Immunolabeling of E14 . 5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1 - resistant Ttyh1 ( Ttyh1res ) expression vector at E13 . 5 using anti - GFP Alexa Fluor 488 - conjugated secondary antibodies . DAPI counterstain is shown in blue . Scale bar : 100 μm . of ( I )

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Immunolabeling of E14.5 brain sections electroporated in utero with shTtyh1 alone or in combination with a shTtyh1-resistant Ttyh1 (Ttyh1res) expression vector at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. The DAPI nuclear counterstain is shown in blue. Scale bar: 100 μm. Quantification of (I)


Two days after E14 . 5 neural progenitor were transduced with retroviral vectors expressing ( A ) Ttyh1 mRNA expression of each indicated Notch target gene were measured by

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Two days after E14.5 primary neural progenitor cells were transduced with retroviral vectors expressing (A) Ttyh1 mRNA expression levels of each indicated Notch target gene were measured by qPCR


Two days after E14 . 5 neural progenitor were transduced with shTtyh1 , mRNA expression of each indicated Notch target gene were measured by

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Two days after E14.5 primary neural progenitor cells were transduced with shTtyh1, mRNA expression levels of each indicated Notch target gene were measured by qPCR


Effects of dominant negative MAML1 ( dnMAML1 ) on Ttyh1 - enhanced Notch target gene expression were tested by qPCR using E14 . 5 neural progenitors at 2 days posttransduction ( n = 4 ) .

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Effects of dominant negative MAML1 (dnMAML1) on Ttyh1-enhanced Notch target gene expression were tested by qPCR using E14.5 primary neural progenitors at 2 days posttransduction (n = 4).


dnMAML1 plasmid was introduced with Ttyh1 vector into embryonic brains at E13 . 5 localization was analyzed at E15 . 5 by anti - GFP immunostaining . GFP DAPI signals are shown in green blue , respectively . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm . E of GFP + positions in ( D ) ( n = 3 ) .

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The dnMAML1 plasmid was introduced with the Ttyh1 vector into embryonic brains at E13.5 and cell localization was analyzed at E15.5 by anti-GFP immunostaining. GFP and DAPI signals are shown in green and blue, respectively. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm. E Quantification of GFP+ cell positions in (D) (n = 3).


NICD plasmid was electroporated with Ttyh1 knockdown vector ( shTtyh1 ) into embryonic brains at E13 . 5 localization was analyzed at E14 . 5 by anti - GFP immunostaining . Scale bar : 100 μm . G of GFP + positions in ( F ) ( n = 3 ) .

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The NICD plasmid was electroporated with the Ttyh1 knockdown vector (shTtyh1) into embryonic brains at E13.5 and cell localization was analyzed at E14.5 by anti-GFP immunostaining. Scale bar: 100 μm. G Quantification of GFP+ cell positions in (F) (n = 3).


Effects of Ttyh1 expression on generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor were analyzed by Western blotting using an anti - NICD antibody . of band

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Effects of Ttyh1 expression on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. Quantification of band intensities


Effects of Ttyh1 knockdown ( C ) on generation of NICD at 2 days posttransduction of E14 . 5 neural progenitor were analyzed by Western blotting using an anti - NICD antibody . ( B , D ) of band

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Effects of Ttyh1 knockdown (C) on the generation of NICD at 2 days posttransduction of E14.5 neural progenitor cells were analyzed by Western blotting using an anti-NICD antibody. (B, D) Quantification of band intensities


Ttyh1 - transduced E14 . 5 neural progenitors were incubated with DAPT for 2 days , then were harvested lysates were used for luciferase ( n = 3 ) .

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Ttyh1-transduced E14.5 neural progenitors were incubated with DAPT for 2 days, then cells were harvested and lysates were used for luciferase assay (n = 3).


Effects of Ttyh1 - knockdown on γ - secretase activity was also assessed using E14 . 5 neural progenitor

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Effects of Ttyh1-knockdown on γ-secretase activity was also assessed using E14.5 neural progenitor cells


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in presence or absence of DAPT , samples were prepared for Western blotting using anti - NICD

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Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for Western blotting using anti-NICD antibody


Ttyh1 - transduced E14 . 5 neural progenitors were incubated for 2 days in presence or absence of DAPT , samples were prepared for qPCR for Notch target genes ( I )

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Ttyh1-transduced E14.5 neural progenitors were incubated for 2 days in the presence or absence of DAPT, and samples were prepared for qPCR for Notch target genes (I)


indicated plasmids were electroporated into E13 . 5 brains , brain samples were harvested at E15 . 5 for anti - GFP immunolabeling . GFP DAPI signals are shown in green blue , respectively . of GFP + localization in ( J ) dnPS1 , dominant negative presenilin 1 ; VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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The indicated plasmids were electroporated into E13.5 brains, and brain samples were harvested at E15.5 for anti-GFP immunolabeling. GFP and DAPI signals are shown in green and blue, respectively. Quantification of GFP+ cell localization in (J) dnPS1, dominant negative presenilin 1; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


Effects of ion selectivity - perturbing R371Q mutation in Ttyh1 E14 . 5 neural progenitor were transduced with retroviral vectors expressing each indicated gene , 2 days posttransduction , were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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Effects of the ion selectivity-perturbing R371Q mutation in Ttyh1 E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Effects of ion selectivity - perturbing R371Q mutation in other Ttyh family members on Notch - dependent transcription . E14 . 5 neural progenitor were transduced with retroviral vectors expressing each indicated gene , 2 days posttransduction , were harvested for qPCR analyses of Notch target mRNAs ( n = 3 ) .

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Effects of the ion selectivity-perturbing R371Q mutation in other Ttyh family members on Notch-dependent transcription. E14.5 neural progenitor cells were transduced with retroviral vectors expressing each indicated gene, and 2 days posttransduction, cells were harvested for qPCR analyses of Notch target mRNAs (n = 3).


Immunolabeling of E15 . 5 brain sections electroporated in utero with indicated plasmids at E13 . 5 using anti - GFP Alexa Fluor 488 - conjugated secondary antibodies . GFP immunofluorescence ( green ) was merged with DAPI - counterstained ( blue ) . ( D ) of GFP + localization in ( C ) ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Immunolabeling of E15.5 brain sections electroporated in utero with indicated plasmids at E13.5 using anti-GFP and Alexa Fluor 488-conjugated secondary antibodies. GFP immunofluorescence (green) was merged with DAPI-counterstained images (blue). (D) Quantification of GFP+ cell localization in (C) (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


series of Ttyh1 mutants with deletions in C - terminal tail region , ( F ) their effects on distribution assessed by intraventricular in utero electroporation at E13 . 5 followed by immunolabeling at E15 . 5 using anti - GFP antibody . Scale bar : 100 μm .

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series of Ttyh1 mutants with deletions in the C-terminal cytoplasmic tail region , and (F) their effects on cell distribution assessed by intraventricular in utero electroporation at E13.5 followed by immunolabeling at E15.5 using anti-GFP antibody. Scale bar: 100 μm.


Representative Western blots showing coimmunoprecipitation ( coIP ) of HA - tagged Rer1 with Myc - tagged Ttyh1 using HEK293T . WCL , whole - lysates .

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Representative Western blots showing coimmunoprecipitation (coIP) of HA-tagged Rer1 with Myc-tagged Ttyh1 using the HEK293T cell line. WCL, whole-cell lysates.


Venus - bimolecular complementation of Ttyh1 Rer1 interaction ( green ) in living COS - 7 by microscopy . Ttyh1 Rer1 were tagged with N - C - terminal fragments of Venus ( VN VC ) , respectively . DsRed2 - ER was cotransfected to label endoplasmic reticulum ( ER , red ) . Scale bar : 25 μm .

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Venus fluorescent protein-based bimolecular fluorescence complementation analysis of Ttyh1 and Rer1 interaction (green) in living COS-7 cells by fluorescence microscopy. Ttyh1 and Rer1 were tagged with N- and C-terminal fragments of the Venus fluorescent protein (VN and VC), respectively. DsRed2-ER was cotransfected to label the endoplasmic reticulum (ER, red). Scale bar: 25 μm.


CoIP assays were performed with anti - Ttyh1 anti - Rer1 antibodies to show endogenous interaction between Ttyh1 Rer1 in U373MG .

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CoIP assays were performed with anti-Ttyh1 and anti-Rer1 antibodies to show endogenous interaction between Ttyh1 and Rer1 in U373MG cells.


Western blot analyses of endogenous Rer1 were performed at 2 days posttransduction using E14 . 5 neural progenitor transduced with retroviral vectors expressing ( D ) Ttyh1 of relative band

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Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing (D) Ttyh1 Quantification of relative band intensities


D - G Western blot analyses of endogenous Rer1 were performed at 2 days posttransduction using E14 . 5 neural progenitor transduced with retroviral vectors expressing shTtyh1 . of relative band

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D-G Western blot analyses of endogenous Rer1 proteins were performed at 2 days posttransduction using E14.5 primary neural progenitor cells transduced with retroviral vectors expressing shTtyh1. Quantification of relative band intensities


extracts from neural progenitor transduced with Ttyh1 retroviruses in presence of DMSO vehicle or a proteasome inhibitor lactacystin ( 10 μM ) , were immunoblotted with an antibody to Rer1 .

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Cell extracts from primary neural progenitor cells transduced with Ttyh1 retroviruses in the presence of DMSO vehicle or a proteasome inhibitor lactacystin (10 μM), were immunoblotted with an antibody to Rer1.


HEK293T were transfected with Ttyh1 - Myc Rer1 - HA expression plasmids , after 24 h , were treated with cycloheximide ( CHX ) ( 100 μg / ml ) then harvested at indicated time intervals . lysates were analyzed by Western blotting using anti - HA antibody . ( J ) of band intensities in ( I ) ( n = 3 ) .

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HEK293T cells were transfected with Ttyh1-Myc and Rer1-HA expression plasmids, and after 24 h, cells were treated with cycloheximide (CHX) (100 μg/ml) and then harvested at the indicated time intervals. Cell lysates were analyzed by Western blotting using anti-HA antibody. (J) Quantification of band intensities in (I) (n = 3).


γ - secretase - dependent luciferase activity was measured using neural progenitor transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction ( n = 3 ) .

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γ-secretase-dependent luciferase activity was measured using primary neural progenitor cells transduced with Ttyh1 with or without Rer1 retroviral vectors at 2 days posttransduction (n = 3).


Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13 . 5 brains subsequent GFP immunofluorescence of E15 . 5 brain sections . GFP DAPI signals are shown in green blue , respectively . ( M ) Distribution of GFP + in each brain layer in ( L ) was quantified ( n = 3 ) . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bar : 100 μm .

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Effects of Rer1 expression on Ttyh1 activity in vivo were examined by in utero electroporation into E13.5 brains and subsequent GFP immunofluorescence of E15.5 brain sections. GFP and DAPI signals are shown in green and blue, respectively. (M) Distribution of GFP+ cells in each brain layer in (L) was quantified (n = 3). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar: 100 μm.


( b , c ) Effects of miR - 212 miR - 132 precursors inhibitors ( anti ) on cardiomyocyte as compared with effects of scrambled ( Scr ) controls ( n = 5 - 13 ) . Representative used for of cardiomyocyte are shown in c .

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(b,c) Effects of miR-212 and miR-132 precursors and inhibitors (anti) on cardiomyocyte cell size as compared with the effects of scrambled (Scr) controls (n=5-13). Representative images used for quantification of cardiomyocyte cell size are shown in c.


( d ) Effects of various pro - hypertrophic stimuli on miR - 212 miR - 132 expression in neonatal cardiomyocytes ( n = 6 - 10 ) .

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(d) Effects of various pro-hypertrophic stimuli on miR-212 and miR-132 expression in neonatal cardiomyocytes (n=6-10).


( e ) miR - 212 miR - 132 expression during pressure - induced left ventricular hypertrophy 3 21 days after transaortic constriction ( TAC ) surgery of mice ( n = 4 ) .

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(e) miR-212 and miR-132 expression levels during pressure-induced left ventricular hypertrophy 3 and 21 days after transaortic constriction (TAC) surgery of mice (n=4).


( f ) Cardiomyocyte diameters after Sham operation or 3 , 14 35 days after TAC ( n = 4 per group ) .

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(f) Cardiomyocyte diameters after Sham operation or 3, 14 and 35 days after TAC (n=4 per group).


( g , h ) miR - 212 miR - 132 expression ( normalized to sno - 202 ) in fractionated cardiomyocytes cardiac fibroblasts derived from adult mice after 3 35 days of TAC . values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . Scale bar in c represents 50 μm . ACTN2 , α - cardiac actinin ; Ang II , angiotensin - 2 ; a . u . , arbitrary unit ; d , day ; DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; NS , no significant difference ; P / I , phenylephrine / isoprenaline .

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(g,h) miR-212 and miR-132 expression (normalized to sno-202 levels) in fractionated cardiomyocytes and cardiac fibroblasts derived from adult mice after 3 and 35 days of TAC. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bar in c represents 50 μm. ACTN2, α-cardiac actinin; Ang II, angiotensin-2; a.u., arbitrary unit; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; NS, no significant difference; P/I, phenylephrine/isoprenaline.


( b ) Expression of miR - 212 miR - 132 in heart samples of wild - type ( WT ) miR - 212 / 132 transgenic ( TG ) mice as assayed by RT - PCR against mature forms of corresponding microRNAs . Rnu6b was used as housekeeping control .

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(b) Expression levels of miR-212 and miR-132 in heart samples of individual wild-type (WT) and miR-212/132 transgenic (TG) mice as assayed by RT-PCR analysis against the mature forms of corresponding microRNAs. Rnu6b was used as housekeeping control.


( c ) Survival rate of two different miR - 212 / 132 TG mouse families ( TG - Fam23 , TG - Fam43 ) versus WT controls was analysed by Kaplan - Meier survival ( n = 87 , 65 53 for WT , TG - Fam23 TG - Fam43 , respectively ) .

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(c) Survival rate of two different miR-212/132 TG mouse families (TG-Fam23, TG-Fam43) versus WT controls was analysed by Kaplan-Meier survival assay (n=87, 65 and 53 for WT, TG-Fam23 and TG-Fam43, respectively).


( d ) of explanted hearts from TG WT mice at 10 after birth . Scale bar , 1 cm .

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(d) Morphology of explanted hearts from TG and WT mice at 10 weeks after birth. Scale bar, 1 cm.


( e , f ) Heart - to - - ratios ( e ) cardiomyocyte diameter ( f ) in 8 - week - old α - MHC - miR - 212 / 132 transgenic mice compared with their wild - type ( n = 5 - 7 ) . Scale bar , 50 μm .

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(e,f) Heart-to-body-weight ratios (e) and cardiomyocyte diameter (f) in 8-week-old α-MHC-miR-212/132 transgenic mice compared with their wild-type littermates (n=5-7). Scale bar, 50 μm.


( g - i ) Echocardiography of cardiac dimensions function for α - MHC - miR - 212 / 132 TG mice WT controls ( n = 16 - 18 ) ( g ) end - systolic , ( h ) end - diastolic , ( i ) fractional shortening . values in e - i represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; WGA , wheat germ agglutinin ( stain ) .

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(g-i) Echocardiography analysis of cardiac dimensions and function for α-MHC-miR-212/132 TG mice and WT controls (n=16-18) (g) end-systolic area, (h) end-diastolic area, (i) fractional shortening. All values in e-i represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin (membrane stain).


( a ) Heart - to - - ratios for 12 - week - old miR - 212 / 132 null ( KO ) wild - type ( WT ) mice ( n = 5 - 6 ) .

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(a) Heart-to-body-weight ratios for 12-week-old miR-212/132 null (KO) and wild-type (WT) mice (n=5-6).


( b ) Cardiomyocyte of neonatal miR - 212 / 132 null WT mice ( n = 5 - 6 isolations ) .

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(b) Cardiomyocyte cell size of neonatal miR-212/132 null and WT mice (n=5-6 isolations).


( c - f ) Heart - to - - ratios ( c ) in Sham - operated WT mice miR - 212 / 132 null WT mice3 after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm .

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(c-f) Heart-to-body-weight ratios (c) in Sham-operated WT mice and miR-212/132 null and WT mice3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.


( c - f ) cardiomyocyte diameter ( d ) , cardiac fibrosis ( e ) in Sham - operated WT mice miR - 212 / 132 null WT mice3 after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm .

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(c-f) cardiomyocyte diameter (d), cardiac fibrosis (e) in Sham-operated WT mice and miR-212/132 null and WT mice3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.


( c - f ) lung wet ( f ) in Sham - operated WT mice miR - 212 / 132 null WT mice 3 after TAC surgery ( n = 4 - 7 ) . Scale bar , 50 μm .

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(c-f) lung wet weight (f) in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC surgery (n=4-7). Scale bar, 50 μm.


( g - i ) Echocardiography of cardiac dimensions function in Sham - operated WT mice miR - 212 / 132 null WT mice 3 after TAC ( n = 4 - 11 ) . ( g ) End - diastolic , ( h ) end - systolic ( i ) fractional shortening . values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 compared with WT TAC ; # # # P0 . 005 compared with WT TAC ; PSR , picrosirius red ( collagen stain ) . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; WGA , wheat germ agglutinin .

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(g-i) Echocardiography analysis of cardiac dimensions and function in Sham-operated WT mice and miR-212/132 null and WT mice 3 weeks after TAC (n=4-11). (g) End-diastolic area, (h) end-systolic area and (i) fractional shortening. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 compared with WT TAC; ###P0.005 compared with WT TAC; PSR, picrosirius red (collagen stain). DAPI, 4′,6-diamidino-2-phenylindole; WGA, wheat germ agglutinin.


( a ) Luciferase activity upon cotransfection of a luciferase construct containing wild - type ( WT ) or mutated ( Mut . ) 3 ′ UTR of FoxO3 with scrambled ( Scr ) control ( Ctrl ) , pre - miR - 132 or pre - miR - 212 ( n = 9 ) .

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

(a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.) 3′UTR of FoxO3 with scrambled (Scr) control (Ctrl), pre-miR-132 or pre-miR-212 (n=9).


( b , c ) Expression of FoxO3 on mRNA ( b ) ( c ) in hearts of WT α - MHC - miR - 212 / - 132 transgenic ( TG ) mice . M : marker ( n = 9 - 13 ) .

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(b,c) Expression levels of FoxO3 on mRNA (b) and protein levels (c) in hearts of WT and α-MHC-miR-212/-132 transgenic (TG) mice. M: Size marker (n=9-13).


( d ) FoxO3 mRNA in neonatal rat cardiomyocytes transfected with Scr Ctrl , anti - miR - 212 anti - miR - 132 after phenylephrine ( PE , 10 μM ) treatment ( n = 6 - 9 ; P - values against Scr + PE ) .

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

(d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6-9; P-values against Scr+PE).


( e - g ) Expression of atrogin - 1 ( e ) Mcip1 ( f ) calcineurin phosphatase activity ( g ) in hearts of WT α - MHC - miR - 212 / 132 TG mice ( n = 5 - 9 ) .

O 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 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

(e-g) Expression levels of atrogin-1 (e) and Mcip1 (f) and calcineurin phosphatase activity levels (g) in hearts of WT and α-MHC-miR-212/132 TG mice (n=5-9).


( h ) Luciferase activity showing NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl , pre - miR - 132 or pre - miR - 212 ( n = 5 ) .

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

(h) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl, pre-miR-132 or pre-miR-212 (n=5).


( i , j ) Mcip1 . 4 ( i ) atrogin - 1 ( j ) mRNA in WT miR - 212 / 132 null ( KO ) mice 3 after transaortic constriction ( TAC ) or Sham operation ( n = 5 - 7 per group ) . values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . # P0 . 05 compared with WT TAC . FC , fold change .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,j) Mcip1.4 (i) and atrogin-1 (j) mRNA levels in WT and miR-212/132 null (KO) mice 3 weeks after transaortic constriction (TAC) or Sham operation (n=5-7 per group). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. #P0.05 compared with WT TAC. FC, fold change.


( a ) mRNA expression of autophagic marker genes in hearts of wild - type ( WT ) α - MHC - miR - 212 / 132 transgenic ( TG ) mice ( n = 9 - 10 ) .

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(a) mRNA expression levels of autophagic marker genes in hearts of wild-type (WT) and α-MHC-miR-212/132 transgenic (TG) mice (n=9-10).


( b , c ) of LC3II to LC3I ( b ) p62 ( c ) in WT , miR - 212 / 132 null ( KO ) α - MHC miR - 212 / 132 TG mice . M : marker ( n = 4 - 12 ) .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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,c) Ratio of LC3II to LC3I (b) and p62 protein levels (c) in WT, miR-212/132 null (KO) and α-MHC miR-212/132 TG mice. M: Size marker (n=4-12).


( d , e ) Representative ( d ) ( e ) of LC3 : mCherry ( in red ) in control miR - 212 / 132 - overexpressing TG H9c2 under normal serum / glucose - deprivation conditions ( n = 30 ) . Nuclei are stained in blue with Hoechst33342 .

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(d,e) Representative images (d) and quantification (e) of LC3:mCherry puncta (in red) in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=30). Nuclei are stained in blue with Hoechst33342.


( f , g ) Representative electron microscopy ( f ) ( g ) of autophagic vacuoles in control miR - 212 / 132 - overexpressing TG H9c2 under normal serum / glucose - deprivation conditions ( n = 20 ) .

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(f,g) Representative electron microscopy images (f) and quantification (g) of autophagic vacuoles in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=20).


( h , i ) Representative FACS plots ( h ) ( i ) of increase of autophagic flux in control miR - 212 / 132 - overexpressing TG H9c2 under normal serum / glucose - deprivation conditions ( n = 3 - 4 experiments ) . values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . Scale bars , 50 μm in d 2 μm in f . FC , fold change .

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(h,i) Representative FACS plots (h) and quantification (i) of percent increase of autophagic flux in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=3-4 experiments). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bars, 50 μm in d and 2 μm in f. FC, fold change.


( a ) Schematic representation of starvation experiment in mice . grey parts on time represent day night phases , respectively . arrow shows start duration of starvation indicated time points of starvation are when are scored for conditioning index .

O O O O O O O O O O O O O O O O O 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) Schematic representation of starvation experiment in mice. The white and grey parts on the time line represent the day and night phases, respectively. The arrow shows the start and duration of the starvation and the indicated time points of starvation are when animals are scored for body conditioning index.


( b ) LC3II to LC3I ratios in wild - type ( WT ) mice fed with normal diet WT , miR - 212 / 132 − / − null α - MHC miR - 212 / 132 transgenic ( TG ) mice under starvation for 31 h ( n = 4 ) .

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 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

(b) LC3II to LC3I ratios in wild-type (WT) mice fed with normal diet and WT, miR-212/132−/− null and α-MHC miR-212/132 transgenic (TG) mice under starvation for 31 h (n=4).


( c ) Electron microscopy from ultrathin sections of resin - embedded heart biopsies of fed starved WT , miR - 212 / 132 null ( KO ) cardiomyocyte - specific miR - 212 / 132 - overexpressing ( TG ) mice . spots around mitochondria ( grey structures ) are autophagic vacuoles . electron - dense black spots shown with arrows are autophagosomes . Scale bars , 4 μm .

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 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

(c) Electron microscopy from ultrathin sections of resin-embedded heart biopsies of fed and starved WT, miR-212/132 null (KO) and cardiomyocyte-specific miR-212/132-overexpressing (TG) mice. White spots around the mitochondria (dark grey structures) are autophagic vacuoles. The electron-dense black spots shown with white arrows are autophagosomes. Scale bars, 4 μm.


( d ) p - mTOR / mTOR ratios in WT miR - 212 / 132 TG H9c2 24 h after normal starvation ( serum / glucose - deprived ) conditions ( n = 6 ) .

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(d) p-mTOR/mTOR ratios in WT and miR-212/132 TG H9c2 cells 24 h after normal and starvation (serum/glucose-deprived) conditions (n=6).


( e ) p - mTOR / mTOR ratios in hearts of WT α - MHC miR - 212 / 132 TG mice fed with normal diet or 31 h after starvation ( n = 4 ) . M , marker . values represent mean ± s . e . m . * * P0 . 01 ; * * * P0 . 005 ; # P = 0 . 11 .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) p-mTOR/mTOR ratios in hearts of WT and α-MHC miR-212/132 TG mice fed with normal diet or 31 h after starvation (n=4). M, size marker. All values represent mean±s.e.m. **P0.01; ***P0.005; #P=0.11.


( a - c ) Heart - to - - ratios ( a ) , cardiomyocyte diameters ( b ) cardiac fibrosis ( c ) in Sham - operated mice mice treated with intravenous injection of either scrambled control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) after TAC . These mice were analysed 3 after TAC ( n = 4 - 11 ) . PSR , picrosirius red .

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

(a-c) Heart-to-body-weight ratios (a), cardiomyocyte diameters (b) and cardiac fibrosis (c) in Sham-operated mice and mice treated with intravenous injection of either scrambled control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC (n=4-11). PSR, picrosirius red staining.


( d - f ) Echocardiography of cardiac dimensions function in Sham - operated mice mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) after TAC . These mice were analysed 3 after TAC for ( d ) fractional shortening , ( e ) end - diastolic ( f ) end - systolic ( n = 4 - 9 ) .

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

(d-f) Echocardiography analysis of cardiac dimensions and function in Sham-operated mice and mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC for (d) fractional shortening, (e) end-diastolic area and (f) end-systolic area (n=4-9).


( g - i ) Cardiac FoxO3 in mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) 3 after TAC treatment ( n = 4 - 8 ) . M : marker . values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 against TAC - control ; # # P0 . 01 against TAC - control ; # # # P0 . 005 against TAC - control . Scale bars , 50 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; WGA , wheat germ agglutinin .

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

(g-i) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.


( g - i ) calcineurin activity ( h ) Mcip1 . 4 mRNA ( i ) in mice treated with intravenous injection of either control ( Scr ) or miR - 132 inhibitors ( Ant - 132 ) 3 after TAC treatment ( n = 4 - 8 ) . M : marker . values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 ; # P0 . 05 against TAC - control ; # # P0 . 01 against TAC - control ; # # # P0 . 005 against TAC - control . Scale bars , 50 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; WGA , wheat germ agglutinin .

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

(g-i) calcineurin activity (h) and Mcip1.4 mRNA levels (i) in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.


( A ) Examples of chromosome arms exhibiting features of telomeric semiconservative ( Semi ) replication , conservative ( Consrv ) replication of entire telomere ( E ) conservative replication of part of telomere ( P ) . Idealized chromosome diagrams ( left ) actual microscopy ( right ) are shown . D - FISH , denaturing FISH ; ND - CO - FISH ( non - denaturing CO - FISH ) . ( B ) Percentages of chromosome arms exhibiting conservative ( Consrv ) replication of entire telomere ( E ) , conservative replication of part of telomere ( P ) conservative replication of either entire telomere of part of it ( E + P ) . NE , U2OS expressing normal of cyclin E ; OE , U2OS overexpressing cyclin E for four days . Bars represent means standard errors of mean from three independent experiments . Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres : P < 0 . 025 for Consrv ( P ) P < 0 . 05 for Consrv ( E + P ) , as calculated using paired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O 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) Examples of chromosome arms exhibiting features of telomeric semiconservative (Semi) replication, conservative (Consrv) replication of the entire telomere (E) and conservative replication of part of the telomere (P). Idealized chromosome diagrams (left) and actual microscopy images (right) are shown. D-FISH, denaturing FISH; ND-CO-FISH (non-denaturing CO-FISH).(B) Percentages of chromosome arms exhibiting conservative (Consrv) replication of the entire telomere (E), conservative replication of part of the telomere (P) and conservative replication of either the entire telomere of part of it (E+P). NE, U2OS cells expressing normal levels of cyclin E; OE, U2OS cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from three independent experiments. Cyclin E overexpression resulted in higher percentages of conservatively replicated telomeres: P<0.025 for Consrv (P) and P<0.05 for Consrv (E+P), as calculated using the paired t test.


( A ) Decrease in fraction of chromosome arms exhibiting conservative ( Consrv ) replication of entire telomere or part of telomere ( E + P ) in U2OS after depletion of POLD3 or POLD4 by siRNA . si Ctrl , control siRNA ; NE , expressing normal of cyclin E ; OE , overexpressing cyclin E for four days . Bars represent means standard errors of mean from two independent experiments . For statistical comparisons effects of PolD3 Pold4 depletion were examined after grouping together data from NE OE . PolD3 depletion reduced fraction of conservatively replicated telomeres at a level of P = 0 . 072 ; whereas for PolD4 depletion decrease was significant at a level of P < 0 . 025 , as calculated using paired t . ( B ) Decreased telomere following PolD3 or PolD4 depletion in U2OS expressing normal of cyclin E ( NE ) or overexpressing cyclin E ( OE ) . were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 , five days later , fixed examined for telomere by Q - FISH . Bars indicate means standard error of mean from more than 2 , 600 telomeres examined per condition . differences between control PolD3 or PolD4 - depleted were significant ( P < 10 - 6 ) for both NE OE , as determined by unpaired 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(A) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests.


( C ) Decreased telomere following PolD3 or PolD4 depletion in parental U2OS . were transfected with control siRNA ( si Ctrl ) or siRNAs targeting POLD3 or POLD4 , five days later , fixed examined for telomere by Q - FISH . Bars indicate means standard error of mean from more than 2 , 800 telomeres examined per condition . differences between control PolD3 or PolD4 - depleted were significant ( P < 10 - 6 ) , as determined by unpaired t .

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

(C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests.


( D ) Increased frequency of chromosome end - to - end fusions following POLD3 or POLD4 depletion . U2OS expressing normal of cyclin E ( NE ) or overexpressing cyclin E for four days ( OE ) were transfected with control siRNA ( siCtrl ) os siRNAs targeting POLD3 or POLD4 . For each condition , 25 metaphases were examined ( representing about 1 , 900 high quality chromosome arms per condition ) ; of chromosome fusions was determined for each metaphase used to calculate means standard errors of mean . For statistical , number of chromosome end - to - end fusions were compared by chi square among various conditions . Cyclin E overexpression led to a higher number of fusions ( P < 0 . 0005 ) ; PolD3 PolD4 depletion also led to a higher number of fusions ( P < 0 . 001 P < 0 . 025 , respectively ) .

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

(D) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).


A . Immunoblotting of Neuro2a ( N2a ) expressing Sig1R - FLAG variants .

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A. Immunoblotting analysis of Neuro2a (N2a) cells expressing Sig1R-FLAG variants.


B . Cycloheximide chase on N2a expressing Sig1R - FLAG . Quantitative data of immunoblotting for of Sig1R - FLAGprotein its variants during cyclohexamide chase were plotted as mean ± of three independent experiments ( upper panel ) . Representative immunoblots for of Sig1R - FLAGproteins were shown ( lower panel ) . * * : p < 0 . 01 , * : p = 0 . 0216 in E102Q vs . wild - type ( WT ) ; # # : p < 0 . 01 , # : p < 0 . 05 in L95fs vs . WT . A one - way ANOVA with subsequent post hoc Tukey ' s .

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B. Cycloheximide chase analysis on N2a cells expressing Sig1R-FLAG. Quantitative data of immunoblotting for the levels of Sig1R-FLAGprotein and its variants during the cyclohexamide chase were plotted as mean ± SEM of three independent experiments (upper panel). Representative immunoblots for the levels of Sig1R-FLAGproteins were shown (lower panel). **: p < 0.01, *: p = 0.0216 in E102Q vs. wild-type (WT); ##: p < 0.01, #: p < 0.05 in L95fs vs. WT. A one-way ANOVA with subsequent post hoc Tukey's test.


C . N2a transfected with Sig1R - FLAG variants were incubated with MG - 132 ( 10 µM ) or combination of E64d pepstatin A ( 5 µg / mL each ) ( E64d / PepA ) for 8 h . relative mean of Sig1R - FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± ( upper panel ) . representative immunoblots were shown ( lower panel ) . * : p < 0 . 05 vs . no inhibitor control . A one - way ANOVA with subsequent post hoc Tukey ' s .

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C. N2a cells transfected with Sig1R-FLAG variants were incubated with MG-132 (10 µM) or the combination of E64d and pepstatin A (5 µg/mL each) (E64d/PepA) for 8 h. The relative mean levels of Sig1R-FLAG variants determined by immunoblotting from three independent experiments were plotted as mean ± SEM (upper panel). The representative immunoblots were shown (lower panel). *: p < 0.05 vs. no inhibitor control. A one-way ANOVA with subsequent post hoc Tukey's test.


E F . Subcellular localization of inositol 1 , 4 , 5 - triphosphate receptor type 1 ( IP3R1 ) type 3 ( IP3R3 ) in N2a stably expressing human IP3R3 ( N2a - IP3R3 , E ) . IP3R1 IP3R3 in isolated fractions were identified by immunoblotting with specific antibodies as indicated . Proper fractionation of these samples was confirmed by fraction specific markers as indicated .

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E and F. Subcellular localization of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) and type 3 (IP3R3) in N2a cells stably expressing human IP3R3 (N2a-IP3R3 cells, E). IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated. Proper fractionation of these samples was confirmed by the fraction specific markers as indicated.


E F . Subcellular localization of inositol 1 , 4 , 5 - triphosphate receptor type 1 ( IP3R1 ) type 3 ( IP3R3 ) in HeLa ( F ) . IP3R1 IP3R3 in isolated fractions were identified by immunoblotting with specific antibodies as indicated . Proper fractionation of these samples was confirmed by fraction specific markers as indicated .

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E and F. Subcellular localization of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) and type 3 (IP3R3) in HeLa cells (F). IP3R1 and IP3R3 in the isolated fractions were identified by immunoblotting with the specific antibodies as indicated. Proper fractionation of these samples was confirmed by the fraction specific markers as indicated.


G . Interaction of IP3R3 with wild - type Sig1R or E102Q ALS - linked Sig1R mutant . Sig1R - FLAG variants were transfected in N2a - IP3R3 , IP3R3 was co - immunoprecipitated using an anti - FLAG antibody identified by immunoblotting with specific antibodies as indicated .

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G. Interaction of IP3R3 with wild-type Sig1R or E102Q ALS-linked Sig1R mutant. Sig1R-FLAG variants were transfected in N2a-IP3R3 cells, and IP3R3 was co-immunoprecipitated using an anti-FLAG antibody and identified by immunoblotting with the specific antibodies as indicated.


H . Suppression of Sig1R by siRNA . Lysates of N2a transfected with control siRNA ( siCtrl ) or siRNA against Sig1R ( siSig1R ) for 24 h were blotted . Similar results were obtained from three independent experiments . Paired t - was used for statistical .

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H. Suppression of Sig1R by siRNA. Lysates of N2a cells transfected with control siRNA (siCtrl) or siRNA against Sig1R (siSig1R) for 24 h were blotted. Similar results were obtained from three independent experiments. Paired t-test was used for statistical analysis.


I J . calcium ( Ca2 + ) flux in N2a or human IP3R3 stably expressing N2a ( N2a - IP3R3 ) . siCtrl or siSig1R was transfected to N2a or N2a - IP3R3 , then cytoplasmicCa2 + flux were determined with fluo - 4 Case12 - mito , respectively . was normalized to in resting state at 0 s , plotted as mean ± SD .

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I and J. Cytoplasmic calcium (Ca2+) flux in N2a or human IP3R3 stably expressing N2a (N2a-IP3R3) cells. siCtrl or siSig1R was transfected to N2a or N2a-IP3R3 cells, then cytoplasmicCa2+ flux were determined with fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized to the intensity in resting state at 0 s, and plotted as mean ± SD.


I J . mitochondrial ( J ) calcium ( Ca2 + ) flux in N2a or human IP3R3 stably expressing N2a ( N2a - IP3R3 ) . siCtrl or siSig1R was transfected to N2a or N2a - IP3R3 , then mitochondrial Ca2 + flux were determined with fluo - 4 Case12 - mito , respectively . was normalized to in resting state at 0 s , plotted as mean ± SD .

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I and J. mitochondrial (J) calcium (Ca2+) flux in N2a or human IP3R3 stably expressing N2a (N2a-IP3R3) cells. siCtrl or siSig1R was transfected to N2a or N2a-IP3R3 cells, then mitochondrial Ca2+ flux were determined with fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized to the intensity in resting state at 0 s, and plotted as mean ± SD.


K L . ( K ) or mitochondrial ( L ) Ca2 + flux in N2a - IP3R3 . siCtrl or siSig1R was transfected with or without Sig1R - FLAG variants . data are obtained plotted as described above . n = 10 each . * * : p < 0 . 01 , * : p < 0 . 05 ( I - L ) . Two - way ANOVA with subsequent post hoc Tukey ' s ( I - J ) .

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K and L. Cytoplasmic (K) or mitochondrial (L) Ca2+ flux in N2a-IP3R3 cells. siCtrl or siSig1R was transfected with or without the Sig1R-FLAG variants. The data are obtained and plotted as described above. n = 10 each. **: p < 0.01, *: p < 0.05 (I-L). Two-way ANOVA with subsequent post hoc Tukey's test (I-J).


A . of Sig1R in lumbar spinal cord ( LSC ) or brain of Sig1R − / − , Sig1R + / − , or Sig1R + / + mice at 5 months old . Representative immunoblots obtained from three independent experiments are shown . Asterisk denotes non - specific bands .

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A. The levels of Sig1R in the lumbar spinal cord (LSC) or brain of Sig1R−/−, Sig1R+/−, or Sig1R+/+ mice at 5 months old. Representative immunoblots obtained from three independent experiments are shown. Asterisk denotes non-specific bands.


B C . Sig1R - deficient SOD1G85Rmice ( SOD1G85R / Sig1R − / − ) exhibited accelerated onset of disease ( B ) shortened survival time ( C ) compared to SOD1G85Rmice with one or two copies of Sig1R ( SOD1G85R / Sig1R + / − , SOD1G85R / Sig1R + / + ) . Open circles indicate Sig1R knockout mice ( Sig1R − / − ) that never developed motor neuron disease but that were sacrificed at 396 days of ( n = 8 ) . Mean onset or survival times of mice with ± SD are shown on top of Kaplan - Meyer curves . * * : p < 0 . 01 , * : p < 0 . 05 , log - rank . n = 14 ( SOD1G85R / Sig1R + / + ) , 29 ( SOD1G85R / Sig1R + / − ) , 17 ( SOD1G85R / Sig1R − / − ) .

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

B and C. Sig1R-deficient SOD1G85Rmice (SOD1G85R/Sig1R−/−) exhibited accelerated onset of the disease (B) and shortened survival time (C) compared to SOD1G85Rmice with one or two copies of Sig1R (SOD1G85R/Sig1R+/−, SOD1G85R/Sig1R+/+). Open circles indicate Sig1R knockout mice (Sig1R−/−) that never developed motor neuron disease but that were sacrificed at 396 days of age (n = 8). Mean onset or survival times of mice with ±SD are shown on the top of Kaplan-Meyer curves. **: p < 0.01, *: p < 0.05, log-rank test. n = 14 (SOD1G85R/Sig1R+/+), 29 (SOD1G85R/Sig1R+/−), 17 (SOD1G85R/Sig1R−/−).


D E . weights were lost earlier both in ( D ) ( E ) of SOD1G85R / Sig1R − / − mice . Data are shown as mean ± SD . * : p < 0 . 05 in SOD1G85R / Sig1R + / + vs . SOD1G85R / Sig1R − / − , † : p < 0 . 05 in SOD1G85R / Sig1R + / − vs . SOD1G85R / Sig1R − / − . n = 8 ( SOD1G85R / Sig1R + / + ) , 13 ( SOD1G85R / Sig1R + / − ) , 8 ( SOD1G85R / Sig1R − / − ) in ( D ) , n = 6 ( SOD1G85R / Sig1R + / + ) , 16 ( SOD1G85R / Sig1R + / − ) , 9 ( SOD1G85R / Sig1R − / − ) in ( E ) . used were same in panels B C . used in panels D E were same in panels B C . * : p < 0 . 05 in SOD1G85R / Sig1R + / + vs . SOD1G85R / Sig1R − / − , † : p < 0 . 05 in SOD1G85R / Sig1R + / − vs . SOD1G85R / Sig1R − / − . Two - way ANOVA with subsequent post hoc Tukey ' s .

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D and E. Body weights were lost earlier both in male (D) and female (E) of SOD1G85R/ Sig1R−/−mice. Data are shown as mean ± SD. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. n = 8 (SOD1G85R/Sig1R+/+), 13 (SOD1G85R/Sig1R+/−), 8 (SOD1G85R/Sig1R−/−) in male (D), and n = 6 (SOD1G85R/Sig1R+/+), 16 (SOD1G85R/Sig1R+/−), 9 (SOD1G85R/Sig1R−/−) in female (E). The used animals were the same in the panels B and C. The animals used in the panels D and E were the same in the panels B and C. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. Two-way ANOVA with subsequent post hoc Tukey's test.


F . Decreased performance on rotarod was observed in SOD1G85R / Sig1R − / − mice . was performed at 0 - 30 rpm with 0 . 1 rpm / s acceleration every week . Data are shown as mean ± SD . * * : p < 0 . 0001 in SOD1G85R / Sig1R + / + vs . SOD1G85R / Sig1R − / − , † † : p < 0 . 0001 in SOD1G85R / Sig1R + / − vs . SOD1G85R / Sig1R − / − . used were same in panels B - E . Two - way ANOVA with subsequent post hoc Tukey ' s .

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F. Decreased performance on the rotarod test was observed in SOD1G85R/ Sig1R−/−mice. The test was performed at 0-30 rpm with 0.1 rpm/s acceleration every week. Data are shown as mean ± SD. **: p < 0.0001 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, † †: p < 0.0001 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. The used animals were the same in the panels B-E. Two-way ANOVA with subsequent post hoc Tukey's test.


G H . Motor function of WT ( Sig1R + / + ) Sig1R knockout ( Sig1R − / − ) mice at 5 or 12 months old was evaluated by rotarod ( 0 - 30 rpm , accelerated at 0 . 1 rpm / s ) . Average time on rotating rod was plotted . Error bars denote SD . Unpaired t - .

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G and H. Motor function of WT (Sig1R+/+) and Sig1R knockout (Sig1R−/−) mice at 5 or 12 months old was evaluated by the rotarod test (0-30 rpm, accelerated at 0.1 rpm/s). Average time on the rotating rod was plotted. Error bars denote SD. Unpaired t-tests.


A B . N2a expressing SOD1WT , SOD1G85R or SOD1G93A ( A ) , or spinal cords of WT or mutant SOD1 transgenic mice at end - stage ( B ) were fractionated as described in Materials methods section ( Cyto : cytoplasm , P1 : nuclei debris , Mito : mitochondria , MAM : mitochondria - associate , P3 : microsomal fraction ) . Proper fractionation was confirmed by fraction specific markers as indicated , also shown in Fig EV1 A - E . An asterisk denotes non - specific bands .

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A and B. N2a cells expressing SOD1WT, SOD1G85R or SOD1G93A (A), or spinal cords of WT or mutant SOD1 transgenic mice at end-stage (B) were fractionated as described in Materials and methods section (Cyto: cytoplasm, P1: nuclei and debris, Mito: mitochondria, MAM: mitochondria-associate membrane, P3: microsomal fraction). Proper fractionation was confirmed by the fraction specific markers as indicated, and also shown in Fig EV1 A-E. An asterisk denotes non-specific bands.


C . Tissue - type specific accumulation of mutant SOD1 at MAM . Brain , liver , or astrocytes from SOD1G93Amice were fractionated blotted as in Figures 4A B ( Upper panel ) . of SOD1 at MAM relative to ones in cytoplasm was performed from immunoblotting data with an anti - human SOD1 antibody in ( B ) top panel of ( C ) , data are plotted as mean ± from three independent experiments ( Lower panel ) . * * : p < 0 . 01 vs . SOD1WTspinal cord . A one - way ANOVA with subsequent post hoc Tukey ' s . Proper fractionation of these samples was confirmed in Fig EV 1F - H .

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C. Tissue and cell-type specific accumulation of mutant SOD1 at the MAM. Brain, liver, or primary astrocytes from SOD1G93Amice were fractionated and blotted as in Figures 4A and B (Upper panel). Quantification of SOD1 proteins at the MAM relative to ones in the cytoplasm was performed from immunoblotting data with an anti-human SOD1 antibody in (B) and the top panel of (C), and the data are plotted as mean ± SEM from three independent experiments (Lower panel). **: p < 0.01 vs. SOD1WTspinal cord. A one-way ANOVA with subsequent post hoc Tukey's test.Proper fractionation of these samples was confirmed in Fig EV 1F-H.


D . Mutant SOD1 prevented association of ER with mitochondria . Isolated ER ( P3 : microsomal fraction ) mitochondria of N2a expressing wild - type ( WT ) or G85R ( 85 ) SOD1 for 48 h were mixed incubated . Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti - PDI ( ER marker ) anti - VDAC ( mitochondrial marker ) , respectively . these results were confirmed by three independent experiments .

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D. Mutant SOD1 prevented association of ER with mitochondria. Isolated ER (P3: microsomal fraction) and mitochondria of N2a cells expressing wild-type (WT) or G85R (85) SOD1 for 48 h were mixed and incubated. Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti-PDI (ER marker) and anti-VDAC (mitochondrial marker), respectively. All these results were confirmed by three independent experiments.


A B . Time course analyses of mutant SOD1 at MAM in SOD1G93Amousespinal cords ( A ) brains ( B ) . Immunoblots show of mutant SOD1 in indicated fractions at various time points ( left panels ) . Quantitative data at right were plotted as mean ± of three independent experiments . Arrows indicate a rapid reduction in SOD1 level at MAM just following onset of disease .

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A and B. Time course analyses of mutant SOD1 levels at the MAM in SOD1G93Amousespinal cords (A) and brains (B). Immunoblots show levels of mutant SOD1 protein in the indicated fractions at various time points (left panels). Quantitative data at the right were plotted as mean ± SEM of three independent experiments. Arrows indicate a rapid reduction in SOD1 level at the MAM just following the onset of disease.


C D . Time course analyses of mutant SOD1 accumulation at MAM in SOD1G85R ( C ) SOD1G37R ( D ) mousespinal cords . Quantitative data from three independent experiments were plotted ( bottom ) . arrows in bottom panel are same as in A B .

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C and D. Time course analyses of mutant SOD1 accumulation at the MAM in SOD1G85R (C) and SOD1G37R (D) mousespinal cords. Quantitative data from three independent experiments were plotted (bottom). The arrows in the bottom panel are the same as in A and B.


E . Time course analyses of MAM - specific at MAM in whole lysates of SOD1G93Amousespinal cords .

E. Time course analyses of MAM-specific proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords.


A - I . Immunofluorescence of spinal cords brains from non - transgenic ( Non - Tg ) , SOD1 transgenic , or Sig1R − / − mice . Transverse sections of mousespinal cords ( A , D - I ) or sagittal sections of mouse brains ( B , C ) were stained using anti - Sig1R ( ) , βIII - tubulin ( red ) IP3R3 ( green ) antibodies . Note that Sig1R IP3R3 are co - localized in motor neurons of anterior horn ( A ) hypoglossal nucleus ( B ) , IP3R3 was not expressed in hippocampal neurons ( C ) . Mutant SOD1 induced aggregation of Sig1R mislocalization of IP3R3 in anterior horn neurons ( D - G ) , while their abnormalities were not observed in SOD1WTmotor neurons ( H ) . Mislocalization of IP3R3 was also observed in Sig1R − / − mouse spinal cords ( I ) . Scale bars : 50 µm .

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A-I. Immunofluorescence staining of spinal cords and brains from non-transgenic (Non-Tg), SOD1 transgenic, or Sig1R−/−mice. Transverse sections of mousespinal cords (A, D-I) or sagittal sections of mouse brains (B, C) were stained using anti-Sig1R (white), βIII-tubulin (red) and IP3R3 (green) antibodies. Note that Sig1R and IP3R3 are co-localized in the motor neurons of the anterior horn (A) and the hypoglossal nucleus (B), and IP3R3 was not expressed in hippocampal neurons (C). Mutant SOD1 induced aggregation of Sig1R and mislocalization of IP3R3 in anterior horn neurons (D-G), while their abnormalities were not observed in SOD1WTmotor neurons (H). Mislocalization of IP3R3 was also observed in Sig1R−/−mouse spinal cords (I). Scale bars: 50 µm.


J K . Representative electron of MAM ( J ) ( double - headed arrows ) in motor neurons of 12 months - old Non - Tg , SOD1G85R or Sig1R − / − mice . Note that ER - mitochondria contacted areas were reduced in both SOD1G85R or Sig1R − / − mice . of mitochondria surface associated to ER was calculated in K . For , 13 - 19 motor neurons 224 - 316 mitochondria with MAM in each ( n = 2 ) were analyzed . Data are expressed as mean ± . Double asterisk means p < 0 . 0001 vs Non - Tg . A one - way ANOVA with subsequent post hoc Tukey ' s . Scale bars : 300 nm .

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

J and K. Representative electron micrographs of the MAM (J) (double-headed arrows) in motor neurons of 12 months-old Non-Tg, SOD1G85R or Sig1R−/−mice. Note that ER-mitochondria contacted areas were reduced in both SOD1G85R or Sig1R−/−mice. Quantification of the mitochondria surface associated to ER was calculated in K. For quantification, 13-19 motor neurons and 224-316 mitochondria with MAM in each animal (n = 2) were analyzed. Data are expressed as mean ± SEM. Double asterisk means p < 0.0001 vs Non-Tg. A one-way ANOVA with subsequent post hoc Tukey's test. Scale bars: 300 nm.


L . for IP3R3 calreticulin were decreased in MAM fractions of Sig1R − / − mouse brains . MAM fractions whole tissue lysates of Sig1R + / + or Sig1R − / − mouse brains were immunoblotted . Representative blots from three independent experiments are shown .

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

L. The levels for IP3R3 and calreticulin were decreased in the MAM fractions of Sig1R−/−mouse brains. MAM fractions and whole tissue lysates of Sig1R+/+ or Sig1R−/−mouse brains were immunoblotted. Representative blots from three independent experiments are shown.


A . N2a were transfected with WT or mutant SOD1 in presence or absence of IP3R3 . Plotted viability of measured by a neurotoxicity revealed that IP3R3 is involved in vulnerability against mutant SOD1 . Data are expressed as mean ± from three independent experiments , triplicated in each experiment . * * : p < 0 . 01 , * : p < 0 . 05 .

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

A. N2a cells were transfected with WT or mutant SOD1 in the presence or absence of IP3R3. Plotted viability of the cells measured by a neurotoxicity assay revealed that IP3R3 is involved in cell vulnerability against mutant SOD1. Data are expressed as mean ± SEM from three independent experiments, triplicated in each experiment. **: p < 0.01, *: p < 0.05.


B - G . Calpain activity ( B - D ) were measured in N2a - IP3R3 expressing Sig1R - FLAG ( B E ) , SOD1 ( C F ) , or both ( D G ) . Mean ± from three independent experiments is plotted . * : p < 0 . 05 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O 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-G. Calpain activity (B-D) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.


B - G . intracellular ATP ( E - G ) were measured in N2a - IP3R3 expressing Sig1R - FLAG ( B E ) , SOD1 ( C F ) , or both ( D G ) . Mean ± from three independent experiments is plotted . * : p < 0 . 05 .

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

B-G. intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.


H I . Calpain activity in vivo was determined by cleavage of spectrin αII . Calpain - 150 kDa fragment of spectrin αII in mouselumbar spinal cord or brain ( H ) was normalized to β - actin ( I ) . Quantitative data in immunoblotting analyses are plotted as mean ± from three independent experiments . * * : p < 0 . 01 .

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

H and I. Calpain activity in vivo was determined by the cleavage of spectrin αII. Calpain-cleaved 150 kDa fragment of spectrin αII in mouselumbar spinal cord or brain (H) was normalized to β-actin (I). Quantitative data in immunoblotting analyses are plotted as mean ± SEM from three independent experiments. **: p < 0.01.


A B . ( A ) or mitochondrial ( B ) Ca2 + flux was measured in N2a - IP3R3 treated with PRE - 084 ( 5 µM ) or NE - 100 ( 5 µM ) for 1 h prior to imaging . mitochondrialCa2 + flux were detected by fluo - 4 Case12 - mito , respectively . was normalized by resting state at 0 s , plotted as mean ± SD . n = 10 each . * * : p < 0 . 01 , * : p < 0 . 05 . Two - way ANOVA with subsequent post hoc Tukey ' s .

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

A and B. Cytoplasmic (A) or mitochondrial (B) Ca2+ flux was measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 1 h prior to fluorescent imaging. Cytoplasmic and mitochondrialCa2+ flux were detected by fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized by the resting state at 0 s, and plotted as mean ± SD. n = 10 each. **: p < 0.01, *: p < 0.05. Two-way ANOVA with subsequent post hoc Tukey's test.


C D . Calpain activity were measured in N2a - IP3R3 treated with PRE - 084 ( 5 µM ) or NE - 100 ( 5 µM ) for 24 h . Data are plotted as mean ± from three independent experiments . * : p < 0 . 05 vs . mock control . A one - way ANOVA with subsequent post hoc Tukey ' s .

O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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 and D. Calpain activity were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.


C D . intracellular ATP were measured in N2a - IP3R3 treated with PRE - 084 ( 5 µM ) or NE - 100 ( 5 µM ) for 24 h . Data are plotted as mean ± from three independent experiments . * : p < 0 . 05 vs . mock control . A one - way ANOVA with subsequent post hoc Tukey ' s .

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 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

C and D. intracellular ATP levels were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.


E F . SOD1G93Amice were intraperitoneally administered with saline or PRE - 084 ( 0 . 25 mg / kg , 3 times per week ) from postnatal day 35 to 95 . Lumbar spinal cord sections were stained by using anti - Sig1R ( ) , IP3R3 ( green ) , βIII - tubulin ( red ) at 95 days old . Arrowheads indicate neurons with normal distribution of Sig1R IP3R3 . Scale bars : 50 µm .

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

E and F. SOD1G93Amice were intraperitoneally administered with saline or PRE-084 (0.25 mg/kg, 3 times per week) from postnatal day 35 to 95. Lumbar spinal cord sections were stained by using anti-Sig1R (white), IP3R3 (green), and βIII-tubulin (red) at 95 days old. Arrowheads indicate the neurons with normal distribution of Sig1R and IP3R3. Scale bars: 50 µm.


A , B , Immunostaining for CD31 + blood vessels in control ( ctrl , A ) Gpr124KO ( B ) forebrains . Note normal vasculature in cortical hem ( asterisk ) of Gpr124KO mice .

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A,B, Immunostaining for CD31+ blood vessels in control (ctrl, A) and Gpr124KO (B) forebrains. Note the normal vasculature in the cortical hem (asterisk) of Gpr124KO mice.


C , D , Immunostaining for Pax6 in control ( C ) Gpr124KO ( D ) forebrains to reveal lateral extension of neocortex ( dashed ) between cortical hem ( arrowhead ) lateral ganglionic eminence ( arrow ) . E , of lateral extension of cortex in control Gpr124KO brains ( mean ± ; N = 10 ; * * * p < 0 . 001 ) .

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

C,D, Immunostaining for Pax6 in control (C) and Gpr124KO (D) forebrains to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). E, Quantification of the lateral extension of the cortex in control and Gpr124KO brains (mean±SEM; N=10; *** p<0.001).


F , G for EdU ( red ) Ki67 ( green ) in control ( F ) Gpr124KO ( G ) cortices at E13 . 5 , 24 hours after EdU injection . H , of Ki67 + NPCs in VZ ( blue ) , SVZ / CP ( grey ) of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control Gpr124KO ( mean ± ; N = 4 ( control ) N = 3 ( Gpr124KO ) ; * p < 0 . 05 , * * p < 0 . 01 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

F,G Staining for EdU (red) and Ki67 (green) in control (F) and Gpr124KO (G) cortices at E13.5, 24 hours after EdU injection. H, Quantification of Ki67+ NPCs in the VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124KO embryos (mean±SEM; N=4 (control) and N=3 (Gpr124KO); * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


I , J , Immunostaining for Tbr2 ( red ) Pax6 ( green ) in control ( I ) Gpr124KO ( J ) cortices . K , of newborn BPs in VZ of control Gpr124KO cortices ( mean ± ; N = 4 ; * p < 0 . 05 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

I,J, Immunostaining for Tbr2 (red) and Pax6 (green ) in control (I) and Gpr124KO (J) cortices. K, Quantification of newborn BPs in the VZ of control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


L , M , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in control ( L ) Gpr124KO ( M ) cortices . N , O of neurogenic ( Ngn1 + ) RGs BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control Gpr124KO cortices ( mean ± ; N = 4 ; * p < 0 . 05 , * * * p < 0 . 001 ) . Note that fraction of total BPs is significantly reduced ( Fig 1K ) while fraction of Ngn1 + BPs is insignificantly reduced ( Fig 1N ) , because proportion of Ngn1 + Tbr2 + within BPs is slightly , though statisically insignificantly higher in Gpr124KObrains ( see Fig S2M ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

L,M, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (L) and Gpr124KO (M) cortices. N,O Quantification of neurogenic (Ngn1+) RGs and BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05, *** p<0.001). Note that the fraction of total BPs is significantly reduced (Fig 1K) while the fraction of Ngn1+BPs is insignificantly reduced (Fig 1N), because the proportion of Ngn1+Tbr2+ cells within all BPs is slightly, though statisically insignificantly higher in the Gpr124KObrains (see Fig S2M). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


P , Q Stainings for Tbr2 ( red ) for Tis21 - GFP expression ( green ) in control ( P ) Gpr124KO ( Q ) cortices . R , S of neurogenic ( Tis21 - GFP + ) RGs BPs ( R ) or expanding ( Tbr2 - Tis21 - GFP - ) RGs ( S ) in control Gpr124KO cortices ( mean ± ; N = 4 ; * * * p < 0 . 001 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

P,Q Stainings for Tbr2 (red) and for Tis21-GFP expression (green) in control (P) and Gpr124KO (Q) cortices. R,S Quantification of neurogenic (Tis21-GFP+) RGs and BPs (R) or expanding (Tbr2- Tis21-GFP-) RGs (S) in control and Gpr124KO cortices (mean±SEM; N=4; *** p<0.001). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A , B , for Isolectin B4 + blood vessels in control ( A ) Gpr124ECcKO ( B ) forebrains . Asterisks mark lateral ventricles .

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

A,B, Staining for Isolectin B4+ blood vessels in control (A) and Gpr124ECcKO (B) forebrains. Asterisks mark the lateral ventricles.


C , D , for pimonidazole ( PIMO , green ) Isolectin B4 + blood vessels ( red ) in ctrl ( C ) Gpr124ECcKO ( D ) cortices .

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

C,D, Staining for pimonidazole (PIMO, green) and Isolectin B4+ blood vessels (red) in ctrl (C) and Gpr124ECcKO (D) cortices.


E , F , Immunostaining for Pax6 in control ( E ) Gpr124ECcKO ( F ) forebrain sections to reveal lateral extension of neocortex ( dashed ) between cortical hem ( arrowhead ) lateral ganglionic eminence ( arrow ) . G , lateral extension of cortex in control Gpr124ECcKObrains ( mean ± ; N = 6 for ctrl N = 8 for Gpr124ECcKO ; * p < 0 . 05 ) .

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

E,F, Immunostaining for Pax6 in control (E) and Gpr124ECcKO (F) forebrain sections to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). G, Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains (mean±SEM; N=6 for ctrl and N=8 for Gpr124ECcKO; * p<0.05).


H , I , for EdU ( red ) Ki67 ( green ) in control ( H ) Gpr124ECcKO ( I ) cortices at E13 . 5 , 24 hours after EdU injection . J , of Ki67 + NPCs in in VZ ( blue ) , SVZ / CP ( grey ) of Ki67 - neurons ( cyan ) generated from EdU - labeled NPCs in control Gpr124ECcKO ( mean ± ; N = 4 ; * p < 0 . 05 , * * P < 0 . 01 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

H,I, Staining for EdU (red) and Ki67 (green) in control (H) and Gpr124ECcKO (I) cortices at E13.5, 24 hours after EdU injection. J, Quantification of Ki67+ NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124ECcKO embryos (mean±SEM; N=4; * p<0.05, ** P<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


K , L , Immunostaining for Tbr2 ( red ) Ngn1 ( green ) in control ( K ) Gpr124KO ( L ) cortices . M - O , of neurogenic ( Ngn1 + ) RGs BPs ( M ) , newborn BPs ( N ) or expanding ( Tbr2 - Ngn1 - ) RGs ( O ) in control Gpr124ECcKO cortices ( mean ± ; N = 4 ; * p < 0 . 05 , * * p < 0 . 01 ) . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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

K,L, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (K) and Gpr124KO (L) cortices. M-O, Quantification of neurogenic (Ngn1+) RGs and BPs (M), newborn BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124ECcKO cortices (mean±SEM; N=4; * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


A , qRT - PCR of NSC - specific markers ( Fabp7 , Hes5 ) neuronal markers ( Dcx , Tubb3 ) in Prom1 + fraction , presented as fold change compared to Prom1 - fraction ) from WT cortices at E14 . 5 ( mean ± ; N = 3 for Dcx ; N = 6 for other genes ) .

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A, qRT-PCR analysis of NSC-specific markers (Fabp7, Hes5) and neuronal markers (Dcx, Tubb3) in the Prom1+ fraction, presented as fold change compared to the Prom1- fraction) from WT cortices at E14.5 (mean±SEM; N=3 for Dcx; N=6 for all other genes).


F , Western blot for HIF - 1 ( top ) , HIF - 2 ( middle ) a - tubulin as loading control ( bottom ) using forebrain lysates from E13 . 5 control ( left ) or Gpr124KOembryos ( middle ) . HIF - 1 / 2 - transfected HEK293 served as positive control ( right ) . G , of HIF - 1 band intensities shown in panel F ( mean ± ; N = 3 ; * * p < 0 . 01 ) .

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

F, Western blot for HIF-1 (top), HIF-2 (middle) and a-tubulin as loading control (bottom) using forebrain lysates from E13.5 control (left) or Gpr124KOembryos (middle). HIF-1/2-transfected HEK293 cells served as positive control (right). G, Quantification of HIF-1 band intensities shown in panel F (mean±SEM; N=3; ** p<0.01).


H , I , Immunostaining for HIF - 1α ( red ) DAPI ( blue ) in control ( H , H ' , H ' ' ) Gpr124KO ( I , I ' , I ' ' ) cortices . Panels H ' , H ' ' , I ' I ' ' are magnifications of boxed areas in panels I J , showing HIF - 1α signal alone ( H ' , I ' ) or together with DAPI ( H ' ' , I ' ' ) . Asterisks indicate autofluorescent blood . dashed indicates basal boundary of VZ . Scale bar = 100 µm . Full , dotted dashed lines indicate basal apical boundaries of cortex or boundaries of cortical zones , respectively . CP , cortical plate ; IZ , intermediate zone ; SVZ , subventricular zone ; VZ , ventricular zone . Scale bar : 100

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H,I, Immunostaining for HIF-1α (red) and DAPI (blue) in control (H, H', H'') and Gpr124KO (I,I',I'') cortices. Panels H',H'',I' and I'' are magnifications of the boxed areas in panels I and J, showing HIF-1α signal alone (H',I') or together with DAPI (H'',I''). Asterisks indicate autofluorescent blood cells. The dashed line indicates the basal boundary of the VZ. Scale bar = 100 µm. Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm


J , qRT - PCR of HIF - 1 target gene expression in VZ from cortex of control Gpr124KO E13 . 5 ( mean ± ; N = 4 ; * * * p < 0 . 001 ) . SVZ , subventricular zone ; VZ , ventricular zone .

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J, qRT-PCR analysis of HIF-1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13.5 embryos (mean±SEM; N=4; *** p<0.001). SVZ, subventricular zone; VZ, ventricular zone.


A - F , for Isolectin B4 ( A - C ) oxidized thiols ( D - F ) in WT cortices at E10 . 5 ( A , D ) , E11 . 5 ( B , E ) E12 . 5 ( C , F ) . Panels D - F are pseudocolor conversions of ( from low [ purple ] to high [ red ] ) . Arrowheads denote position of vascular front .

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A-F, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front.


G - I , Immunostaining for HIF - 1α ( red ) DAPI ( blue ) in WT cortices at E10 . 5 ( G - G ' ' ) , E11 . 5 ( H - H ' ' ) E12 . 5 ( I - I ' ' ) . Panels G ' , G ' ' , H ' , H ' ' , I ' I ' ' are magnifications of boxed areas in their respective original panels . Arrows point to border of lateral dorsal cortex .

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G-I, Immunostaining for HIF-1α (red) and DAPI (blue) in WT cortices at E10.5 (G-G''), E11.5 (H-H'') and E12.5 (I-I''). Panels G',G'',H',H'',I' and I'' are magnifications of the boxed areas in their respective original panels. Arrows point to the border of lateral and dorsal cortex.


J - L , Immunostaining for Glut1 of WT cortices at E10 . 0 ( J ) , E11 . 5 ( K ) E12 . 5 ( L ) . Dashed lines indicate apical border of cortex . Scale bar : 100 µm ( D , F , G , H , I , J - L ) or 50 µm ( I ' ' ) , respectively .

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J-L, Immunostaining for Glut1 of WT cortices at E10.0 (J), E11.5 (K) and E12.5 (L). Dashed lines indicate the apical border of the cortex. Scale bar: 100 µm (D,F,G,H,I, J-L) or 50 µm (I''), respectively.


A , qRT - PCR of HIF - 1 HIF - 1 target gene expression in cortices from E13 . 5 WT HIF - 1αCC + / - ( mean ± ; N = 8 for WT N = 5 for HIF - 1αCC + / - ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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A, qRT-PCR analysis of HIF-1 and HIF-1 target gene expression in cortices from E13.5 WT and HIF-1αCC+/-embryos (mean±SEM; N=8 for WT and N=5 for HIF-1αCC+/-; * p<0.05, ** p<0.01, *** p<0.001).