n=3 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01.


A ) Left panels : representative of ORO on ACM HC C - MSC in AM supplemented or not with 150µg / ml oxLDL . Middle panel : image ( n = 11 biological replicates ; Two - Way Anova ) . Right panels : representative of Western Blot of CD36 , PPARγ GAPDH expression of ACM HC C - MSC extracts in AM supplemented or not with 150µg / ml oxLDL ( n = 5 biological replicates ) d . a . normalized on house - keeping GAPDH ( Two - Way Anova ) . Data information : mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 150µg/ml oxLDL. Middle panel: image quantification (n=11 biological replicates; Two-Way Anova). Right panels: representative images of Western Blot of CD36, PPARγ and GAPDH protein expression of ACM and HC C-MSC protein extracts in AM supplemented or not with 150µg/ml oxLDL (n=5 biological replicates) and d.a. normalized on the house-keeping GAPDH (Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


B ) Left panels : representative of ORO on ACM HC C - MSC in AM supplemented or not with 20μg / ml 13HODE , 5mmol / L NAC or both . Right panel : image ( n = 13 biological replicates ; Two - Way Anova ) . Data information : mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panels: representative images of ORO staining on ACM and HC C-MSC in AM supplemented or not with 20μg/ml 13HODE, 5mmol/L NAC or both. Right panel: image quantification (n=13 biological replicates; Two-Way Anova). Data information: mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.