( D ) Genomic PCR showing Cre - mediated recombination of BraffloxAVKA allele in vivo using primer pair E14fwd E15rev ( indicated as blue arrows in A ) .

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(D) Genomic PCR showing Cre-mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev (indicated as blue arrows in A).


( A ) at weaning of BrafAVKA mice from two different founders showed no significant differences . 215 : n = WT = 24 / Heterozygous = 32 / AVKA = 19 ; 273 : n = WT = 114 / Heterozygous = 117 / AVKA = 52 , mean + , t -

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(A) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences. 215: n = WT = 24/ Heterozygous = 32/ AVKA = 19; 273: n = WT = 114/ Heterozygous = 117/ AVKA = 52, mean + SEM, t-test


( B ) Homozygous BrafAVKA mice show reduced splenic cellularity . n = 7 , mean + , t - , * p < 0 . 05 .

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(B) Homozygous BrafAVKA mice show reduced splenic cellularity. n = 7, mean + SEM, t-test, * p < 0.05.


( C - E ) Relative number of CD8 + T . FACS shows an increase in naïve ( C ) , whereas differentiated ( D ) Tregs ( E ) reveal a reduction in BrafAVKA mice compared to WT . n = 8 , mean + , t - , * p < 0 . 05 , * * p < 0 . 01 .

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(C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01.


( F ) B of BrafAVKA mice show reduced CD69 surface expression upon LPS anti - IgM stimulation . n = 3 , mean + , , * * p < 0 . 01 .

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(F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01.


( G ) B also show reduced proliferation upon stimulation compared with WT B . n = 3 , mean + , , * * p < 0 .

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(G) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells. n = 3, mean + SEM, test, ** p < 0.01


( A ) Facial appearance of BrafAVKA mice .

(A) Facial appearance of BrafAVKA mice.


( B ) Top : In vitro kinase ( IVK ) of B - Raf complexes purified from either WT or BrafAVKA knock - in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates . Botttom : of IVK differentials : # 215 samples indicated in black , # 273 samples in grey .

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(B) Top: In vitro kinase (IVK) assay of B-Raf complexes purified from either WT or BrafAVKA knock-in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates. Botttom: Quantification of IVK differentials: #215 samples indicated in black, #273 samples in grey.


( C ) Western blot showing reduced pMEK pERK in BrafAVKA brains . n = 5 , mean + , t - , * * < 0 . 01 .

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(C) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains. n = 5, mean + SEM, t-test, ** < 0.01.


( D ) Immunohistochemistry showing reduced pERK in Purkinje of homozygous BrafAVKA mice . Scale bar indicates 10x magnification .

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(D) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice. Scale bar indicates 10x magnification.


( A ) Total brain lysates of WT BrafAVKA mice were subject to immunoprecipitation ( IP ) using either anti - B - Raf antibodies or non - specific IgG as negative control . co - purified were visualized using indicated antibodies quantified . Ksr1 is significantly decreased in B - RafAVKA complexes while other are equally bound to B - RafWT or B - RafAVKA . n = 3 , mean + , t - , * p < 0 . 05 .

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(A) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation (IP) using either anti-B-Raf antibodies or non-specific IgG as negative control. The co-purified proteins were visualized using the indicated antibodies and quantified. Ksr1 is significantly decreased in B-RafAVKA complexes while the other proteins are equally bound to B-RafWT or B-RafAVKA. n = 3, mean + SEM, t-test, * p < 0.05.


( B ) Anti - Myc IP of Plat - E transfected with Myc - tagged Raf - 1 different HA - B - Raf constructs . Western blot showed that B - RafAVKA is not impaired in its ability to interact with Raf - 1 .

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(B) Anti-Myc IP of Plat-E cells transfected with Myc-tagged Raf-1 and different HA-B-Raf constructs. Western blot analysis showed that B-RafAVKA is not impaired in its ability to interact with Raf-1.


( C ) Anti - Myc IP of Plat - E expressing different Myc - HA - B - Raf . Note that B - RafAVKA is not impaired in its ability to interact with other B - Raf , while homo - dimerization of B - RafEVKD is enhanced .

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(C) Anti-Myc IP of Plat-E cells expressing different Myc- and HA-B-Raf proteins. Note that B-RafAVKA is not impaired in its ability to interact with other B-Raf proteins, while homo-dimerization of B-RafEVKD is enhanced.


( D ) Top : Exemplary Western blot of EGF stimulated astrocytes . Mutant astrocytes show drastic reduction in basic stimulated of pMEK pERK . Bottom : of Western blot samples . WT : n = 7 , AVKA : n = 6 , mean + , t - , * p < 0 . 05 , * * p < 0 . 01 .

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(D) Top: Exemplary Western blot analysis of EGF stimulated primary astrocytes. Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK. Bottom: Quantification of all Western blot samples. WT: n = 7, AVKA: n= 6, mean + SEM, t-test, * p <0.05, ** p < 0.01.


( A ) MEFs expressing B - RafWT or B - RafAVKA were stimulated with EGF for indicated time points ( min ) analyzed by Western blotting .

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(A) MEFs expressing B-RafWT or B-RafAVKA were stimulated with EGF for the indicated time points (min) and analyzed by Western blotting.


( B ) IP of EGF stimulated MEFs expressing B - RafWT or B - RafAVKA . Sorafenib was used to increase build up .

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(B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up.


( C ) Proliferation of two different MEF pools expressing either B - RafWT or B - RafAVKA . MEFs expressing B - RafAVKA show a strong reduction in growth rate . n = 3 , mean + , t - , * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) Proliferation of two different MEF pools expressing either B-RafWT or B-RafAVKA. MEFs expressing B-RafAVKA show a strong reduction in growth rate. n = 3, mean + SEM, t-test, ** p < 0.01, *** p < 0.001.


( D ) of B - RafAVKA expressing MEFs are enlarged present with abnormal

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(D) Cell morphology of B-RafAVKA expressing MEFs are enlarged and present with abnormal nuclei


( E ) MEFs expressing B - RafAVKA show a significant increase in caspase - 3 activity . n = 3 , mean + , t - , * * p < 0 . 01 .

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(E) MEFs expressing B-RafAVKA show a significant increase in caspase-3 activity. n = 3, mean + SEM, t-test, ** p < 0.01.


( F ) Decay of TAg expression in MEFs following dox withdrawal .

(F) Decay of TAg expression in MEFs following dox withdrawal.


( G ) of MEFs expressing B - Rafwt or B - RafAVKA in presence or absence of TAg .

(G) Morphology of MEFs expressing B-Rafwt or B-RafAVKA in the presence or absence of TAg.


( A ) MEFs were then infected with retroviral vectors encoding indicated B - Raf . Giemsa revealed transformed hyper - proliferated by purple colour . ( B ) of Focus formation potential of B - Raf mutants used in ( A ) . n = 3 , mean + , t - , * * * p < 0 . 001 .

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(A) MEFs were then infected with retroviral vectors encoding the indicated B-Raf proteins. Giemsa staining revealed transformed and hyper-proliferated cells by dark purple colour. (B) Quantification of Focus formation potential of the B-Raf mutants used in (A). n = 3, mean + SEM, t-test, *** p < 0.001.


A . Example of littermate of indicated genotypes 26 days post partum . Additional examples are provided in Appendix Fig S2 .

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A. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2.


B . Weights of mice of indicated genotypes from 18 to 120 days post partum . Boxplots indicate 5 - 95 percentile . A minimum of 8 is plotted at each . Asterisks denote statistical significance ( NS , not significant , * P value < 0 . 05 , * * P value < 0 . 01 * * * P value < 0 . 001 ) determined by unpaired two - way Wilcoxon rank - sum .

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B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test.


C . Example of dissected brains ( upper panel ) coronal sections ( left right panels ) stained with H & amp ; amp ; E of indicated genotypes . Note severe hydrocephaly ventricular dilation of lateral ventricle ( LV ) third ventricle ( TV ) in a Gemc1 - / - mouse ( bottom right panel ) compared to a Gemc1 + / + littermate ( bottom left panel ) . Periventricular malacia ( asterisks ) hippocampal atrophy ( arrow ) is also evident in Gemc1 - / - sample . Scale bars = 500 µm .

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C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&amp;E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm.


D . Kaplan Meyer survival curve of a cohort of mice of indicated genotypes numbers ( n ) . study was terminated at 16 months mice were examined for histology . 97 % ( 31 / 32 ) of Gemc1 - / - mice examined to date exhibited hydrocephaly ( additional histological examples in Appendix Fig S2 ) .

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D. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2).


E . RT - QPCR of Gemc1 expression in murine tissues from Gemc1 + / + mice using a Taqman probe . Data is compiled from duplicate samples of 3 mice per tissue mean standard deviation are plotted .

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E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted.


F . RT - QPCR of Gemc1 in trachea , oviduct ovary tissue from Gemc1 + / + Gemc1 - / - mice shows that knockout reduces Gemc1 mRNA to undetectable . Mean standard deviation of duplicate samples from 2 mice are plotted . Additional examples from other Gemc1 - / - tissues are shown in Appendix Fig S1 .

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F. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.


. Example of dissected testes from littermate mice of indicated genotypes at 2 months of .

. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age.


B . Testes from Gemc1 - / - mice have strongly reduced cellularity compared to Gemc1 + / + or Gemc1 + / - , as measured by disaggregation of tissue counting with a Neubauer Chamber ( n = 3 ) . Mean standard deviation are indicated .

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B. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated.


C . Histological of tubules from 6 - week - old Gemc1 - / - mice revealed thinning of spermatogenic layer decreased numbers of elongated spermatids . Scale bars = 100 µm ( left panels ) 50 µm ( right panels ) .

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C. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels).


D . Sperm from cauda epididymis of wild type Gemc1 - / - mice revealed no sperm in mice lacking GEMC1 ( n = 3 ) . Mean standard deviation are indicated . E . Examples of sperm from spermatocyte spreads from 2 month old mice . Scale bars = 10 µm .

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D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm.


F G . Histological evaluation of caput epididymis in sagittal ( F ) coronal ( G ) orientation from 2 4 month old , respectively . Note absence of luminal spermatozoa in Gemc1 - / - mice compared to wild type . Scale bars = 100 µm ( F , left panels ) 50 µm ( F , right panels G ) .

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F and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).


A . Examples of ovaries from Gemc1 + / + ( top panels ) Gemc1 - / - mice ( bottom panels ) at 6 of . Ovaries were smaller in Gemc1 - / - mice contained primarily degenerated antral follicles . A histologically normal antral follicle is indicated in Gemc1 + / + ovary ( top right , black arrow ) degenerated antral follicles with high of are indicated in Gemc1 - / - ovaries ( bottom right , red arrows ) . Scale bars = 200 µm ( left panels ) ) 100 µm ( right top panels right bottom panel respectively ) . B . Morphological abnormalities in oviduct of 3 - week - old Gemc1 - / - mice ( right panels ) compared to Gemc1 + / + ( left panels ) . A clear lack of cilia ( bottom panels ) in Gemc1 - / - oviducts is observed by H & amp ; amp ; E . Scale bars = 50 µm ( top panels ) .

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A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&amp;E staining. Scale bars = 50 µm (top panels).


C . of frozen sections of oviduct with antibodies for Ac - tubulin ( cilia ) γ - tubulin ( basal bodies ) reveals normal cilia organization in wild type oviduct ( left ) while basal bodies cilia are not detectable in Gemc1 - / - oviducts ( right ) . D . FOXJ1 E . RFX3 that is characteristic of MCCs was clearly evident in Gemc1 + / + oviducts absent in Gemc1 - / - mice . Scale bars = 50 µm ( C - E ) .

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C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).


A . H & amp ; amp ; E of nasal mucosa of E17 . 5 of indicated genotype . Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1 + / + compared to attenuated , flat columnar to squamous epithelium of Gemc1 - / - . Scale bars = 20 µm .

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A. H&amp;E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1+/+ embryos compared to the attenuated, flat columnar to squamous epithelium of Gemc1-/- embryos. Scale bars = 20 µm.


B . PAS ( purple ) of nasal mucosa of E18 . 5 of indicated genotype . Normal PAS positive goblet ( inset , red arrows ) are interspersed between MCCs ( inset , black arrows ) with their characteristic goblet or cup - like shape in Gemc1 + / + . Gemc1 - / - mucosa has abundant PAS positive materials that accumulate in apical borders of almost every columnar epithelial ( inset , red arrows ) lacks typical goblet . Scale bars = 20 µm .

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B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm.


C . Transmission electron of cross - sections of adult trachea from Gemc1 + / + revealed presence of secretory ( SC ) multiciliated ( MCC ) . apical of MCCs accumulated basal bodies ( BB ) nucleating multiple cilia ( C ) microvilli ( MV ) . In Gemc1 - / - airway epithelium , no MCCs are identifiable only secretory - like with MV at apical surface are detectable . Scale bars = 5µm . Higher magnifications of boxed regions are shown below , scale bars = 0 . 5µm .

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C. Transmission electron micrographs of cross-sections of adult trachea from Gemc1+/+ animals revealed the presence of secretory cells (SC) and multiciliated cells (MCC). The apical membrane of MCCs accumulated basal bodies (BB) nucleating multiple cilia (C) and microvilli (MV). In the Gemc1-/- airway epithelium, no MCCs are identifiable and only secretory-like cells with MV at the apical surface are detectable. Scale bars = 5µm. Higher magnifications of the boxed regions are shown below, scale bars = 0.5µm.


D . Lack of Ac - tubulin ( top panel ) RFX3 ( second panel from top ) in trachea of 1 month old mice .

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D. Lack of Ac-tubulin (top panel) and RFX3 (second panel from top) staining in the trachea of 1 month old mice.


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

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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 F . RT - QPCR of genes related to MCC formation in oviduct ( E ) trachea ( F ) of Gemc1 - / - mice compared to wild type , 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 ) 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 DP1 . Western blot of HA or FLAG tagged expression in input lysates are shown in top panel . FLAGimmunoprecipiation ( IP ) of GEMC1co - IPs DP1 ( α - HA , lane 5 ) E2F4 ( α - HA , lane 6 ) . interaction is enhanced upon co - expression of HA tagged E2F4 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 ) E2F5 - DP1 ( α - HA , lane 6 ) but not E2F1 or E2F1 - DP1 ( α - HA , lanes 2 5 ) . In FLAG - IP panels , a short ( top ) long ( second from top ) exposure of 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 domain required for E2F4 interaction . GFP tagged GEMC1 mutant forms were expressed IPs were performed against GFP . Wild type GEMC1 ( lane 5 ) or GEMC1 lacking CC domain ( lane 6 ) pulled down E2F4 - DP1 . A GEMC1 lacking 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 single acid analogous that identified in MCIDAS in human RGMC ( G313D ) impairs interaction with E2F4 - DP1 ( compare land 5 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 TIRT domain pull down Myc - tagged Multicilin ( anti - Myc blot , lanes 4 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 of FOXJ1 MCIDAS expression ( RT - QPCR ) in HEK293T . Results for MCIDAS are graphed on a smaller scale in right panel : V = vector G = GEMC1 . mean standard deviation of 3 experiments in HEK293T are graphed . Similar induction of FOXJ1 was observed in U2OS 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 CC TIRT domain of GEMC1 are required to activate FOXJ1 ( RT - QPCR ) . A corresponding Western blot showing relative expression of mutant GFP tagged GEMC1 variants is shown from HEK293T . mean standard deviation of 3 experiments in HEK293T 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 co - expression of Geminin ( compare 3rd 4th lanes ) . Example Western blot is shown below . Results graphed are mean standard deviation of 3 experiments in HEK293T .

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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 Multicilin results in similar of FOXJ1 expression ( measured by RT - QPCR ) as Multicilin alone in HEK293T . mean standard deviation of 4 experiments in HEK293T 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 F . activation of FOXJ1 by GEMC1 is partially dependent on Multicilin . HEK293T were transfected with a control siRNA ( siC ) or 2 different siRNAs against MCIDAS ( si1 si2 ) prior to being transfected with GFP or GFP - GEMC1 expression vectors . Relative MCIDAS ( E ) FOXJ1 ( F ) expression were assessed by RT - QPCR . mean 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 mean , ± S . D . n = 13 11 independent blood donors for nigericin CPPD , respectively . IL - 18 ELISA of supernatants from MDMs treated as in A . Bars represent mean , ± S . D . n = 11 independent blood donors .

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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 mean of LDH release relative to total lysed , ± S . D . n = 10 11 independent blood donors for nigericin 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 lysates from MDMs treated as in A . Bands in 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 ) 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 mRNA expression of IL - 1β , IL - 18 , ASC , CASP1 NLRP3 in MDMs treated with P22077 ( 2 . 5 µM ) / or LPS ( 1 μg / mL ; 4 hrs . ) , as indicated . Bars represent 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 relative abundance of NLRP3 , ASC , pro - caspase - 1 , pro - IL - 1β , pro - IL - 18 , USP7 USP47 in lysates of MDMs treated with P22077 ( 2 . 5 µM ) / 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 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 lysates from MDMs as treated in C . Bands in figure represent : Pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) 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 mean of LDH release relative to total 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 lysates . PMA differentiated THP1 were LPS - primed ( 1 μg / mL , 4 hrs . ) pre - incubated for 15 min with either 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) . followed by treatment with nigericin ( 10 μM , 45 mins . ) . ' input ' represents abundance of ASC in lysates prior to isolation of 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 . 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.


from an immunofluorescence experiment showing ASC - speck formation in THP1 treated as in A . show that were stained with an anti - ASC antibody . are representative of 3 independent experiments . Scale bar 20µm . of ASC - speck formation in depicted in C . number of ASC specks was quantified expressed as of 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 indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 min . ) . Digitonin - solubilised lysates were analysed by native - PAGE or SDS - PAGE NLRP3 western - blotting . Western blots of supernatants lysates for IL - 1β are also shown as a control for P22077 inflammasome inhibition . Bands in figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) NLRP3 ( 120 kDa ) . β - actin is shown as a loading control . Blots are representative of 4 independent murine donors .

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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 indicated concentrations ) for 15 mins . before treatment with ATP ( 5 mM , 30 mins . ) . Endogenous NLRP3 was immunoprecipitated ( IP ) with anti - NLRP3 probed with either anti - ubiquitin ( Poly - Ub ) , anti - K63 - ubiquitin ( Poly - K63 - Ub ) or anti - NLRP3 . As a negative control , lysate made from LPS - primed 10 μM P22077 treated was immunoprecipitated with a nonspecific IgG . Total of NLRP3 in lysates ( input ) were also probed . Western blots of supernatants lysates for IL - 1β are also shown as a control for P22077 inhibition . Bands in figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) NLRP3 ( 120kDa ) . β - actin is shown as a loading control . Blots are representative of 2 independent murine donors for 2 . 5µM P22077 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 USP47 upon treatment with nigericin . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) then treated with nigericin ( 10 μM ) for differing lengths of time , as indicated . Relative , activity - labelling of USP7 USP47 was analysed by western blot after incubation with ABPs . Higher panels show lysates incubated with ABPs ( labelled ) , with active DUB bound to ABP indicated by suffix ' - VME ' . Lower panels , without probe labelling , show total USP7 or USP47 . ( Unlabelled ) . Western blots are representative of 3 independent MDM donors .

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

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 USP47 upon treatment with CPPD . MDMs were either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) 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 are representative of 3 independent MDM donors .

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

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 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 are representative of 3 independent MDM donors .

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 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

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

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

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 lysates from THP - 1 engineered by CRISPR / Cas9 to induce USP7 / or USP47 KOs . Deficiency of both USP7 USP47 was induced by doxycycline ( Doxy ) treatment ( 1 µg / mL ; 3 days ) , as indicated . were differentiated with PMA either unprimed or LPS - primed ( 1 μg / mL , 4 hrs . ) treated with nigericin ( 10 μM , 45 mins . ) , as indicated . Bands in 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 USP47 . β - actin is shown as a loading control . Blots are representative of at least 3 independent experiments .

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

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 LPS - primed ( 1 μg / mL , 4 hrs . ) , nigericin activated ( 10 μM , 45 mins . ) THP - 1 , which contain indicated sgRNA . Bars represent mean of IL - 1β release relative to their respective non - doxycycline treated control , ± S . D . LDH - release measured from as treated in B . Bars represent mean of LDH release relative to their respective non - doxycycline treated control , ± 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 luciferase . variants found in SHOX are indicated in red . Luciferase assays to impact of SHOX mutations on its transcriptional activity were performed on FGFR3 promoter in U2OS ( n = 4 ) . Experiments were performed in triplicates . pcDNA4 - TO empty vector was used as control . R153L represents a mutation known to affect SHOXprotein activity was used as positive control ( Schneider et al . , 2005 ) . Homeodomain , binding domain ; Src Homology 3 domain ; OAR , OtpAristalessRax domain . RLU , Relative Units .

O O B-MEASURED_VAR O O O O O 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 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 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

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

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

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 expression of CYP26C1 in human chondrocytes on level . Overexpression of CYP26C1 in U2OS was carried out to compare sizes . + , cDNA ; - , water ; M , marker ; HPC , human 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 expression of CYP26C1 mRNA in human 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 reference genes SDHA HPRT in human 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 , 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 of SHOX promoter identified three putative RXRa binding sites which were mutated to their direct effect on SHOX expression upon treatment with ATRA 250 nM in U2OS ( n = 4 ) . Experiments were performed in triplicates . RLU , Relative Units .

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

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

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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 injected with control MO or with shox MO . C . Expression of col2a1 at 55 hpf was examined by in situ hybridization in wild type injected with control MO with shox MO . Arrow dotted indicate pectoral fin .

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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 injected with control MO or with shox MO . D . Pectoral fin was measured by imageJ ( n = 30 ) .

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

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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 injected with control MO or with cyp26c1 MO . ( C , D ) Expression of col2a1 ( C ) shox ( D ) at 55 hpf was examined by in situ hybridization in injected with control MO or with cyp26c1 MO . ( C ) Dorsal view magnification on pectoral fins of col2a1expression . Arrow dotted indicate pectoral fin . ( D ) Dorsal view magnification on pectoral fins of shox expression . Arrow dotted indicate 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 was measured by imageJ ( n = 30 ) .

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


F . Cignal - RARE system luciferase testing cyp26c1 MO on RA acid in zebrafishembryos ( n = 5 replicates ) . were separated in groups of 20 - 30 luciferase assayed . RLU , Relative 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 b - actin in zebrafishembryos injected with control MO or cyp26c1 MO ( n = 4 ) . was extracted from 10 - 15 injected 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 b - actin in zebrafishembryos treated with 100 nM ATRA ( n = 4 ) . were collected at 24 hpf treated with mock control or ATRA . was extracted from 10 - 15 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 injected with control MO , subphenotypic doses of shox MO , cyp26c1 MO or a combination of shox + cyp26c1 MOs . ( A ) Dorsal views of at 55 hours post fertilization ( hpf ) . Dotted , pectoral fins . shox + cyp26c1 double morphants show smaller fins compared to control single MOs ( n = 30 ) .

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

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

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

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 was measured by imageJ ( n = 30 ) .

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


E . Relative expression of shox mRNA normalized to reference genes eef1a b - actin in zebrafishembryos injected with control MO , shox MO , cyp26c1 MO , or shox + cyp26c1 MOs ( n = 4 ) . was extracted from 10 - 15 injected 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 of presenilin expression endoproteolysis in lysates of untransfected HEK293 / sw endogenously expressing presenilin single clones overexpressing PS1 WT or indicated PS1 mutants .

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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 of total Aβ secretion production of different Aβ species ( shown in low high immunoblot exposures ) in conditioned media of

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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 of Aβ species immunoprecipitated from conditioned media of

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


Immunoblot of C99 - PS1 NTF or full - ( FL ) crosslinks ( CL ) of 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 of crosslink efficiencies at positions T48 L49 compared to corresponding controls ( n = 4 - 5 biological replicates ) . Changes in of crosslink efficiencies at positions K54 V44 compared to corresponding controls ( n = 4 - 5 biological replicates ) .

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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 of Aβ species in conditioned medium of HEK293 / sw treated with RO7019009 or vehicle ( DMSO ) . Aβ 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 measured using 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 Notch cleavage by RO7019009 in different lines . Comparison of in vitro potencies for Aβ42 inhibition in HEK293 / sw , IC50 = 14 nM ( n = 3 ) ( as in ( C ) ) with those in H4 / sw , IC50 = 5 nM ( n = 7 ) N2a / sw , IC50 = 2 nM ( n = 6 ) together with that of Notch cleavage inhibition in a HEK293 - Notch1 luciferase reporter , IC50 > 10 µM ( n = 2 ) ( 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 of total Aβ in conditioned media of HEK293 / sw treated with RO7019009 or vehicle ( DMSO ) . Total APPs were analyzed to control for normal APP secretion equal sample loading . Right panel : 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 of Aβ species in conditioned media of RO7019009 treated HEK293 / sw after separation by Tris - Bicine urea SDS PAGE ; synthetic Aβ peptides were loaded as 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 of Aβ species in conditioned media of HEK293 / sw overexpressing WT or mutant PS1 treated with RO7019009 or vehicle ( DMSO ) showing secretion of Aβ38 ( A ) , Aβ43 ( B ) Aβ42 ( C ) compared to total Aβ ( Aβ38 + Aβ40 + Aβ42 + Aβ43 ) ( n = 5 biological replicates ) . Note that in some experiments Aβ38 generation was below 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 of Aβ species immunoprecipitated from conditioned media of HEK293 / sw 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 of C99 V44Bpa crosslinking ( CL ) to PS1 WT or NTFs ( circle ) of normally endoproteolysed mutants in presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to WT or mutant NTF in ( A ) . Immunoblot of C99 V44Bpa crosslinking to full - ( FL ) PS1 ( triangle ) of endoproteolysis - deficient mutants corresponding M292D control in presence of vehicle ( DMSO ) or RO7019009 . Quantitation of crosslink efficiencies of C99 V44Bpa to 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 . ( C ) shows a magnification of box represented in ( B ) . Bar : 20 μm . D Colocalization analyses along 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 ER marker AtCNX1 - mCherry . ( F ) shows a magnification of box represented in ( E ) . Bar : 20 μm . G Colocalization analyses along 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 free Ca2 + in root expressing NES - YC3 . 6 . Superimposition of representative normalized variations for wild - type , oscrt3 - 1 , complemented transgenic pOsCRT3 root . rectangle represents regions of interest ( ROIs ) for measurements . 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.


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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 of interaction between OsCRT3 OsCIPK7 .

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


E Localization of OsCIPK7 - GFP in N . benthamiana leaf epidermal . OFP - HDEL was used as an ER marker . Bar : 20 μm . F Magnification of box represented in ( E ) . Colocalization analyses along 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 yeast on selective medium ( SD / - Leu - Trp , SD / - Leu - Trp - His - Ade ) 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 OsCBL8 in N . benthamiana leaf epidermal . Pictures were taken after 2 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 complementation ( BiFC ) confirming interaction between OsCBL7 / 8 OsCIPK7 in N . benthamiana leaf epidermal . 19K was used as a negative control . Bar : 50 μm .

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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 OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three - hybrid .

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


E Constructs OsCRT3 expression level in ( D ) .

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


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

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


H Pull - down showing that interaction between OsCIPK7 OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3 . MBP was used as a negative control . Relative expression 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 . loaded is indicated by red arrows . Autoradiograph of γ - 32P - labeled OsCIPK7 indicates autophosphorylation of OsCIPK7 .

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 stained with Coomassie brilliant blue ( CBB ) are shown in top panel indicated by red arrows . Autoradiograph of γ - 32P - labeled myelin basic OsCIPK7 are shown in bottom panel .

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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 kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 . SDS - PAGE gel showing CBB - stained OsCRT3 , myelin basic , OsCIPK7 ( top panel ) . γ - 32P - labeled myelin basic OsCIPK7 substrate phosphorylation ( bottom panel ) . amount of OsCRT3 added was 0 μg , 0 . 2 μg , 0 . 4 μg , 0 . 6 μg , 0 . 8 μg in 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 SVZ of three different Ctrl FIP200GFAP cKO mice treated with chloroquine , 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 DAPI in SVZ DG of Ctrl FIP200GFAP cKO mice at P28 ( n = 3 p62 for each ) . Dotted lines indicate boundaries of SVZ granular zone ( GZ ) . arrows arrowheads mark larger smaller p62 + , respectively , in striatum ( ST ) 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 ) of mitochondria in SVZ tissue from Ctrl FIP200GFAP cKO mice at P28 P56 ( n = 2 mice for each , > 30 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 ) of mitochondria in cultured neurospheres from Ctrl FIP200GFAP cKO mice ( n = 2 mice for each , > 30 for each mouse ) . Arrows mark mitochondria .

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(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 DAPI in dentate gyrus ( e ) SVZ ( f ) of Ctrl FIP200GFAP cKO mice at P28 ( n = 5 mice for each ) . Dotted lines indicate boundaries of GZ ( e ) SVZ ( f ) . Arrows mark in SGZ ( e ) SVZ ( f ) , arrowheads mark in surround regions ( GZ in e ST in f ) . LV , lateral ventricle ; ML , molecular layer . Full - 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 eosin of dentate gyrus ( DG ) SVZ from Ctrl FIP200GFAP cKO mice at P28 . Lines indicate boundaries of DG SVZ . Arrows mark in 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 DAPI in DG ( b , c ) from Ctrl FIP200GFAP cKO mice at P28 . boxed areas in c - e are shown in more detail in insets ( c ) . Dotted lines indicate boundaries of granular zone ( GZ ) SGZ ( b ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( b , c ) , arrowheads mark GFAP + nestin − 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 DAPI in SVZ ( d , e ) from Ctrl FIP200GFAP cKO mice at P28 . boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of SVZ ( d , e ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( b , c ) , arrowheads mark GFAP + nestin − 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 DAPI in SVZ ( d , e ) from Ctrl FIP200GFAP cKO mice at P28 . boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of SVZ ( d , e ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( b , c ) , arrowheads mark GFAP + nestin − 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 RMS ( boxed areas ) , SVZ DG of Ctrl FIP200GFAP cKO mice at P28 . Lines indicate boundaries of DG SVZ . Arrows mark PSA - NCAM + in RMS , SVZ 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 olfactory bulb ( OB ) DG of Ctrl FIP200GFAP cKO mice at P31 . Lines indicate layers of OB boundaries of DG . Boxed areas in top panels are shown in more detail for of NeuN in insets . CC , corpus callosum ; E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral layer ; EPL , external plexiform layer ; GL , glomerular layer .

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(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 ) ( 1st ) secondary ( 2nd ) neurospheres from Ctrl FIP200GFAP cKO mice at P0 P28 . Representative P0 phase contrast are shown in a . Mean ± s . e . m . of number ( b , d ) ( c , e ) of 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 ) DAPI of SVZ dentate gyrus ( DG ) from Ctrl FIP200GFAP cKO mice at P28 . Lines indicate boundaries of SVZ . Numbered boxed areas are shown in more detail as insets for of TUNEL DAPI in SVZ 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 SVZ DG of Ctrl FIP200GFAPcKO mice at P28 . Dotted lines indicate boundaries of SVZ granular zone ( GZ ) . Boxed areas are shown in more detail in insets . Lines indicate boundaries of SVZ GZ . Arrows mark BrdU + . 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 SVZ dentate gyrus of Ctrl FIP200GFAP cKO mice at P31 . Lines indicate boundaries of SVZ GZ . Arrows mark BrdU + . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 .

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

(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 2cKO mice analyzed by western blot using antibodies to p53 actin .

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

(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 Stat3 in neurospheres of Ctrl , FIP200GFAP cKO , 2cKO Trp53GFAP cKO mice are shown ( n = 3 mice for each ) .

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

(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 number ( c ) ( d ) of secondary neurospheres from Ctrl , FIP200GFAP cKO , 2cKO 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).