E . Cilia ( Ac - tubulin staining ) are not detectable in the ependymal cells of 2 month old Gemc1 - / - mice ( right panel ) . Choroid plexus ( CP ) and lateral ventricles ( LV ) are indicated . Scale bars = 20

E. Cilia (Ac-tubulin staining) are not detectable in the ependymal cells of 2 month old Gemc1-/- mice (right panel). Choroid plexus (CP) and lateral ventricles (LV) are indicated. Scale bars = 20 µm


E and F . RT - QPCR analysis of individual genes related to MCC formation in the oviduct ( E ) and trachea ( F ) of Gemc1 - / - mice compared to wild type littermates , using 18S rRNA as a reference ( n = 2 ) . Genes examined generally fall into 3 classes : centriole duplication ( Cep152 , Plk4 , Cetn2 ) , Deuterosome mediated centriole expansion ( Deup1 , Ccdc78 , Ccno ) and transcription of genes required for MCC differentiation ( FoxJ1 , Myb , Mcidas ) .

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E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).


A . GEMC1 interacts with E2F4 and DP1 . Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel . FLAGimmunoprecipiation ( IP ) of GEMC1co - IPs DP1 ( α - HA , lane 5 ) and E2F4 ( α - HA , lane 6 ) . The interaction is enhanced upon co - expression of HA tagged E2F4 and DP1 ( α - HA , lane 7 ) .

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A. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAGimmunoprecipiation (IP) of GEMC1co-IPs DP1 (α-HA, lane 5) and E2F4 (α- HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7).


B . GEMC1 interacts with E2F5 but not E2F1 . Co - IP of E2F5 ( α - HA , lane 3 ) and E2F5 - DP1 ( α - HA , lane 6 ) but not E2F1 or E2F1 - DP1 ( α - HA , lanes 2 and 5 ) . In the FLAG - IP panels , a short ( top ) and long ( second from top ) exposure of the Western are shown .

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B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown.


C . Identification of the domain required for E2F4 interaction . GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP . Wild type GEMC1 ( lane 5 ) or GEMC1 lacking the CC domain ( lane 6 ) pulled down E2F4 - DP1 . A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4 - DP1 ( lanes 7 , 8 ) .

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C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8).


D . Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients ( G313D ) impairs the interaction with E2F4 - DP1 ( compare land 5 and 6 ) .

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D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6).


E . GEMC1 interacts with Multicilin through its CC domain . Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc - tagged Multicilin ( anti - Myc blot , lanes 4 and 6 ) while a CC domain mutant does not ( anti - Myc blot , lane 5 ) .

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E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5).


A . Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression ( RT - QPCR ) in HEK293T cells . Results for MCIDAS are graphed on a smaller scale in the right panel : V = vector and G = GEMC1 . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar induction of FOXJ1 was observed in U2OS and T98G ( Fig EV3 ) .

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A. Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression (RT-QPCR) in HEK293T cells. Results for MCIDAS are graphed on a smaller scale in the right panel: V = vector and G = GEMC1. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar induction of FOXJ1 was observed in U2OS and T98G (Fig EV3).


B . Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 ( RT - QPCR ) . A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar results were also observed in U2OS ( Fig EV3 ) .

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B. Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 (RT-QPCR). A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar results were also observed in U2OS (Fig EV3).


C . Activation of FOXJ1 by GEMC1 is inhibited by the co - expression of Geminin ( compare 3rd and 4th lanes ) . Example Western blot is shown below . Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells .

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C. Activation of FOXJ1 by GEMC1 is inhibited by the co-expression of Geminin (compare 3rd and 4th lanes). Example Western blot is shown below. Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells.


D . Co - expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression ( measured by RT - QPCR ) as Multicilin alone in HEK293T cells . The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed .

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D. Co-expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression (measured by RT-QPCR) as Multicilin alone in HEK293T cells. The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed.


E and F . The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin . HEK293T cells were transfected with a control siRNA ( siC ) or 2 different siRNAs against MCIDAS ( si1 and si2 ) prior to being transfected with GFP or GFP - GEMC1 expression vectors . Relative MCIDAS ( E ) and FOXJ1 ( F ) expression levels were assessed by RT - QPCR . The mean and standard deviation of a representative experiment performed in triplicate is graphed . An additional experiment is presented in Fig EV3 .

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E and F. The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin. HEK293T cells were transfected with a control siRNA (siC) or 2 different siRNAs against MCIDAS (si1 and si2) prior to being transfected with GFP or GFP-GEMC1 expression vectors. Relative MCIDAS (E) and FOXJ1 (F) expression levels were assessed by RT-QPCR. The mean and standard deviation of a representative experiment performed in triplicate is graphed. An additional experiment is presented in Fig EV3.


IL - 1β ELISA of supernatants from LPS - primed ( 1 μg / mL , 4hrs . ) MDMs pre - incubated with 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) 15 mins . before treatment with either nigericin ( 10 μM , 45 mins . ) or CPPD crystals ( 250 μg / mL , 2 hrs . ) . Bars represent the mean , ± S . D . n = 13 and 11 independent blood donors for nigericin and CPPD , respectively . IL - 18 ELISA of supernatants from MDMs treated as in A . Bars represent the mean , ± S . D . n = 11 independent blood donors .

O O O O O O 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 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 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

IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors.


LDH - release from MDMs treated as in A . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 10 and 11 independent blood donors for nigericin and CPPD , respectively .

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LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively.


Western blots of supernatants and cell lysates from MDMs treated as in A . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) , pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) and mature caspase - 1 ( mCasp - 1 ; 20 kDa ) . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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Western blots of supernatants and cell lysates from MDMs treated as in A. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa), pro-caspase-1 (pro-Casp-1; 45 kDa) and mature caspase-1 (mCasp-1; 20 kDa). β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


Quantitative RT - PCR showing the mRNA expression levels of IL - 1β , IL - 18 , ASC , CASP1 and NLRP3 in MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ; 4 hrs . ) , as indicated . Bars represent the mean fold increase from an untreated ( UT )

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Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) control


Western blots showing the relative abundance of NLRP3 , ASC , pro - caspase - 1 , pro - IL - 1β , pro - IL - 18 , USP7 and USP47 in cell lysates of MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ) for 4 hrs . , as indicated . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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Western blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


IL - 18 ELISA of supernatants from LPS - primed ( 1 μg / mL , 10 mins . ) MDMs pre - incubated with either vehicle ( 0 . 1 % DMSO ) or P22077 ( 2 . 5 µM ) for 15 mins . before treatment with nigericin ( 10 μM , 45 mins . ) . Bars represent the mean , ± S . D . n = 9 independent blood donors .

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IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors.


Western blots of supernatants and cell lysates from MDMs as treated in C . Bands in the figure represent : Pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) and β - actin as a loading control . Blots are representative of at least 3 independent blood donors .

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Western blots of supernatants and cell lysates from MDMs as treated in C. Bands in the figure represent: Pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa) and β-actin as a loading control. Blots are representative of at least 3 independent blood donors.


LDH - release from MDMs as treated in C . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 9 independent blood donors .

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LDH-release from MDMs as treated in C. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 9 independent blood donors.


Western blots showing ASC - oligomerisation after DSS - mediated crosslinking in insoluble fractions from THP1 cell lysates . PMA differentiated THP1 cells were LPS - primed ( 1 μg / mL , 4 hrs . ) and pre - incubated for 15 min with either 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) . followed by treatment with nigericin ( 10 μM , 45 mins . ) . The ' input ' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction . Blots show membranes that were probed with an anti - ASC antibody . Data is representative of at least three independent experiments .

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Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in insoluble fractions from THP1 cell lysates. PMA differentiated THP1 cells were LPS-primed (1 μg/mL, 4 hrs.) and pre-incubated for 15 min with either 0.1% DMSO or P22077 (2.5 µM). followed by treatment with nigericin (10 μM, 45 mins.). The 'input' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction. Blots show membranes that were probed with an anti-ASC antibody. Data is representative of at least three independent experiments.


Western blots showing ASC - oligomerisation after DSS - mediated crosslinking in supernatants from THP1s treated as in A . Blots show membranes that were probed with an anti - ASC antibody . The input represents non - crosslinked supernatant . Data is representative of at least three independent experiments .

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Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in supernatants from THP1s treated as in A. Blots show membranes that were probed with an anti-ASC antibody. The input represents non-crosslinked supernatant. Data is representative of at least three independent experiments.


Images from an immunofluorescence experiment showing ASC - speck formation in THP1 cells treated as in A . All images show cells that were stained with an anti - ASC antibody . Images are representative of 3 independent experiments . Scale bar 20µm . Quantification of ASC - speck formation in images depicted in C . The number of ASC specks was quantified and expressed as the percentage of cells containing specks .

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Images from an immunofluorescence experiment showing ASC-speck formation in THP1 cells treated as in A. All images show cells that were stained with an anti-ASC antibody. Images are representative of 3 independent experiments. Scale bar 20µm. Quantification of ASC-speck formation in images depicted in C. The number of ASC specks was quantified and expressed as the percentage of cells containing specks.


NLRP3 oligomerisation analysed by native PAGE . LPS - primed ( 1 μg / mL , 4 hrs . ) murine BMDMs pre - incubated with either 0 . 1 % DMSO or P22077 ( at the indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 min . ) . Digitonin - solubilised cell lysates were analysed by native - PAGE or SDS - PAGE and NLRP3 western - blotting . Western blots of supernatants and cell lysates for IL - 1β are also shown as a control for P22077 inflammasome inhibition . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) and NLRP3 ( 120 kDa ) . β - actin is shown as a loading control . Blots are representative of 4 independent murine donors .

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

NLRP3 oligomerisation analysed by native PAGE. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 min.). Digitonin-solubilised cell lysates were analysed by native-PAGE or SDS-PAGE and NLRP3 western-blotting. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120 kDa). β-actin is shown as a loading control. Blots are representative of 4 independent murine donors.


NLRP3 ubiquitination analysed by western blot . LPS - primed ( 1 μg / mL , 4 hrs . ) murine BMDMs pre - incubated with either 0 . 1 % DMSO or P22077 ( at the indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 mins . ) . Endogenous NLRP3 was immunoprecipitated ( IP ) with anti - NLRP3 and probed with either anti - ubiquitin ( Poly - Ub ) , anti - K63 - ubiquitin ( Poly - K63 - Ub ) or anti - NLRP3 . As a negative control , cell lysate made from LPS - primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG . Total levels of NLRP3 in cell lysates ( input ) were also probed . Western blots of supernatants and cell lysates for IL - 1β are also shown as a control for P22077 inhibition . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) and NLRP3 ( 120kDa ) . β - actin is shown as a loading control . Blots are representative of 2 independent murine donors for 2 . 5µM P22077 and 4 independent murine donors for 10 µM .

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NLRP3 ubiquitination analysed by western blot. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 mins.). Endogenous NLRP3 was immunoprecipitated (IP) with anti-NLRP3 and probed with either anti-ubiquitin (Poly-Ub), anti-K63-ubiquitin (Poly-K63-Ub) or anti-NLRP3. As a negative control, cell lysate made from LPS-primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120kDa). β-actin is shown as a loading control. Blots are representative of 2 independent murine donors for 2.5µM P22077 and 4 independent murine donors for 10 µM.


Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and then treated with nigericin ( 10 μM ) for differing lengths of time , as indicated . Relative , activity - based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs . Higher panels show lysates incubated with ABPs ( labelled ) , with the active DUB bound to ABP indicated by the suffix ' - VME ' . Lower panels , without probe labelling , show total USP7 or USP47 levels . ( Unlabelled ) . Western blots are representative of 3 independent MDM donors .

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Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors.


Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and then pre - incubated with either 0 . 1 % DMSO or P22077 ( 2 . 5 μM ) prior to incubation with CPPD crystals ( 250 µg / mL , 3 hrs . ) . Blots are labelled as in B and are representative of 3 independent MDM donors .

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Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then pre-incubated with either 0.1% DMSO or P22077 (2.5 μM) prior to incubation with CPPD crystals (250 µg/mL, 3 hrs.). Blots are labelled as in B and are representative of 3 independent MDM donors.


Western blots showing activity changes in both USP7 and USP47 in LPS - primed ( 1 μg / mL , 4 hrs . ) MDMs pre - incubated with different P22077 concentrations , as indicated , prior to activation by nigericin treatment ( 10 μM ; 45 mins . ) . Blots are labelled as in B and are representative of 3 independent MDM donors .

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Western blots showing activity changes in both USP7 and USP47 in LPS-primed (1 μg/mL, 4 hrs.) MDMs pre-incubated with different P22077 concentrations, as indicated, prior to activation by nigericin treatment (10 μM; 45 mins.). Blots are labelled as in B and are representative of 3 independent MDM donors.


Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium ( High K + ) conditions . MDMs were LPS - primed ( 1 μg / mL , 4 hrs . ) and then changed to media that either had normal or high concentrations of K + , and subsequently treated with nigericin ( 10 μM ; 45 mins . ) . Blots are labelled as in B and are representative of at least 2 independent MDM donors . Pro - IL - 1β ( 31 kDa ) and mature IL - 1β ( mIL - 1β , 17 kDa ) in the supernatants of these cells are shown as a control . Non - specific bands are highlighted with an asterisk .

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Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium (High K+) conditions. MDMs were LPS-primed (1 μg/mL, 4 hrs.) and then changed to media that either had normal or high concentrations of K+, and subsequently treated with nigericin (10 μM; 45 mins.). Blots are labelled as in B and are representative of at least 2 independent MDM donors. Pro-IL-1β (31 kDa) and mature IL-1β (mIL-1β, 17 kDa) in the supernatants of these cells are shown as a control. Non-specific bands are highlighted with an asterisk.


Western blots of supernatants and cell lysates from THP - 1 cells engineered by CRISPR / Cas9 to induce USP7 and / or USP47 KOs . Deficiency of both USP7 and USP47 was induced by doxycycline ( Doxy ) treatment ( 1 µg / mL ; 3 days ) , as indicated . All cells were differentiated with PMA and either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) and treated with nigericin ( 10 μM , 45 mins . ) , as indicated . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) ; mature caspase - 1 ( mCasp - 1 ; 20 kDa ) ; USP7 and USP47 . β - actin is shown as a loading control . Blots are representative of at least 3 independent experiments .

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Western blots of supernatants and cell lysates from THP-1 cells engineered by CRISPR/Cas9 to induce USP7 and/or USP47 KOs. Deficiency of both USP7 and USP47 was induced by doxycycline (Doxy) treatment (1 µg/mL; 3 days), as indicated. All cells were differentiated with PMA and either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and treated with nigericin (10 μM, 45 mins.), as indicated. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-caspase-1 (pro-Casp-1; 45 kDa); mature caspase-1 (mCasp-1; 20 kDa); USP7 and USP47. β-actin is shown as a loading control. Blots are representative of at least 3 independent experiments.


IL - 1β release measured from doxycycline treated ( 1 µg / mL ; 3 days ) PMA - differentiated and LPS - primed ( 1 μg / mL , 4 hrs . ) , nigericin activated ( 10 μM , 45 mins . ) THP - 1 cells , which contain the indicated sgRNA . Bars represent the mean percentage of IL - 1β release relative to their respective non - doxycycline treated control cells , ± S . D . LDH - release measured from cells as treated in B . Bars represent the mean percentage of LDH release relative to their respective non - doxycycline treated control cells , ± S . D .

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IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D.


A . SHOXprotein representation and luciferase assay . The variants found in SHOX are indicated in red . Luciferase assays to test the impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS cells ( n = 4 ) . Experiments were performed in triplicates . pcDNA4 - TO empty vector was used as control . R153L represents a mutation known to affect SHOXprotein activity and was used as positive control ( Schneider et al . , 2005 ) . Homeodomain , DNA binding domain ; Src Homology 3 domain ; OAR , OtpAristalessRax domain . RLU , Relative Light Units .

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A. SHOXprotein representation and luciferase assay. The variants found in SHOX are indicated in red. Luciferase assays to test the impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS cells (n = 4). Experiments were performed in triplicates. pcDNA4-TO empty vector was used as control. R153L represents a mutation known to affect SHOXprotein activity and was used as positive control (Schneider et al., 2005). Homeodomain, DNA binding domain; Src Homology 3 domain; OAR, OtpAristalessRax domain. RLU, Relative Light Units.


B . CYP26C1protein representation and luciferase assay . Variants found in CYP26C1 are indicated in green . Cignal - RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all - trans retinoic acid ( ATRA ) for 24 hours ( n = 4 ) . Experiments were performed in triplicates . pIRES2 - EGFP empty vector was used as control . The residue C459 represents the Iron binding residue ( Q6VOL0 , UniProtKB ) and was mutated to Ala and used as a positive control . TM , Transmembrane helix ; P450 , cytochrome p450 domain . RLU , Relative Light Units .

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B. CYP26C1protein representation and luciferase assay. Variants found in CYP26C1 are indicated in green. Cignal-RARE system luciferase assays to test the impact of CYP26C1 variants on its RA degradation activity were performed in U2OS cells treated with 250 nM all-trans retinoic acid (ATRA) for 24 hours (n = 4). Experiments were performed in triplicates.pIRES2-EGFP empty vector was used as control. The residue C459 represents the Iron binding residue (Q6VOL0, UniProtKB) and was mutated to Ala and used as a positive control. TM, Transmembrane helix; P450, cytochrome p450 domain. RLU, Relative Light Units.


A . Right panel , western blot showing the expression of CYP26C1 in human primary chondrocytes on protein level . Overexpression of CYP26C1 in U2OS cells was carried out to compare protein sizes . + , cDNA ; - , water ; M , marker ; HPC , human primary chondrocytes .

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A. Right panel, western blot showing the expression of CYP26C1 in human primary chondrocytes on protein level. Overexpression of CYP26C1 in U2OS cells was carried out to compare protein sizes. +, cDNA; -, water; M, marker; HPC, human primary chondrocytes.


A . Left panel , RT - PCR showing the expression of CYP26C1 mRNA in human primary chondrocytes . SOX9 was used as chondrocyte marker .

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A. Left panel, RT-PCR showing the expression of CYP26C1 mRNA in human primary chondrocytes. SOX9 was used as chondrocyte marker.


B . Relative expression of SHOX mRNA normalized to the reference genes SDHA and HPRT in human primary chondrocytes treated with ATRA 100 nM for 6h ( n = 5 ) . One outlier with high relative expression ( 2 . 3 fold ) was excluded from ATRA 100 nM treatment ( two - sided Grubb ' s test , Z value 2 . 34 , p - value < 0 . 05 ) .

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B. Relative expression of SHOX mRNA normalized to the reference genes SDHA and HPRT in human primary chondrocytes treated with ATRA 100 nM for 6h (n = 5). One outlier with high relative expression (2.3 fold) was excluded from ATRA 100 nM treatment (two-sided Grubb's test, Z value 2.34, p-value < 0.05).


C . SHOX promoter was cloned in pGL3basic for luciferase reporter experiments as previously described ( Verdin et al , 2015 ) . In silico analysis of SHOX promoter identified three putative RXRa binding sites which were mutated to test their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS cells ( n = 4 ) . Experiments were performed in triplicates . RLU , Relative Light Units .

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C. SHOX promoter was cloned in pGL3basic for luciferase reporter experiments as previously described (Verdin et al, 2015). In silico analysis of SHOX promoter identified three putative RXRa binding sites which were mutated to test their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS cells (n = 4). Experiments were performed in triplicates. RLU, Relative Light Units.


A - C . Wild type embryos injected with control MO or with shox MO . ( A ) Lateral views of the embryos at 55 hours post fertilization ( hpf ) . ( B ) Dorsal view and magnification on the lateral view of the embryos . Dotted line , pectoral fins . shox morphants show smaller fins compared to controls ( n = 30 embryos ) .

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A-C. Wild type embryos injected with control MO or with shox MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins. shox morphants show smaller fins compared to controls (n = 30 embryos).


A - C . Wild type embryos injected with control MO or with shox MO . C . Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type embryos injected with control MO and with shox MO . Arrow and dotted line indicate the pectoral fin .

A-C. Wild type embryos injected with control MO or with shox MO. C. Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type embryos injected with control MO and with shox MO. Arrow and dotted line indicate the pectoral fin.


A - C . Wild type embryos injected with control MO or with shox MO . D . Pectoral fin area was measured by imageJ ( n = 30 embryos ) .

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A-C. Wild type embryos injected with control MO or with shox MO. D. Pectoral fin area was measured by imageJ (n = 30 embryos).


A - D . Wild type embryos injected with control MO or with cyp26c1 MO . ( A ) Lateral views of the embryos at 55 hours post fertilization ( hpf ) . ( B ) Dorsal view and magnification on the lateral view of the embryos . Dotted line , pectoral fins ; * , otic vesicle . cyp26c1 morphants show smaller fins compared to controls ( n = 30 embryos ) .

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A-D. Wild type embryos injected with control MO or with cyp26c1 MO. (A) Lateral views of the embryos at 55 hours post fertilization (hpf). (B) Dorsal view and magnification on the lateral view of the embryos. Dotted line, pectoral fins; *, otic vesicle. cyp26c1 morphants show smaller fins compared to controls (n = 30 embryos).


A - D . Wild type embryos injected with control MO or with cyp26c1 MO . ( C , D ) Expression of col2a1 ( C ) and shox ( D ) at 55 hpf was examined by in situ hybridization in embryos injected with control MO or with cyp26c1 MO . ( C ) Dorsal view and magnification on the pectoral fins of col2a1expression . Arrow and dotted line indicate the pectoral fin . ( D ) Dorsal view and magnification on the pectoral fins of shox expression . Arrow and dotted line indicate the pectoral fin .

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A-D. Wild type embryos injected with control MO or with cyp26c1 MO. (C, D) Expression of col2a1(C) and shox (D) at 55 hpf was examined by in situ hybridization in embryos injected with control MO or with cyp26c1 MO. (C) Dorsal view and magnification on the pectoral fins of col2a1expression. Arrow and dotted line indicate the pectoral fin. (D) Dorsal view and magnification on the pectoral fins of shox expression. Arrow and dotted line indicate the pectoral fin.


E . Pectoral fin area was measured by imageJ ( n = 30 embryos ) .

E. Pectoral fin area was measured by imageJ (n = 30 embryos).


F . Cignal - RARE system luciferase assay testing cyp26c1 MO on RA acid levels in zebrafishembryos ( n = 5 replicates ) . Embryos were separated in groups of 20 - 30 and luciferase assayed . RLU , Relative Light Units .

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F. Cignal-RARE system luciferase assay testing cyp26c1 MO on RA acid levels in zebrafishembryos (n = 5 replicates). Embryos were separated in groups of 20-30 and luciferase assayed. RLU, Relative Light Units.


G . Relative shox mRNA expression normalized to reference genes eef1a and b - actin in zebrafishembryos injected with control MO or cyp26c1 MO ( n = 4 ) . RNA was extracted from 10 - 15 injected embryos at 55 hours post fertilization .

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G. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafishembryos injected with control MO or cyp26c1 MO (n = 4). RNA was extracted from 10-15 injected embryos at 55 hours post fertilization.


H . Relative shox mRNA expression normalized to reference genes eef1a and b - actin in zebrafishembryos treated with 100 nM ATRA ( n = 4 ) . Embryos were collected at 24 hpf and treated with mock control or ATRA . RNA was extracted from 10 - 15 embryos after 6 hours treatment .

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H. Relative shox mRNA expression normalized to reference genes eef1a and b-actin in zebrafishembryos treated with 100 nM ATRA (n = 4). Embryos were collected at 24 hpf and treated with mock control or ATRA. RNA was extracted from 10-15 embryos after 6 hours treatment.


A - C . Wild type embryos injected with control MO , subphenotypic doses of shox MO , cyp26c1 MO or a combination of shox + cyp26c1 MOs . ( A ) Dorsal views of the embryos at 55 hours post fertilization ( hpf ) . Dotted line , pectoral fins . shox + cyp26c1 double morphants show smaller fins compared to control and single MOs ( n = 30 embryos ) .

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A-C. Wild type embryos injected with control MO, subphenotypic doses of shox MO, cyp26c1 MO or a combination of shox + cyp26c1 MOs. (A) Dorsal views of the embryos at 55 hours post fertilization (hpf). Dotted line, pectoral fins. shox + cyp26c1 double morphants show smaller fins compared to control and single MOs (n = 30 embryos).


A - C . Wild type embryos injected with control MO , subphenotypic doses of shox MO , cyp26c1 MO or a combination of shox + cyp26c1 MOs . ( B , C ) Dorsal view and magnification on the pectoral fins of col2a1expression at 55 hpf . Arrow and dotted line indicate the pectoral fin .

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A-C. Wild type embryos injected with control MO, subphenotypic doses of shox MO, cyp26c1 MO or a combination of shox + cyp26c1 MOs. (B, C) Dorsal view and magnification on the pectoral fins of col2a1expression at 55 hpf. Arrow and dotted line indicate the pectoral fin.


D . Pectoral fins area was measured by imageJ ( n = 30 embryos ) .

D. Pectoral fins area was measured by imageJ (n = 30 embryos).


E . Relative expression of shox mRNA normalized to reference genes eef1a and b - actin in zebrafishembryos injected with control MO , shox MO , cyp26c1 MO , or shox + cyp26c1 MOs ( n = 4 ) . RNA was extracted from 10 - 15 injected embryos at 55 hours post fertilization .

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E. Relative expression of shox mRNA normalized to reference genes eef1a and b-actin in zebrafishembryos injected with control MO, shox MO, cyp26c1 MO, or shox + cyp26c1 MOs (n = 4). RNA was extracted from 10-15 injected embryos at 55 hours post fertilization.


Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293 / sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants .

Immunoblot analysis of presenilin expression and endoproteolysis in cell lysates of untransfected HEK293/sw cells endogenously expressing presenilin and single cell clones overexpressing PS1 WT or the indicated PS1 mutants.


Immunoblot analysis of total Aβ secretion and the production of different Aβ species ( shown in low and high immunoblot exposures ) in conditioned media of cell

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Immunoblot analysis of total Aβ secretion and the production of different Aβ species (shown in low and high immunoblot exposures) in conditioned media of cell lines


Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of cell lines


Immunoblot analysis of C99 - PS1 NTF or full - length ( FL ) crosslinks ( CL ) of the indicated mutants . Samples that were not UV - irradiated were loaded to control for specificity .

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Immunoblot analysis of C99-PS1 NTF or full-length (FL) crosslinks (CL) of the indicated mutants. Samples that were not UV-irradiated were loaded to control for specificity.


Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls ( n = 4 - 5 biological replicates ) . Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls ( n = 4 - 5 biological replicates ) .

Changes in the ratio of crosslink efficiencies at positions T48 and L49 compared to corresponding controls (n = 4-5 biological replicates). Changes in the ratio of crosslink efficiencies at positions K54 and V44 compared to corresponding controls (n = 4-5 biological replicates).


ELISA analysis of Aβ species in conditioned medium of HEK293 / sw cells treated with RO7019009 or vehicle ( DMSO ) . Aβ levels are shown relative to DMSO vehicle control ( n = 4 biological replicates ) .

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ELISA analysis of Aβ species in conditioned medium of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Aβ levels are shown relative to DMSO vehicle control (n = 4 biological replicates).


Dose response curve of RO7019009 in HEK293 / sw cells measured using the MSD sandwich - immunoassay ( n = 3 biological replicates ) .

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Dose response curve of RO7019009 in HEK293/sw cells measured using the MSD sandwich-immunoassay (n = 3 biological replicates).


Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines . Comparison of in vitro potencies for Aβ42 inhibition in HEK293 / sw cells , IC50 = 14 nM ( n = 3 ) ( as in ( C ) ) with those in H4 / sw cells , IC50 = 5 nM ( n = 7 ) and N2a / sw cells , IC50 = 2 nM ( n = 6 ) together with that of Notch cleavage inhibition in a HEK293 - based Notch1 luciferase reporter cell line , IC50 > 10 µM ( n = 2 ) ( all n numbers represent biological replicates ) .

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Dose response curves for inhibition of Aβ42 generation and Notch cleavage by RO7019009 in different cell lines. Comparison of in vitro potencies for Aβ42 inhibition in HEK293/sw cells, IC50 = 14 nM (n = 3) (as in (C)) with those in H4/sw cells, IC50 = 5 nM (n = 7) and N2a/sw cells, IC50 = 2 nM (n = 6) together with that of Notch cleavage inhibition in a HEK293-based Notch1 luciferase reporter cell line, IC50 > 10 µM (n = 2) (all n numbers represent biological replicates).


Left panel : Immunoblot analysis of total Aβ in conditioned media of HEK293 / sw cells treated with RO7019009 or vehicle ( DMSO ) . Total APPs levels were analyzed to control for normal APP secretion and equal sample loading . Right panel : Quantification of relative Aβ amounts in ( E ) ( n = 2 biological replicates ) .

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Left panel: Immunoblot analysis of total Aβ in conditioned media of HEK293/sw cells treated with RO7019009 or vehicle (DMSO). Total APPs levels were analyzed to control for normal APP secretion and equal sample loading. Right panel: Quantification of relative Aβ amounts in (E) (n = 2 biological replicates).


Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293 / sw cells after separation by Tris - Bicine urea SDS PAGE ; synthetic Aβ peptides were loaded as size markers .

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Immunoblot analysis of Aβ species in conditioned media of RO7019009 treated HEK293/sw cells after separation by Tris-Bicine urea SDS PAGE; synthetic Aβ peptides were loaded as size markers.


ELISA analysis of Aβ species in conditioned media of HEK293 / sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle ( DMSO ) showing the secretion of Aβ38 ( A ) , Aβ43 ( B ) and Aβ42 ( C ) compared to total Aβ ( Aβ38 + Aβ40 + Aβ42 + Aβ43 ) ( n = 5 biological replicates ) . Note that in some experiments Aβ38 generation was below the detection limit for some mutants . Data are presented as mean ± SD .

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ELISA analysis of Aβ species in conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with RO7019009 or vehicle (DMSO) showing the secretion of Aβ38 (A), Aβ43 (B) and Aβ42 (C) compared to total Aβ (Aβ38 + Aβ40 + Aβ42 + Aβ43) (n = 5 biological replicates). Note that in some experiments Aβ38 generation was below the detection limit for some mutants. Data are presented as mean ± SD.


Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293 / sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle ( DMSO ) .

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Mass spectrometry analysis of Aβ species immunoprecipitated from conditioned media of HEK293/sw cells overexpressing WT or mutant PS1 treated with 500 nM RO7019009 or vehicle (DMSO).


Immunoblot analysis of C99 V44Bpa crosslinking ( CL ) to PS1 WT or NTFs ( circle ) of normally endoproteolysed mutants in the presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in ( A ) . Immunoblot analysis of C99 V44Bpa crosslinking to full - length ( FL ) PS1 ( triangle ) of endoproteolysis - deficient mutants and the corresponding M292D control in the presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in ( C ) .

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Immunoblot analysis of C99 V44Bpa crosslinking (CL) to PS1 WT or NTFs (circle) of normally endoproteolysed mutants in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the WT or mutant NTF in (A). Immunoblot analysis of C99 V44Bpa crosslinking to full-length (FL) PS1 (triangle) of endoproteolysis-deficient mutants and the corresponding M292D control in the presence of vehicle (DMSO) or RO7019009. Quantitation of crosslink efficiencies of C99 V44Bpa to the M292 or FAD mutant FL PS1 in (C).


B , C Colocalization of N1 - 54 : mVenus : OsCRT3 with OFP - HDEL . OFP - HDEL is an ER lumen marker harboring a C - terminal HDEL ER - retention sequence . ( C ) shows a magnification of the box represented in ( B ) . Bar : 20 μm . D Colocalization analyses along the white arrow in ( C ) .

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B, C Colocalization of N1-54:mVenus:OsCRT3 with OFP-HDEL. OFP-HDEL is an ER lumen marker harboring a C-terminal HDEL ER-retention sequence. (C) shows a magnification of the box represented in (B). Bar: 20 μm. D Colocalization analyses along the white arrow in (C).


E , F Colocalization of N1 - 54 : mVenus : OsCRT3 with the ER membrane marker AtCNX1 - mCherry . ( F ) shows a magnification of the box represented in ( E ) . Bar : 20 μm . G Colocalization analyses along the white arrow in ( F ) .

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E, F Colocalization of N1-54:mVenus:OsCRT3 with the ER membrane marker AtCNX1-mCherry. (F) shows a magnification of the box represented in (E). Bar: 20 μm. G Colocalization analyses along the white arrow in (F).


C Cold response of cytosolic free Ca2 + concentration in root cells expressing NES - YC3 . 6 . Superimposition of representative normalized ratio variations for wild - type , oscrt3 - 1 , and the complemented transgenic pOsCRT3 root cells . The rectangle represents the regions of interest ( ROIs ) for the ratio measurements . The blue background indicates duration of cold treatment . Bar : 50 μm .

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C Cold response of cytosolic free Ca2+ concentration in root cells expressing NES-YC3.6. Superimposition of representative normalized ratio variations for wild-type, oscrt3-1, and the complemented transgenic pOsCRT3 root cells. The rectangle represents the regions of interest (ROIs) for the ratio measurements. The blue background indicates duration of cold treatment. Bar: 50 μm.


B Yeast two - hybrid analysis to test the OsCRT3 - OsCIPK7 interaction . The interactions between OsCRT3 , OsCRT3N , OsCRT3P or OsCRT3C with OsCIPK7 , OsCIPK7N , OsCIPK7C , OsCIPK7NAF , or OsCIPK7PPI were verified .

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B Yeast two-hybrid analysis to test the OsCRT3-OsCIPK7 interaction. The interactions between OsCRT3, OsCRT3N, OsCRT3P or OsCRT3C with OsCIPK7, OsCIPK7N, OsCIPK7C, OsCIPK7NAF, or OsCIPK7PPI were verified.


C Pull - down assay of the interaction between OsCRT3 and OsCIPK7 .

C Pull-down assay of the interaction between OsCRT3 and OsCIPK7.


E Localization of OsCIPK7 - GFP in N . benthamiana leaf epidermal cells . OFP - HDEL was used as an ER marker . Bar : 20 μm . F Magnification of the box represented in ( E ) . Colocalization analyses along the white arrow are shown . Bar : 20 μm .

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E Localization of OsCIPK7-GFP in N. benthamiana leaf epidermal cells. OFP-HDEL was used as an ER marker. Bar: 20 μm. F Magnification of the box represented in (E). Colocalization analyses along the white arrow are shown. Bar: 20 μm.


A OsCIPK7 interacts with OsCBLs in a yeast two - hybrid system . Interactions were verified by growing the yeast on selective medium ( SD / - Leu - Trp , SD / - Leu - Trp - His - Ade ) and conducting β - Galactosidase assays .

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A OsCIPK7 interacts with OsCBLs in a yeast two-hybrid system. Interactions were verified by growing the yeast on selective medium (SD/-Leu-Trp, SD/-Leu-Trp-His-Ade) and conducting β-Galactosidase assays.


B Subcellular localization of OsCBL7 and OsCBL8 in N . benthamiana leaf epidermal cells . Pictures were taken after 2 and 3 days of expression . Bar : 50 μm .

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B Subcellular localization of OsCBL7 and OsCBL8 in N. benthamiana leaf epidermal cells. Pictures were taken after 2 and 3 days of expression. Bar: 50 μm.


C Bimolecular fluorescence complementation ( BiFC ) assay confirming the interaction between OsCBL7 / 8 and OsCIPK7 in N . benthamiana leaf epidermal cells . 19K was used as a negative control . Bar : 50 μm .

C Bimolecular fluorescence complementation (BiFC) assay confirming the interaction between OsCBL7/8 and OsCIPK7 in N. benthamiana leaf epidermal cells. 19K was used as a negative control. Bar: 50 μm.


D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three - hybrid assay .

D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three-hybrid assay.


E Constructs and OsCRT3 protein expression level in ( D ) .

E Constructs and OsCRT3 protein expression level in (D).


F Luciferase Complementation Imaging assay ( LCI ) to detect the interaction between OsCIPK7 - nluc and OsCBL7 - cluc or OsCBL8 - cluc , with co - expression of N1 - 54 : mVenus : OsCRT3 ( mVenus - OsCRT3 ) or GFP , respectively . Quantification of luciferase activity was measured and shown . Values are mean ± SD . At least 6 leaves were used for each sample . * P < 0 . 05 , Student ' s t - test . Experiments were repeated at least twice with similar results .

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F Luciferase Complementation Imaging assay (LCI) to detect the interaction between OsCIPK7-nluc and OsCBL7-cluc or OsCBL8-cluc, with co-expression of N1-54:mVenus:OsCRT3 (mVenus-OsCRT3) or GFP, respectively. Quantification of luciferase activity was measured and shown. Values are mean ± SD. At least 6 leaves were used for each sample. *P < 0.05, Student's t-test. Experiments were repeated at least twice with similar results.


G Immunoblot confirming the expression of OsCIPK7 , OsCBL7 , OsCBL8 , and OsCRT3 in ( F ) respectively .

G Immunoblot confirming the expression of OsCIPK7, OsCBL7, OsCBL8, and OsCRT3 in (F) respectively.


H Pull - down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3 . MBP was used as a negative control . Relative expression analysis was conducted using Image J software .

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H Pull-down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3. MBP was used as a negative control. Relative expression analysis was conducted using Image J software.


A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro . The loaded protein is indicated by red arrows . Autoradiograph of γ - 32P - labeled OsCIPK7 indicates autophosphorylation of OsCIPK7 .

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A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro. The loaded protein is indicated by red arrows. Autoradiograph of γ-32P-labeled OsCIPK7 indicates autophosphorylation of OsCIPK7.


B OsCIPK7 didn ' t phosphorylate OsCBL7 or OsCBL8 in vitro . Loaded proteins stained with Coomassie brilliant blue ( CBB ) are shown in the top panel and indicated by red arrows . Autoradiograph of γ - 32P - labeled myelin basic protein and OsCIPK7 are shown in the bottom panel .

B OsCIPK7 didn't phosphorylate OsCBL7 or OsCBL8 in vitro. Loaded proteins stained with Coomassie brilliant blue (CBB) are shown in the top panel and indicated by red arrows. Autoradiograph of γ-32P-labeled myelin basic protein and OsCIPK7 are shown in the bottom panel.


C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein . SDS - PAGE gel showing CBB - stained OsCRT3 , myelin basic protein , and OsCIPK7 ( top panel ) . γ - 32P - labeled myelin basic protein and OsCIPK7 substrate phosphorylation ( bottom panel ) . The amount of OsCRT3 added was 0 μg , 0 . 2 μg , 0 . 4 μg , 0 . 6 μg , 0 . 8 μg in the SDS - PAGE gel lanes , respectively .

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C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein. SDS-PAGE gel showing CBB-stained OsCRT3, myelin basic protein, and OsCIPK7 (top panel). γ-32P-labeled myelin basic protein and OsCIPK7 substrate phosphorylation (bottom panel). The amount of OsCRT3 added was 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg in the SDS-PAGE gel lanes, respectively.


( a ) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine , and then analyzed by western blot using antibodies to LC3 ( top ) , p62 ( middle ) or vinculin ( bottom ) .

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(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).


( b ) Immunofluorescence for p62 and DAPI in the SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 ( n = 3 p62 for each ) . Dotted lines indicate boundaries of the SVZ and granular zone ( GZ ) . The arrows and arrowheads mark larger and smaller p62 + aggregates , respectively , in the striatum ( ST ) and SVZ .

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(b) Immunofluorescence for p62 and DAPI in the SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 (n = 3 p62 for each). Dotted lines indicate boundaries of the SVZ and granular zone (GZ). The arrows and arrowheads mark larger and smaller p62+ aggregates, respectively, in the striatum (ST) and SVZ.


( c ) TEM of mitochondria in SVZ tissue from Ctrl and FIP200GFAP cKO mice at P28 and P56 ( n = 2 mice for each , > 30 cells for each mouse ) . Arrows mark mitochondria .

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(c) TEM of mitochondria in SVZ tissue from Ctrl and FIP200GFAP cKO mice at P28 and P56 (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria.


( d ) TEM of mitochondria in cultured neurospheres from Ctrl and FIP200GFAP cKO mice ( n = 2 mice for each , > 30 cells for each mouse ) . Arrows mark mitochondria .

(d) TEM of mitochondria in cultured neurospheres from Ctrl and FIP200GFAP cKO mice (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria.


( e , f ) DHE and DAPI fluorescence in the dentate gyrus ( e ) and SVZ ( f ) of Ctrl and FIP200GFAP cKO mice at P28 ( n = 5 mice for each ) . Dotted lines indicate the boundaries of the GZ ( e ) and SVZ ( f ) . Arrows mark cells in the SGZ ( e ) and SVZ ( f ) , and arrowheads mark cells in surround regions ( the GZ in e and the ST in f ) . LV , lateral ventricle ; ML , molecular layer . Full - length blots are presented in Supplementary Figure 11 .

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(e,f) DHE and DAPI fluorescence in the dentate gyrus (e) and SVZ (f) of Ctrl and FIP200GFAP cKO mice at P28 (n = 5 mice for each). Dotted lines indicate the boundaries of the GZ (e) and SVZ (f). Arrows mark cells in the SGZ (e) and SVZ (f), and arrowheads mark cells in surround regions (the GZ in e and the ST in f). LV, lateral ventricle; ML, molecular layer. Full-length blots are presented in Supplementary Figure 11.


( a ) Hematoxylin and eosin staining of the dentate gyrus ( DG ) and SVZ from Ctrl and FIP200GFAP cKO mice at P28 . Lines indicate the boundaries of the DG and SVZ . Arrows mark cells in the SVZ .

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(a) Hematoxylin and eosin staining of the dentate gyrus (DG) and SVZ from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark cells in the SVZ.


( b - e ) Immunofluorescence for GFAP , nestin or SOX2 and DAPI in the DG ( b , c ) from Ctrl and FIP200GFAP cKO mice at P28 . The boxed areas in c - e are shown in more detail in insets ( c ) . Dotted lines indicate boundaries of the granular zone ( GZ ) and SGZ ( b ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( b , c ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes .

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(b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the DG (b,c) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in insets (c). Dotted lines indicate boundaries of the granular zone (GZ) and SGZ (b). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.


( b - e ) Immunofluorescence for GFAP , nestin and DAPI in the SVZ ( d , e ) from Ctrl and FIP200GFAP cKO mice at P28 . The boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of the SVZ ( d , e ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( b , c ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes .

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(b-e) Immunofluorescence for GFAP, nestin and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.


( b - e ) Immunofluorescence for GFAP , nestin or SOX2 and DAPI in the SVZ ( d , e ) from Ctrl and FIP200GFAP cKO mice at P28 . The boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of the SVZ ( d , e ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( b , c ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes .

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(b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.


( f ) Immunofluorescence for PSA - NCAM in the RMS ( boxed areas ) , SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 . Lines indicate the boundaries of the DG and SVZ . Arrows mark PSA - NCAM + cells in the RMS , SVZ and DG .

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(f) Immunofluorescence for PSA-NCAM in the RMS (boxed areas), SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark PSA-NCAM+ cells in the RMS, SVZ and DG.


( g ) Immunofluorescence for NeuN in the olfactory bulb ( OB ) and DG of Ctrl and FIP200GFAP cKO mice at P31 . Lines indicate the layers of the OB and boundaries of the DG . Boxed areas in top panels are shown in more detail for staining of NeuN in insets . CC , corpus callosum ; E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer .

(g) Immunofluorescence for NeuN in the olfactory bulb (OB) and DG of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the layers of the OB and boundaries of the DG. Boxed areas in top panels are shown in more detail for staining of NeuN in insets. CC, corpus callosum; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.


( a - e ) Primary ( 1st ) and secondary ( 2nd ) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28 . Representative P0 phase contrast images are shown in a . Mean ± s . e . m . of number ( b , d ) and size ( c , e ) of primary and secondary neurospheres from three independent experiments are shown ( n = 3 mice for each ) .

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(a-e) Primary (1st) and secondary (2nd) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28. Representative P0 phase contrast images are shown in a. Mean ± s.e.m. of number (b,d) and size (c,e) of primary and secondary neurospheres from three independent experiments are shown (n = 3 mice for each).


( f ) Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) and DAPI staining of the SVZ and dentate gyrus ( DG ) from Ctrl and FIP200GFAP cKO mice at P28 . Lines indicate the boundaries of the SVZ . Numbered boxed areas are shown in more detail as insets for staining of TUNEL and DAPI in the SVZ and DG .

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(f) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DAPI staining of the SVZ and dentate gyrus (DG) from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the SVZ. Numbered boxed areas are shown in more detail as insets for staining of TUNEL and DAPI in the SVZ and DG.


( g ) Short - term BrdU incorporation in the SVZ and DG of Ctrl and FIP200GFAPcKO mice at P28 . Dotted lines indicate the boundaries of the SVZ and granular zone ( GZ ) . Boxed areas are shown in more detail in the insets . Lines indicate the boundaries of the SVZ and GZ . Arrows mark BrdU + cells . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 .

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(g) Short-term BrdU incorporation in the SVZ and DG of Ctrl and FIP200GFAPcKO mice at P28. Dotted lines indicate the boundaries of the SVZ and granular zone (GZ). Boxed areas are shown in more detail in the insets. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.


( h ) Long - term retention of BrdU in the SVZ and dentate gyrus of Ctrl and FIP200GFAP cKO mice at P31 . Lines indicate the boundaries of the SVZ and GZ . Arrows mark BrdU + cells . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 .

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(h) Long-term retention of BrdU in the SVZ and dentate gyrus of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.


( a ) Lysates extracted from neurospheres of Ctrl , FIP200GFAP cKO , Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin .

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(a) Lysates extracted from neurospheres of Ctrl, FIP200GFAP cKO, Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin.


( b ) Mean ± s . e . m . of relative mRNA level ( normalized to Ctrl mice as 1 ) of Cdkn1a , Bbc3 , Fas , Pax6 and Stat3 in neurospheres of Ctrl , FIP200GFAP cKO , 2cKO and Trp53GFAP cKO mice are shown ( n = 3 mice for each ) .

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(b) Mean ± s.e.m. of relative mRNA level (normalized to Ctrl mice as 1) of Cdkn1a, Bbc3, Fas, Pax6 and Stat3 in neurospheres of Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each).


( c , d ) Mean ± s . e . m . of the number ( c ) and size ( d ) of primary and secondary neurospheres from Ctrl , FIP200GFAP cKO , 2cKO and Trp53GFAP cKO mice at P28 ( n = 3 mice for each ) .

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(c,d) Mean ± s.e.m. of the number (c) and size (d) of primary and secondary neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice at P28 (n = 3 mice for each).


( e - h ) Analysis of immunofluorescence or staining on frozen - sectioned neurospheres for nestin , Ki67 , TUNEL , PAX6 , phosphorylated STAT3 ( pSTAT3 ) and DAPI , as indicated . Mean ± s . e . m . of the percentage of Ki67 + ( e ) , BrdU + ( f ) and TUNEL + ( g ) cells in neurospheres from Ctrl , FIP200GFAP cKO , 2cKO and Trp53GFAP cKO mice are shown ( n = 3 mice for each , counting of > 500 cells for each mouse ) . Representative images are shown in h . * P 0 . 01 . Full - length blots are presented in Supplementary Figure 11 .

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(e-h) Analysis of immunofluorescence or staining on frozen-sectioned neurospheres for nestin, Ki67, TUNEL, PAX6, phosphorylated STAT3 (pSTAT3) and DAPI, as indicated. Mean ± s.e.m. of the percentage of Ki67+ (e), BrdU+ (f) and TUNEL+ (g) cells in neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each, counting of >500 cells for each mouse). Representative images are shown in h. *P 0.01. Full-length blots are presented in Supplementary Figure 11.


( a - c ) Hematoxylin and eosin staining of the dentate gyrus and dentate gyrus ( DG ) of P28mice ( a ) . Arrows mark cells in the SVZ . Mean ± s . e . m . of the SVZ cellularity ( b ) and DG area ( c ) per section are shown . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hematoxylin and eosin staining of the dentate gyrus and dentate gyrus (DG) of P28mice (a). Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( d - m ) Immunofluorescence of the DG ( d - h ) of P28mice ( d , e , i , j ) . Boxed areas are shown in more detail in insets ( e , j ) and / or panels below ( i , j ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( d , e ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . Mean ± s . e . m . of the number of GFAP + nestin + and GFAP + SOX2 + radial glia ( f , h ) , and NSCs ( k , m ) , and GFAP + nestin − astrocytes ( g , l ) per section are shown . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-m) Immunofluorescence of the DG (d-h) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( d - m ) Immunofluorescence of the SVZ ( i - m ) of P28mice ( d , e , i , j ) . Boxed areas are shown in more detail in insets ( e , j ) and / or panels below ( i , j ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( d , e ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . Mean ± s . e . m . of the number of GFAP + nestin + and GFAP + SOX2 + radial glia ( f , h ) , and NSCs ( k , m ) , and GFAP + nestin − astrocytes ( g , l ) per section are shown . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

(d-m) Immunofluorescence of the SVZ (i-m) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( n , o ) Mean ± s . e . m . of the number of TUNEL + cells per 100 SVZ cells ( n ) or per 1 mm2 DG area ( o ) of P28 mice .

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

(n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28 mice.


( p - r ) BrdU retention in the SVZ and DG of P31mice . BrdU + cells are marked by arrows ( p ) . Mean ± s . e . m . of the number of BrdU + cells per section are shown ( q , r ) . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-r) BrdU retention in the SVZ and DG of P31mice. BrdU+ cells are marked by arrows (p). Mean ± s.e.m. of the number of BrdU+ cells per section are shown (q,r). Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( s , t ) Mean ± s . e . m . of percentage of GFAP + nestin + BrdU + cells of total BrdU retained cells in the SVZ ( s ) and SGZ ( t ) of P31mouse . n = 5 mice , ≥ 4 sections per mouse , > 500 cells counted per mouse in n , > 20 BrdU + cells counted per mouse .

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

(s,t) Mean ± s.e.m. of percentage of GFAP+nestin+BrdU+ cells of total BrdU retained cells in the SVZ (s) and SGZ (t) of P31mouse. n = 5 mice, ≥4 sections per mouse, >500 cells counted per mouse in n, >20 BrdU+ cells counted per mouse.


( a - f ) Immunofluorescence for PSA - NCAM ( a - c ) or NeuN ( d - f ) in the dentate gyrus ( DG ) , SVZ , RMS and olfactory bulb ( OB ) of P28mice . Lines indicate boundaries of the DG ( a , d ) , SVZ ( a ) , and layers of the OB ( d ) . Boxed areas in a indicate the RMS . Arrows mark PSA - NCAM + cells . Mean ± s . e . m . of the number of PSA - NCAM + cells ( b , c ) or NeuN + cells ( e , f ) per section are shown . LV , lateral ventricle ; ML , molecular layer ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer . NS , not significant ; * P 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28mice. Lines indicate boundaries of the DG (a,d), SVZ (a), and layers of the OB (d). Boxed areas in a indicate the RMS. Arrows mark PSA-NCAM+ cells. Mean ± s.e.m. of the number of PSA-NCAM+ cells (b,c) or NeuN+ cells (e,f) per section are shown. LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.


( g - i ) Immunofluorescence of BrdU retention cells in the DG and OB of P31 mice . Boxed areas in left column of g are shown in more detail in right column . Dotted lines indicate the boundaries of the DG ( left ) and GZ ( right ) . Arrows mark NeuN + BrdU + cells and arrowheads mark NeuN − BrdU + cells in the DG . Mean ± s . e . m . of the percentage of NeuN + BrdU + cells of total BrdU retained cells in the DG ( h ) and OB ( i ) are shown . ( j , k ) Mean ± s . e . m . of percentage of GFAP + nestin − BrdU + cells among total BrdU + retained cells in the SVZ ( j ) and SGZ ( k ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of BrdU retention cells in the DG and OB of P31 mice. Boxed areas in left column of g are shown in more detail in right column. Dotted lines indicate the boundaries of the DG (left) and GZ (right). Arrows mark NeuN+BrdU+ cells and arrowheads mark NeuN−BrdU+ cells in the DG. Mean ± s.e.m. of the percentage of NeuN+BrdU+ cells of total BrdU retained cells in the DG (h) and OB (i) are shown. (j,k) Mean ± s.e.m. of percentage of GFAP+nestin−BrdU+ cells among total BrdU+ retained cells in the SVZ (j) and SGZ (k).


( l , m ) DHE and DAPI fluorescence at P28 in the DG ( l ) and SVZ ( m ) from FIP200GFAP cKO mice with or without NAC treatment , 2cKO mice and Trp53GFAP cKO mice . Dotted lines indicate the boundaries of the granular zone ( GZ ) ( l ) and SVZ ( m ) . Arrows mark cells in the SGZ ( l ) and SVZ ( m ) and arrowheads mark cells in surrounding regions ( the GZ in l and the striatum ( ST ) in m ) . n = 5 mice , ≥ 4 sections per mouse , > 20 ( h , j , k ) or > 200 ( i ) BrdU + cells counted per mouse . cKO ( n ) , NAC non - responder cKO mice ; LV , lateral ventricle ; ML , molecular layer ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer . NS , not significant ; * P 0 . 05 .

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

(l,m) DHE and DAPI fluorescence at P28 in the DG (l) and SVZ (m) from FIP200GFAP cKO mice with or without NAC treatment, 2cKO mice and Trp53GFAP cKO mice. Dotted lines indicate the boundaries of the granular zone (GZ) (l) and SVZ (m). Arrows mark cells in the SGZ (l) and SVZ (m) and arrowheads mark cells in surrounding regions (the GZ in l and the striatum (ST) in m). n = 5 mice, ≥4 sections per mouse, >20 (h,j,k) or >200 (i) BrdU+ cells counted per mouse. cKO(n), NAC non-responder cKO mice; LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.


( a - c ) Hematoxylin and eosin staining ( a ) of the SVZ and dentate gyrus ( DG ) of P28mice treated by NAC or vehicle ( Veh ) control . Dotted lines indicate boundaries for the SVZ and DG . Arrows mark cells in the SVZ .

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

(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ.


( a - c ) Hematoxylin and eosin staining ( a ) of the SVZ and dentate gyrus ( DG ) of P28 mice treated by NAC or vehicle ( Veh ) control . Dotted lines indicate boundaries for the SVZ and DG . Arrows mark cells in the SVZ . Mean ± s . e . m . of the SVZ cellularity ( b ) and DG area ( c ) per section are shown .

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

(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28 mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown.


( d - m ) Immunofluorescence of the DG ( d - h ) and SVZ ( i - m ) of P28 mice treated by NAC or vehicle control . Representative images are shown in d , g ( DG ) and k , m ( SVZ ) . Dotted lines indicate boundaries of the granular zone ( GZ ) ( d , g ) and SVZ ( k , m ) . Boxed areas in g , k , m are shown in more detail in insets ( g , m ) and / or panels below ( k , m ) . Lines indicate boundaries of the GZ ( d , g ) and SVZ ( k , m ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( d , g ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . Mean ± s . e . m . of the number of GFAP + nestin + and GFAP + SOX2 + radial glia ( e , h ) , and NSCs ( i , l ) , and GFAP + nestin − astrocytes ( f , j ) per section are shown . ( n , o ) Mean ± s . e . m . of the number of TUNEL + cells per 100 SVZ cells ( n ) or per 1 mm2 DG area ( o ) of P28mice treated by NAC or vehicle control . n = 5 mice , ≥ 4 sections per mouse , > 500 cells counted per NAC in n . cKO ( n ) , NAC non - responder cKO mice ; E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . * * P 0 . 01 .

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

(d-m) Immunofluorescence of the DG (d-h) and SVZ (i-m) of P28 mice treated by NAC or vehicle control. Representative images are shown in d,g (DG) and k,m (SVZ). Dotted lines indicate boundaries of the granular zone (GZ) (d,g) and SVZ (k,m). Boxed areas in g,k,m are shown in more detail in insets (g,m) and/or panels below (k,m). Lines indicate boundaries of the GZ (d,g) and SVZ (k,m). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,g), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (e,h), and NSCs (i,l), and GFAP+nestin− astrocytes (f,j) per section are shown. (n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28mice treated by NAC or vehicle control. n = 5 mice, ≥4 sections per mouse, >500 cells counted per NAC in n. cKO(n), NAC non-responder cKO mice; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. **P 0.01.


( a - f ) Immunofluorescence for PSA - NCAM ( a - c ) or NeuN ( d - f ) in the dentate gyrus ( DG ) , SVZ , RMS and olfactory bulb ( OB ) of P28 mice treated with NAC or vehicle ( Veh ) control . Representative images are shown for PSA - NCAM ( a ) and NeuN ( d ) . Dotted lines indicate boundaries of the DG ( a , d ) , SVZ ( a ) and layers of the OB ( d ) . Arrows mark PSA - NCAM + cells in a . Mean ± s . e . m . of the number of PSA - NCAM + cells in SVZ ( b ) and DG ( normalized to 1 mm2 area ) ( c ) , and the number of NeuN + cells in DG ( e ) and IGL of OB ( per mm2 ) ( f ) per section are shown ( n = 5 mice for each ; ≥ 4 sections for each mouse ) . cKO ( n ) , NAC non - responder cKO mice ; GZ , granular zone ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer . * * P 0 . 01 .

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

(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice treated with NAC or vehicle (Veh) control. Representative images are shown for PSA-NCAM (a) and NeuN (d). Dotted lines indicate boundaries of the DG (a,d), SVZ (a) and layers of the OB (d). Arrows mark PSA-NCAM+ cells in a. Mean ± s.e.m. of the number of PSA-NCAM+ cells in SVZ (b) and DG (normalized to 1 mm2 area) (c), and the number of NeuN+ cells in DG (e) and IGL of OB (per mm2) (f) per section are shown (n = 5 mice for each; ≥4 sections for each mouse). cKO(n), NAC non-responder cKO mice; GZ, granular zone; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. **P 0.01.


A Lysates of HeLa cells stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( shown a mean value , * P < 0 . 05 , * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (shown a mean value, *P < 0.05, **P < 0.01; N = 3).


B Lysates of HeLa cells stably expressing GFP alone or 3xGFP - Nup85 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( SE , short exposure , LE , long exposure ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of HeLa cells stably expressing GFP alone or 3xGFP-Nup85 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (SE, short exposure, LE, long exposure) (shown a mean value, *P < 0.05; N = 3).


C Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot . The arrow points to the heavy chain of IgG ( IgG HC ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot. The arrow points to the heavy chain of IgG (IgG HC) (shown a mean value, *P < 0.05; N = 3).


D HeLa cells were treated with indicated siRNAs , synchronized by double thymidine block , and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor ( LBR ) to label the NE , and FXR1 .

O O O O O O O O O O O O O O O O O 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 HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1.


E HeLa cells stably expressing GFP - FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414 , which labels FG - Nups . The magnified framed regions are shown in the corresponding numbered panels . The arrowheads indicate NE and cytoplasmic localization of GFP - FXR1 .

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

E HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1.


F HeLa cells stably expressing GFP - Nup107 were synchronized by double thymidine block and released for 12 hours , permeabilized with Triton / SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus , respectively , and analysed by immunofluorescence microscopy .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 hours, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus, respectively, and analysed by immunofluorescence microscopy.


A HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy . The magnified framed regions are shown in the corresponding numbered panels .

O O O O O O O O O O O O O O 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 HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


B HeLa cells stably expressing GFP , GFP - FXR1 wild type ( WT ) and GFP - FXR1 mutated in the sequence recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with the indicated siRNAs , synchronized by double thymidine block , released for 24 hours and then analysed by immunofluorescence microscopy . The percentage of cells with cytoplasmic nucleoporin granules was quantified , 1000 cells were analysed for each graph ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 , N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3).


HeLa cells were treated with the indicated siRNAs , synchronized by double thymidine block , released for 12 hours and analysed by immunofluorescence microscopy . Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right . Additional or complementary representative images and channels of cells depicted in ( C )

HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C)