Ser1660 is phosphorylated in response to damage required for interaction of separase with γH2AX . Hek293 were arrested in G2 phase by sequential thymidine - RO - 3306 treatment , DRB - ( + ) or mock treated ( - ) , then analysed as indicated ( C ) damage - induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition . G2 - enriched Hek293 were treated with KU - 55933 ( 0 . 3 μM ) / or DRB analyzed by ( IP - ) Western 12 h thereafter using indicated

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Ser1660 is phosphorylated in response to DNA damage and required for the interaction of separase with γH2AX. Hek293 cells were arrested in G2 phase by sequential thymidine- and RO-3306 treatment, DRB- (+) or mock treated (-), and then analysed as indicated (C) DNA damage-induced Ser1660 phosphorylation of endogenous separase is largely blocked by ATM inhibition. G2-enriched Hek293 cells were treated with KU-55933 (0.3 μM) and/or DRB and analyzed by (IP-)Western 12 h thereafter using the indicated antibodies


( D ) Preventing NES phosphorylation spoils localization of separase in response to DSBs . HeLaK expressing N - terminally NLS - eGFP - tagged separase variants were treated with DRB or carrier solvent ( − DRB ) for 4 h then subjected to IFM using anti - γH2AX anti - Nup153 to visualize sites of damage pore complexes , respectively . Transgenic separase was detected on eGFP autofluorescence . Note that due to their relatively high co - localization of separase - WT - S1660D with γH2AX - foci is not discernable . Scale bar = 5 μm

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(D) Preventing NES phosphorylation spoils nuclear localization of separase in response to DSBs. HeLaK cells expressing N-terminally NLS-eGFP-tagged separase variants were treated with DRB or carrier solvent (− DRB) for 4 h and then subjected to IFM using anti-γH2AX and anti-Nup153 to visualize sites of DNA damage and nuclear pore complexes, respectively. Transgenic separase was detected based on the eGFP autofluorescence. Note that due to their relatively high nuclear concentration co-localization of separase-WT and -S1660D with γH2AX-foci is not discernable. Scale bar = 5 μm


( E ) In vitro phosphorylation of separase on Ser1660 by ATM kinase . Incubation of GST - p53 ( 9 - 22 ) , GST , separase - WT , or separase - S1660 with recombinant ATM - WT , ATM - KD ( kinase dead ) , / or KU - 55933 in presence of [ γ - 33P ] - ATP was followed by immunoblotting

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(E) In vitro phosphorylation of separase on Ser1660 by ATM kinase. Incubation of GST-p53 (amino acids 9-22), GST, separase-WT, or separase-S1660 with recombinant ATM-WT, ATM-KD (kinase dead), and/or KU-55933 in presence of [γ-33P]-ATP was followed by immunoblotting and autoradiography


( F ) Arg - methylation of RG - repeats mediates recruitment of separase to DSB - containing . Myc - separase - WT or - KG expressing were treated with DRB as indicated analysed by ( IP - ) Western Coomassie

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(F) Arg-methylation of RG-repeats mediates recruitment of separase to DSB-containing chromatin. Myc-separase-WT or -KG expressing cells were treated with DRB as indicated and analysed by (IP-)Western and Coomassie staining


( G ) In vitro Arg - methylation of separase ' s RG - repeats by PRMT1 . Incubation of histone H4 , separase - WT , or separase - KG with recombinant PRMT1 or reference buffer in presence of S - adenosyl - L - [ methyl - 3H ] - methionine was followed by Coomassie autoradiography

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(G) In vitro Arg-methylation of separase's RG-repeats by PRMT1. Incubation of histone H4, separase-WT, or separase-KG with recombinant PRMT1 or reference buffer in presence of S-adenosyl-L-[methyl-3H]-methionine was followed by Coomassie staining and autoradiography


( A ) Persistence of γH2AX - MDC1 - positive foci in absence of NES - phosphorylation or RG - methylation of separase . Transfected Hek293T were siRNA - Dox - treated to deplete endogenous separase induce expression of indicated , siRNA resistant Myc - tagged separase variants , respectively . Then , they were constitutively ( − washout ) or transiently exposed to DRB ( + washout ) finally quantitatively assessed by IFM for γH2AX - MDC1 - positive foci . Shown are averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 each . Scale bar = 5

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(A) Persistence of γH2AX- and MDC1-positive foci in absence of NES-phosphorylation or RG-methylation of separase. Transfected Hek293T cells were siRNA- and Dox-treated to deplete endogenous separase and induce expression of the indicated, siRNA resistant Myc-tagged separase variants, respectively. Then, they were constitutively (− washout) or transiently exposed to DRB (+ washout) and finally quantitatively assessed by IFM for γH2AX- and MDC1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm


( C ) Recruitment of separase - WT - S1660D but not separase - superNES , - S1660A , - KG to DSBs . from ( A ) were treated as indicated analysed by IFM for co - localization of Myc - separase with γH2AX - foci . Scale bar = 5 μm

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(C) Recruitment of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG to DSBs. Cells from (A) were treated as indicated and analysed by IFM for co-localization of Myc-separase with γH2AX-foci. Scale bar = 5 μm


( D ) Interaction of separase - WT - S1660D but not separase - superNES , - S1660A , - KG with γH2AX . from ( A ) were subjected to ( IP - ) Western as indicated . Lanes that illustrate of cyclin A2 γH2AX after DRB - washout those that analyse interaction of Myc - separase variants with γH2AX in presence of DRB are labeled by red arrows blue arrow heads ,

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(D) Interaction of separase-WT and -S1660D but not separase-superNES, -S1660A, and -KG with γH2AX. Cells from (A) were subjected to (IP-)Western analysis as indicated. Lanes that illustrate cellular levels of cyclin A2 and γH2AX after DRB-washout and those that analyse interaction of Myc-separase variants with γH2AX in presence of DRB are labeled by red arrows and blue arrow heads, respectively


Separase is sumoylated in response to DSBs . Hek293T transfected to express His6 - separase ( A were treated with DRB or carrier solvent ( − DRB ) then subjected to denaturing IMAC followed by immunoblotting of input samples eluates using indicated

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Separase is sumoylated in response to DSBs. Hek293T cells transfected to express His6-separase (A were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodies


Separase is sumoylated in response to DSBs . Hek293T transfected to expres His6 - Sumo2 ( B ) were treated with DRB or carrier solvent ( − DRB ) then subjected to denaturing IMAC followed by immunoblotting of input samples eluates using indicated

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Separase is sumoylated in response to DSBs. Hek293T cells transfected to expres His6-Sumo2 (B) were treated with DRB or carrier solvent (− DRB) and then subjected to denaturing IMAC followed by immunoblotting of input samples and eluates using the indicated antibodies


Lys - 1034 is a major target of DSB - induced sumoylation of separase . Hek293T expressing His6 - Sumo2 ( C or - K1034R ( D ) were DRB - or mock - treated subjected to denaturing IMA Input samples eluates were immunoblotted using indicated

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Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressing His6-Sumo2 (C or -K1034R (D) were DRB- or mock-treated and subjected to denaturing IMA Input samples and eluates were immunoblotted using the indicated antibodies


Lys - 1034 is a major target of DSB - induced sumoylation of separase . Hek293T expressin Myc - separase - WT or - K1034R ( D ) were DRB - or mock - treated subjected t Myc - I Input samples eluates were immunoblotted using indicated

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Lys-1034 is a major target of DSB-induced sumoylation of separase. Hek293T cells expressin Myc-separase-WT or -K1034R (D) were DRB- or mock-treated and subjected t Myc-I Input samples and eluates were immunoblotted using the indicated antibodies


( E ) Mms21 - dependent in vitro sumoylation of separase at Lys - 1034 . Myc beads were loaded with separase - WT or - K1034R or left empty ( last lane ) , combined with recombinant Ubc9 / or Mms21 , as indicated , incubated in presence of His6 - Sumo2 , Sae1 - Sae2 ATP . Supernatant washed beads were analysed by Coomassie ( lower panels ) immunoblotting ( upper panels ) , respectively

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(E) Mms21-dependent in vitro sumoylation of separase at Lys-1034. Myc beads were loaded with separase-WT or -K1034R or left empty (last lane), combined with recombinant Ubc9 and/or Mms21, as indicated, and incubated in presence of His6-Sumo2, Sae1-Sae2 and ATP. Supernatant and washed beads were analysed by Coomassie staining (lower panels) and immunoblotting (upper panels), respectively


( F ) expressing indicated separase variants were DRB - or mock - treated then lysed . Lysates pelleted therefrom were assessed by immunoblotting Coomassie , as indicated . Note that lanes shown in upper panels , although not directly juxtaposed , nevertheless originate from same gel

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(F) Cells expressing the indicated separase variants were DRB- or mock-treated and then lysed. Lysates and chromatin pelleted therefrom were assessed by immunoblotting and Coomassie staining, as indicated. Note that the lanes shown in the upper panels, although not directly juxtaposed, nevertheless originate from the same gel


( G ) Preventing sumoylation of Lys - 1034 compromises separase ' s ability to support DSB repair . indicated variants were probed for their ability to functionally replace endogenous separase in HDR as described in figures 6A 6B . Shown are averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 each

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(G) Preventing sumoylation of Lys-1034 compromises separase's ability to support DSB repair. The indicated variants were probed for their ability to functionally replace endogenous separase in HDR as described in figures 6A and 6B. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each


( A , B ) SEPARASE + / + SEPARASE + / - MEFs were constitutively ( − washout ) or transiently exposed to DRB ( + washout ) then analysed by IF for with γH2AX - 53BP1 - positive foci . Shown are averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100 each . Scale bar = 5 μm . ( C ) lysates from one experiment in ( A ) were analysed by immunoblotting using indicated antibodies

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(A, B) SEPARASE+/+ and SEPARASE+/- MEFs were constitutively (− washout) or transiently exposed to DRB (+ washout) and then analysed by IF for cells with γH2AX- and 53BP1-positive foci. Shown are averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each. Scale bar = 5 μm. (C) Cell lysates from one experiment in (A) were analysed by immunoblotting using the indicated antibodies


SEPARASE + / + SEPARASE + / - MEFs were treated with DRB ( + ) or carrier solvent ( − ) for indicated times then analysed by immunoblotting of total lysates (

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SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed by immunoblotting of total cell lysates (D


SEPARASE + / + SEPARASE + / - MEFs were treated with DRB ( + ) or carrier solvent ( − ) for indicated times then analysed b IPs using anti - Rad21 or unspecific IgG ( E ) . lower right two lanes show in vitro - expressed mouse Rad21 treated with hyperactive ( SA ) separase or a protease - dead ( PD )

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SEPARASE+/+ and SEPARASE+/- MEFs were treated with DRB (+) or carrier solvent (−) for the indicated times and then analysed b IPs using anti-Rad21 or unspecific IgG (E). The lower right two lanes show in vitro-expressed mouse Rad21 treated with hyperactive (SA) separase or a protease-dead (PD) variant


( A ) Transformation susceptibility of MEFs . SEPARASE + / + versus SEPARASE + / - MEFs were infected to express E1A hRasV12 then subjected to a colony formation . Shown are representative ( left ) graphical representation of number of transformed foci ( right ) from two independent experiments ( bars = standard deviations )

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(A) Transformation susceptibility of primary MEFs. SEPARASE+/+ versus SEPARASE+/- MEFs were infected to express E1A and hRasV12 and then subjected to a colony formation assay. Shown are representative images (left) and the graphical representation of the number of transformed foci (right) from two independent experiments (bars = standard deviations)


( B ) Two - stage DMBA ( initiation ) plus TPA ( promotion ) skin carcinogenesis . number of skin papillomas are plotted against time for each genotype : SEPARASE + / + ( n = 8 ) versus SEPARASE + / - ( n = 11 )

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(B) Two-stage DMBA (initiation) plus TPA (promotion) skin carcinogenesis. The number and size of skin papillomas are plotted against time for each genotype: SEPARASE+/+ (n=8) versus SEPARASE+/- (n= 11)


( C ) Representative hematoxylin - eosin - stained sections from SEPARASE + / - biopsies . ( i ) glandular hyperplasia ; ( ii ) benign papilloma ; ( iii ) squamous carcinoma . Scale bars are 200 µm in ( i ) 500 µm in ( ii ) ( iii ) . ( D ) Quantitative histological assessment of DMBA - TPA skin cancerogenesis . Given are numbers of total , corresponding in brackets

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(C) Representative hematoxylin- and eosin-stained sections from SEPARASE+/- biopsies. (i) glandular hyperplasia; (ii) benign papilloma; (iii) squamous cell carcinoma. Scale bars are 200 µm in (i) and 500 µm in (ii) and (iii). (D) Quantitative histological assessment of the DMBA-TPA skin cancerogenesis assay. Given are numbers of total, and corresponding percentage in brackets


( A ) Flow cytometry of l ( 2 ) mbn starved for 2 h ( 2h S ) or 4 h ( 4h S ) showed an increase in LTG ( x axis ) compared with control in full - nutrient medium ( C ) . gate shown on histogram represents LTGhigh population . ( B ) Representative of GFP - LC3 in control 2 - h starved l ( 2 ) mbn . An increase in GFP - LC3 was observed in starved ( left ) . Bar , 10 μm . ( C ) Flow cytometry of 4 - h starved were incubated with 3MA ( 4h S + 3MA ) Baf ( 4h S + Baf ) . Both autophagy inhibitors reduced LTG compared with starved ( 4h S ) . Control in nutrient - full medium ( C ) are represented by brown . ( D ) Both autophagy inhibitors , 3MA Baf , reduced LTGhigh population significantly . ( 3MA , P = 0 . 00001 ; Baf , P = 0 . 00006 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O

(A) Flow cytometry analysis of l(2)mbn cells starved for 2 h (2h S) or 4 h (4h S) showed an increase in LTG fluorescence levels (x axis) compared with control cells in full-nutrient medium (C). The gate shown on the histogram represents the LTGhigh population. (B) Representative images of GFP-LC3 puncta in control and 2-h starved l(2)mbn cells. An increase in GFP-LC3 puncta was observed in the starved cells (left). Bar, 10 μm. (C) Flow cytometry analysis of 4-h starved cells were incubated with 3MA (4h S + 3MA) and Baf (4h S + Baf). Both autophagy inhibitors reduced the LTG fluorescence levels compared with starved cells (4h S). Control cells in nutrient-full medium (C) are represented by the brown line. (D) Both autophagy inhibitors, 3MA and Baf, reduced the LTGhigh population significantly. (3MA, P = 0.00001; and Baf, P = 0.00006).


( E ) RNAi of representative Atg genes decreased LTR compared with control . ( Atg1 , P = 0 . 01 ; Atg5 , P = 0 . 01 ; Atg7 , P = 0 . 002 ; Atg8a , P = 0 . 008 ; Atg8b , P = 0 . 006 ; Atg12 , P = 0 . 02 ) .

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(E) RNAi of representative Atg genes decreased the LTR fluorescence levels compared with control. (Atg1, P = 0.01; Atg5, P = 0.01; Atg7, P = 0.002; Atg8a, P = 0.008; Atg8b, P = 0.006; and Atg12, P = 0.02).


( F ) RNAi of tested genes in TOR - PI3K pathways had a statistically significant effect on LTG . Known negative regulators of autophagy are shown with gray bars ; positive regulators are shown with bars . ( Pten , P = 0 . 007 ; Tsc1 , P = 0 . 027 ; Tsc2 , P = 0 . 025 ; RheB , P = 0 . 005 ; Tor , P = 0 . 016 ; S6k , P = 0 . 012 ) . dsRNA corresponding to a human gene ( Hs ) was used as a negative control in E F . Results represent mean value ± SD from at least three independent experiments .

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(F) RNAi of all tested genes in the TOR-PI3K pathways had a statistically significant effect on LTG fluorescence levels. Known negative regulators of autophagy are shown with gray bars; positive regulators are shown with white bars. (Pten, P = 0.007; Tsc1, P = 0.027; Tsc2, P = 0.025; RheB, P = 0.005; Tor, P = 0.016; and S6k, P = 0.012). dsRNA corresponding to a human gene (Hs) was used as a negative control in E and F. Results represent the mean value ± SD from at least three independent experiments.


( A ) of LTGhigh was reduced by hid - RNAi ( P = 0 . 006 ) but not by rpr , grim , skl - RNAi .

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(A) The percentage of LTGhigh cells was reduced by hid-RNAi (P = 0.006) but not by rpr, grim, and skl-RNAi.


( B ) Knockdown of Ras , phl , rl expression by RNAi resulted in an increase in of LTGhigh . ( Ras , P = 0 . 003 ; phl , P = 0 . 001 ; rl , P = 0 . 028 ) .

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(B) Knockdown of Ras, phl, and rl expression by RNAi resulted in an increase in the percentage of LTGhigh cells. (Ras, P = 0.003; phl, P = 0.001; and rl, P = 0.028).


( C ) th - RNAi treatment ( 24 h ) had no significant effect on LTG ; in contrast , RNAi of Bruce resulted in an increase in LTG ( P = 0 . 01 ) .

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(C) th-RNAi treatment (24 h) had no significant effect on LTG levels; in contrast, RNAi of Bruce resulted in an increase in LTG fluorescence levels (P = 0.01).


( D ) Reduction of debcl , Buffy , or p53 expression by RNAi resulted in a decrease in LTG . ( debcl , P = 0 . 018 ; Buffy , P = 0 . 006 ; p53 , P = 0 . 004 ) .

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(D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004).


( E ) RNAi of effector caspase Dcp - 1 resulted in a significant decrease in LTGhigh population ( P = 0 . 001 ) .

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(E) RNAi of effector caspase Dcp-1 resulted in a significant decrease in the LTGhigh population (P = 0.001).


( F ) Representative of GFP - LC3 in treated with indicated RNAi after a 2 - h starvation treatment . Bar , 10 μm .

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(F) Representative images of GFP-LC3 puncta in cells treated with the indicated RNAi after a 2-h starvation treatment. Bar, 10 μm.


( G ) of with GFP - LC3 after RNAi treatment . with more than three GFP - LC3 punctate dots were considered to be GFP - LC3 - positive . treated with RNAi indicated here showed a significant difference ( P < 0 . 05 ) in of GFP - LC3 - positive compared with human ( Hs ) RNAi control . ( Pten , P = 0 . 006 ; Tor , P = 0 . 034 ; Buffy , P = 0 . 005 ; debcl , P = 0 . 003 ; Bruce , P = 0 . 003 ; Dcp - 1 , P = 0 . 007 ; hid , P = 0 . 002 ; Ras , P = 0 . 006 ; phl , P = 0 . 050 ) . Results represent mean value ± SD from three independent experiments .

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(G) Quantification of cells with GFP-LC3 puncta after RNAi treatment. Cells with more than three GFP-LC3 punctate dots were considered to be GFP-LC3-positive cells. Cells treated with the RNAi indicated here all showed a significant difference (P < 0.05) in the percentage of GFP-LC3-positive cells compared with the human (Hs) RNAi control. (Pten, P = 0.006; Tor, P = 0.034; Buffy, P = 0.005; debcl, P = 0.003; Bruce, P = 0.003; Dcp-1, P = 0.007; hid, P = 0.002; Ras, P = 0.006; and phl, P = 0.050). Results represent the mean value ± SD from three independent experiments.


( A ) GFP - LC3 were expressed in nurse ( NC ) but not in follicle ( FC ) by using UASp / nanos Gal4 system . DAPI of nuclei is shown in blue .

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(A) GFP-LC3 proteins were expressed in nurse cells (NC) but not in follicle cells (FC) by using the UASp/nanos Gal4 system. DAPI staining of nuclei is shown in blue.


( B ) UASp - GFP - LC3 ; nanos GAL4 flies were conditioned on yeast paste had a diffuse GFP - LC3 pattern . Numerous GFP - LC3 ( green ) at region 2 within germarium were observed in nutrient - deprived flies . Ovaries were stained with LTR in w1118 flies . Germarium of nutrient - deprived w1118 flies had an increase in punctate LTR ( red ) compared with well - fed germarium .

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(B) UASp-GFP-LC3; nanos GAL4 flies were conditioned on yeast paste and had a diffuse GFP-LC3 pattern. Numerous GFP-LC3 puncta (green) at region 2 within germarium were observed in nutrient-deprived flies. Ovaries were stained with LTR in w1118 flies. Germarium of nutrient-deprived w1118 flies had an increase in punctate LTR staining (red) compared with well-fed germarium.


( C ) Degenerating stage 8 egg chambers ( arrows ) had numerous GFP - LC3 ( green ) an increase in LTR - positive dots ( red ) compared with healthy egg chambers ( arrowheads ) . DAPI ( blue ) of nuclei is shown in two panels on right .

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(C) Degenerating stage 8 egg chambers (arrows) had numerous GFP-LC3 puncta (green) and an increase in LTR-positive dots (red) compared with healthy egg chambers (arrowheads). DAPI (blue) staining of nuclei is shown in the two panels on the right.


( D ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) showed a dramatic decrease in LTR . DAPI of nuclei is shown in blue .

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(D) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14) showed a dramatic decrease in LTR staining. DAPI staining of nuclei is shown in blue.


( A ) Germaria of nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR compared with nutrient - deprived wild - type flies shown in Fig . 3 B . DAPI of nuclei is shown in blue . ( B ) Degenerating stage 8 egg chambers ( arrows ) of nutrient - deprived Dcp - 1Prev flies showed a dramatic decrease in LTR compared with nutrient - deprived wild - type flies shown in Fig . 3 C .

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(A) Germaria of the nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 B. DAPI staining of nuclei is shown in blue. (B) Degenerating stage 8 egg chambers (arrows) of nutrient-deprived Dcp-1Prev flies showed a dramatic decrease in LTR staining compared with nutrient-deprived wild-type flies shown in Fig. 3 C.


( C ) Lack of Dcp - 1 function ( UASp - GFP - LC3 Dcp - 1Prev / Dcp - 1Prev ; nanos - GAL4 / + ) resulted in uniform diffuse of GFP - LC3 rather than punctate pattern observed in wild - type degenerating stage 8 egg chambers shown in Fig 3 C . ( D ) Dying egg chambers ( arrows ) of NGT / + ; nanos - GAL4 / UASp - fl - Dcp - 1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR ( red ) compared with healthy egg chambers ( arrowheads ) . ( E ) Expression of activated Dcp - 1 ( a truncated form ) GFP - LC3 in germ ( UASp - GFP - LC3 / + ; nanos - GAL4 / nanos - GAL4 UASp - tDcp - 1 ) resulted in abundant degenerating stage 8 egg chambers ( arrows ) with numerous GFP - LC3 ( green ) . DAPI of nuclei is shown in blue .

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(C) Lack of Dcp-1 function (UASp-GFP-LC3 Dcp-1Prev/Dcp-1Prev; nanos-GAL4/+) resulted in uniform diffuse staining of GFP-LC3 rather than the punctate pattern observed in wild-type degenerating stage 8 egg chambers shown in Fig 3 C. (D) Dying egg chambers (arrows) of NGT/+; nanos-GAL4/UASp-fl-Dcp-1 flies that were conditioned on yeast paste showed a significant increase in punctate LTR staining (red) compared with healthy egg chambers (arrowheads). (E) Expression of activated Dcp-1 (a truncated form) and GFP-LC3 in the germ line (UASp-GFP-LC3/+; nanos-GAL4/nanos-GAL4 UASp-tDcp-1) resulted in abundant degenerating stage 8 egg chambers (arrows) with numerous GFP-LC3 puncta (green). DAPI staining of nuclei is shown in blue.


( A ) germarium in well - fed BruceE81 flies showed an increase in LTR ( red ) compared with wild - type well - fed flies shown in Fig . 3 B ( top right ) . DAPI ( ) of nuclei is shown on right . ( B ) In well - fed wild - type flies , mid - oogenesis nurse is a rare event . Lack of Bruce function resulted in an increase in dying stage 8 egg chambers ( arrows ) in ovaries under well - fed conditions , these degenerating stage 8 egg chambers had numerous LTR ( red ) punctate dots . DAPI ( ) is shown on right . Bars : ( A ) 20 μm ; ( B ) 50 μm .

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(A) The germarium in well-fed BruceE81 flies showed an increase in LTR staining (red) compared with wild-type well-fed flies shown in Fig. 3 B (top right). DAPI staining (white) of nuclei is shown on the right. (B) In well-fed wild-type flies, mid-oogenesis nurse cell death is a rare event. Lack of Bruce function resulted in an increase in dying stage 8 egg chambers (arrows) in ovaries under well-fed conditions, and these degenerating stage 8 egg chambers had numerous LTR (red) punctate dots. DAPI staining (white) is shown on the right. Bars: (A) 20 μm; (B) 50 μm.


( A ) Ovaries were stained with TUNEL ( green ) to detect fragmentation . Clusters of cysts with TUNEL were observed in region 2 in nutrient - deprived w1118 files . In Dcp - 1Prev flies , fewer TUNEL - positive cysts in region 2 were observed . Under well - fed conditions , numerous TUNEL - positive cysts were observed in BruceE81 flies . DAPI of nuclei is shown in . ( B ) Numerous degenerating stage 8 egg chambers ( arrows ) with Bruce - positive ( green ) were observed in well - fed BruceE81 flies . DAPI of nuclei ( ) is shown on right .

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(A) Ovaries were stained with TUNEL (green) to detect DNA fragmentation. Clusters of cysts with TUNEL staining were observed in region 2 in nutrient-deprived w1118 files. In Dcp-1Prev flies, fewer TUNEL-positive cysts in region 2 were observed. Under well-fed conditions, numerous TUNEL-positive cysts were observed in BruceE81 flies. DAPI staining of nuclei is shown in white. (B) Numerous degenerating stage 8 egg chambers (arrows) with Bruce-positive staining (green) were observed in well-fed BruceE81 flies. DAPI staining of nuclei (white) is shown on the right.


( A ) TUNEL - positive was observed in dying stage 8 egg chambers ( arrows ) of starved control flies ( CG5335d30 / Atg7d14 ) . DAPI of nuclei ( ) is shown on right . ( B ) In nutrient - deprived Atg7 mutants ( Atg7d77 / Atg7d14 ) , degenerating stage 8 egg chambers ( arrows ) showed no or low of TUNEL . condensation , detected by DAPI , was still observed . ( C ) Dying stage 8 egg chambers ( arrows ) from nutrient - deprived control siblings ( Atg1Δ3D / TM3 ) generated from same cross in D had abundant TUNEL - positive . ( D ) In nutrient - deprived Atg1 GLCs , degenerating stage 8 egg chambers ( arrows ) showed no or low of TUNEL . condensation ( DAPI , right ) in degenerating egg chambers appeared to occur as in controls .

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(A) TUNEL-positive staining was observed in dying stage 8 egg chambers (arrows) of starved control flies (CG5335d30/Atg7d14). DAPI staining of nuclei (white) is shown on the right. (B) In nutrient-deprived Atg7 mutants (Atg7d77/Atg7d14), degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation, detected by DAPI, was still observed. (C) Dying stage 8 egg chambers (arrows) from nutrient-deprived control siblings (Atg1Δ3D/TM3) generated from the same cross in D had abundant TUNEL-positive staining. (D) In nutrient-deprived Atg1 GLCs, degenerating stage 8 egg chambers (arrows) showed no or low levels of TUNEL staining. Nuclear DNA condensation (DAPI, right) in degenerating egg chambers appeared to occur as in the controls.


CSTB gene expression in hCOs , starting from day ( d ) 16 until d140 . For every time point , at least 3 different samples were analyzed ; each sample was made by a pool of 3 to 4 hCOs . Data are represented as mean ± . Unpaired t - ( * P < 0 . 05 , * * P < 0 . 01 ) .

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CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).


of sections of control hCOs immunostained with CSTB antibody at different time points in culture . dashed lines represent apical surface of cavities / ventricles ( V - like ) . cortical plate like side is indicated ( CP - like ) . Nuclei ( blue ) are stained with DAPI .

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Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.


Real time qPCR results of PAX6 , NEUN , NESTIN , CSTB gene expression in PAX6 - NEUN - FACS sorted nuclei from f - CTRL d135 hCOs . Data are represented as mean ± . Statistical significance was on T - student ( * P < 0 . 05 , * * P < 0 . 01 ) . For PAX6 , 10 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei 5 for Neun + sorted Nuclei ; for NEUN , NESTIN CSTB , 9 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei 4 for Neun + sorted Nuclei .

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Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.


Western blot relative quantifications ( arbitrary units , a . u . ) on extracts from cortical ( Cx ) ganglionic eminence ( GE ) tissues for Cstb using Actb as a loading control . Cx GE tissues from mouse at different time of development as indicated in figure .

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Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.


Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb acetylated tubulin ( acTub ) as marker for stabilized microtubules . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles . Nuclei ( blue ) are stained with DAPI .

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Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - empty vector control or GFP - CSTB analyzed 5 dpe . Sections were then immunostained for GFP KI67 . dashed lines represent apical surface of cavities / ventricles ( V - like ) . cortical plate like side is indicated ( CP - like ) . of total number of proliferating KI67 + / ( µm2 ) of V - like structures transfected with GFP - empty vector control or GFP - CSTB in ( A ) .

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Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb analyzed 2 dpe immunostained with GFP , Ki67 BrdU 30 minutes after BrdU injection . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles . of total number of proliferating Ki67 + / ( µm2 ) ( D ) Ki67 + Brdu + / ( µm2 ) ( E ) of ventricles transfected with GFP - empty vector or GFP - Cstb in ( C ) .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb , analyzed 2 dpe immunostained with GFP Phospho - Histone H3 ( PH3 ) ( F ) with GFP , Pax6 Tbr2 ( G ) . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.


Distribution of proliferating Ki67 + / BrdU + ( H ) mitotic progenitor PH3 + ( I ) in mouse cortex . cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side Bin5 to pial side of cortex PH3 + or Ki67 + / BrdU + calculated in each Bin .

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Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.


Distribution of Tbr2 + intermediate progenitors ( J ) Pax6 + Tbr2 + ( K ) in mouse cortex . cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side Bin5 to pial side of cortex Tbr2 + or Pax6 + Tbr2 + calculated in each Bin .

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Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.


Western blot for CSTB in CSF from 2 different E14 mouse ( A ) , in CM from E14 cortical in culture for 4

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Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 days


Western blot for CSTB in CM from hCOs after 4 days in culture ( C ) . CM was collected from hCOs at different time points in culture ( d19 , d38 , d60 , d80 ) . extract ( PE ) from 60 days hCOs was used as a positive control . GAPDH immunostaining was used as a negative control on hCOs CM . Ponceau red is shown to indicate presence of at comparable in CM

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

Western blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector HA - empty vector or - Cstb , analyzed 3 dpe . Immunostaining with RFP to identify electroporated GFP to identify migrating interneurons in GAD65 - GFP transgenic mouse . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles . Distribution of GFP + interneurons in 5 equal Bins of mouse developing cortex - Bin1 corresponded to apical side Bin5 to pial side . Ventricle ( V ) is indicated . dashed lines represent apical surface of ventricles .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-ORGANISM O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE O O O B-GENEPROD O B-CELL_TYPE O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry HA - Cstb expressing vector , analyzed 3 dpe . Immunostaining with RFP to identify electroporated GFP to identify migrating interneurons in GAD67 - GFP transgenic mouse . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles . Distribution of GFP + interneurons in 5 equal Bins of mouse developing cortex comparing electroporated sides with ones in same section to avoid different distribution of interneurons rostro - caudally .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-ORGANISM O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE O O O B-GENEPROD O B-CELL_TYPE O O O O O B-ORGANISM O B-TISSUE O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O O O O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - miR - neg vector ( CTRL ) GFP - miRNA - Cstb ( KD ) , analyzed 3 dpe immunostained with GFP . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles . Distribution of electroporated GFP + in mouse cortex . cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side Bin5 to pial side of cortex GFP + calculated in each Bin . Nuclei ( blue ) are stained with DAPI .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY B-GENEPROD O O B-ORGANISM B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-miR-neg vector (CTRL) and GFP-miRNA-Cstb (KD), analyzed 3 dpe and immunostained with GFP. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin. Nuclei (blue) are stained with DAPI.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - empty vector control GFP - miRNA - Cstb , analyzed 3 dpe immunostained with GFP Ki67 . Ventricle ( V ) is indicated . dashed lines represent apical surface of ventricles . of total number of proliferating Ki67 + / ( µm2 ) of ventricular structures transfected with GFP - miR - neg vector or GFP - miRNA - Cstb in ( C ) . Nuclei ( blue ) are stained with DAPI .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-empty vector control and GFP-miRNA-Cstb, analyzed 3 dpe and immunostained with GFP and Ki67. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricular structures transfected with GFP-miR-neg vector or GFP-miRNA- Cstb in (C). Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - CSTB expressing vector or GFP - R68X mutant analyzed 5 dpe . Sections were immunostained for GFP Ki67 . dashed lines represent apical surface of cavities / ventricles ( V - like ) . cortical plate like side is indicated ( CP - like ) . of total number of proliferating KI67 + / ( µm2 ) ( F ) Ki67 + GFP + / ( µm2 ) ( G ) of ventricles like structures transfected with GFP - empty vector control , GFP - CSTB or GFP - R68X mutant in ( E ) .

B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

Micrograph sections of d40 hCOs electroporated with GFP-CSTB expressing vector or GFP-R68X mutant and analyzed 5 dpe. Sections were immunostained for GFP and Ki67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area(µm2)(F)and Ki67+GFP+ cells/area(µm2)(G) of ventricles like structures transfected with GFP-empty vector control, GFP-CSTB or GFP-R68X mutant in (E).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X , analyzed 2 dpe immunostained with GFP Phospho - Histone H3 ( PH3 ) . Ventricle ( V ) cortical plate ( CP ) are indicated . dashed lines represent apical surface of ventricles . Distribution of electroporated GFP + in mouse cortex . cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side Bin5 to pial side of cortex GFP + calculated in each Bin .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY B-GENEPROD O O B-ORGANISM B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD O O O O O O

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin.


Distribution of mitotic progenitor PH3 + in mouse cortex . cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side Bin5 to pial side of cortex PH3 + calculated in each Bin .

O O O O O O O B-ORGANISM B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O

Distribution of mitotic progenitor PH3+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells calculated in each Bin.


K . of total number of proliferating PH3 + / ( µm2 ) of ventricular structures transfected with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant in ( H ) .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

K.Quantification of the total number of proliferating PH3+ cells/area(µm2)of ventricular structures transfected with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant in (H).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant , analyzed 2 dpe immunostained with GFP Tbr2 . Ventricle ( V ) is indicated . dashed lines represent apical surface of ventricles . of total number of Tbr2 + / cortical ( µm2 ) in ( L ) . Data shown as Z - scores relative to mean of GFP control vector .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant, analyzed 2 dpe and immunostained with GFP and Tbr2. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of Tbr2+ cells/ cortical area (µm2) in (L). Data shown as Z-scores relative to the mean of GFP control vector.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector HA - Cstb or R68X , analyzed 3 dpe . Immunostaining with RFP to identify electroporated GFP to identify migrating interneurons in GAD65 - GFP transgenic mouse . Ventricle ( V ) is indicated . dashed lines represent apical surface of ventricles . of total number of GAD65 - GFP interneurons / cortical ( µm2 ) in ( N ) . Data shown as Z - scores relative to mean of GFP control vector . Nuclei ( blue ) are stained with DAPI .

B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE I-TISSUE B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-ORGANISM O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-Cstb or R68X, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of GAD65-GFP interneurons/ cortical area (µm2) in (N). Data shown as Z-scores relative to the mean of GFP control vector. Nuclei (blue) are stained with DAPI.


Left panel shows representative of hCOs from different iPSC lines at analyzed time points . Right panel shows difference of areas of f - CTRL , b - CTRL , UL1 UL4 hCOs at different time in culture . Data shown as Z - scores relative to mean of b - CTRL .

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Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.


Micrograph sections of d30 b - CTRL , UL1 UL4 hCOs immunostained for proliferation markers KI67 PH3 . dashed lines represent apical surface of cavities / ventricles . of total number of proliferating Ki67 + / ( µm2 ) of ventricles like structures of number of PH3 + facing ventricle / apical surface ( µm ) . Data shown as Z - scores relative to mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 UL4 hCOs immunostained for proliferation markers KI67 PH3 . dashed lines represent apical surface of cavities / ventricles . of total number of proliferating KI67 + / of ventricles like structures of number of PH3 + facing ventricle / apical surface ( µm ) . Data shown as Z - scores relative to mean of CTRL . Nuclei ( blue ) are stained with DAPI .

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Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.


Scheme of experiment with conditioned medium exchange between b - CTRL UL4 hCOs of Ki67 + by FACS on 36 days old dissociated hCOs . Four samples were analyzed as biological replicates .

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Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.


Micrograph sections of d30 b - CTRL , UL1 UL4 hCOs immunostained for neuronal marker DCX . dashed lines represent apical surface of cavities / ventricles . of DCX + . of DCX + was calculated related to total ( µm2 ) of ventricular structure . Data shown as Z - scores relative to mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

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Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 UL4 hCOs immunostained for neuronal marker DCX . dashed lines represent apical surface of cavities / ventricles . of DCX + . ( µm2 ) of DCX + was calculated related to total of ventricular structure . Data shown as Z - scores relative to mean of b - CTRL . of number of neuronal DCX + by FACS sorting on 60 days old dissociated hCOs . Data shown as Z - scores relative to mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d30 fused - organoid ; ventral GFP - f - CTRL dorsal UL1 .

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Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.


of sections from d30 fused organoids , showing ventral GFP + in dorsal . migrating GFP + were counted starting from 100 µm far from ventral / dorsal border . of ventrally - derived GFP + in dorsal / thickness of counted ( µm3 ) . counted was always 100 µm far from border between ventral dorsal as indicated in Fig . EV5D . Every dot represents a different hCOs different areas were counted for each hCO . Nuclei ( blue ) are stained with DAPI .

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Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.


Volcano plot representing identified by high - resolution nanoLC MS / MS . Up - down - regulated ( - 0 . 3 ≥ log2FC ≥ 0 . 3 ) in human cerebral organoids ( UL1 vs b - CTRL ) are shown in red green , respectively .

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Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.


A ) TUBB1 is expressed in developing human thyroid at 8 gestational ( GW ) , 10 GW , 12 GW in adult human thyroid : quantitative PCR results normalized to one of three thyroid tissues at 8 GW value ( in black ) peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus 8 GW ( in black ) using ANOVA showed no significant

O O B-GENEPROD O B-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW, and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differences


B ) Tubb1 is expressed in developing mouse thyroid at E13 . 5 , E15 . 5 , E17 . 5 in adult mouse thyroid : quantitative PCR results normalized to one of three thyroid tissues at E13 . 5 value peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus E13 . 5 ( black ) , * * p < 0 . 01 using ANOVA

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B) Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5, and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus E13.5 (black), **p<0.01 using ANOVA test


C ) Sorted mouse thyroid , Tubb1 expression in endothelial ( Pecam - positive ) , epithelial ( Epcam ) , fibroblasts ( Pdgfra ) , leucocytes ( CD45 ) at E17 . 5 adulthood : quantitative PCR results ( normalized to Epcam - positive at adult stage peptidylprolyl isomerase A ) . Mean ± of three independent - sorting experiments . Statistical comparisons versus epithelial ( in black ) at each stage , * p < 0 . 05 using ANOVA

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C) Sorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam-positive cells), epithelial cells (Epcam), fibroblasts (Pdgfra), and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to Epcam-positive cells at adult stage and peptidylprolyl isomerase A). Mean±SEM of three independent cell-sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *p<0.05 using ANOVA test


D ) Immunohistofluorescence of β1 - tubulin ( in red ) , thyroglobulin ( TG , in green ) , both merged ( from left to right ) at 12 GW in human thyroid tissue . boxes delineate enlarged areas shown

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D) Immunohistofluorescence of β1-tubulin (in red), thyroglobulin (TG, in green), and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown below


F ) Localization of transfected Myc - tagged wild - type mutant ( P160L ) β1 - tubulin in Nthy . Immunostaining with anti - Myc antibody ( in green ) microtubule cytoskeleton stained with α - tubulin antibody ( in red ) . Note that β1 - tubulin - P160L is found throughout cytoplasm showing appearance , whereas wild - type β1 - tubulin colocalizes with

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F) Localization of transfected Myc-tagged wild-type and mutant (P160L) β1-tubulin in Nthy cells. Immunostaining with anti-Myc antibody (in green) and microtubule cytoskeleton stained with α-tubulin antibody (in red). Note that β1-tubulin -P160L is found throughout the cytoplasm showing puncta appearance, whereas wild-type β1-tubulin colocalizes with the microtubules


A ) Thyroid was investigated using Nkx2 - 1 at E9 . 5 E11 . 5 ( sagittal sections ) at E13 . 5 , E15 . 5 , E17 . 5 ( transverse sections ) in wild - type ( wt ) Tubb1 - / - . Nkx2 - 1was used as a marker of early thyrocyte progenitors differentiated thyrocytes . Delays in thyroid migration in fusion of median anlage to ultimobranchial bodies were observed at E11 . 5 E13 . 5 ( arrow ) , respectively , in Tubb1 - / - mice . At E11 . 5 , stained migrating thyroid were visible along tract only in Tubb1 - / - ( arrow ) . At E17 . 5 , a pyramidal lobe was visible in Tubb1 - / - ( arrow ) . tr : trachea ; ub : ultimobranchial . Scale bar : 50

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A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μ


B ) Total thyroid surface ( μm2 ) was quantified using Nkx2 - 1 at each embryonic stage . Top : thyroid surface normalised for of each . Bottom : thyroid surface in μm2 . Right : proliferation calculated as proportion of Nkx2 - 1 - positive labelled with Ki67 at E9 . 5 in wt Tubb1 - / - thyroid anlages . ma : median anlage . Note that thyroid ma is larger proliferation higher at E . 9 . 5 in Tubb1 - / - mice , whereas from E13 . 5 onwards Tubb1 - / - thyroids were hypoplastic . Wt in black , KO , Tubb1 - / - in

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B) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in gre


C ) Thyroid marker expression by quantitative PCR in thyroid tissue at E15 . 5 E17 . 5 in Tubb1 - / - versus wt mice normalised for peptidylprolyl isomerase A . Note decreases in thyroid differentiation markers at E15 . 5 E17 . 5 also in thyroid transcription factors at E17 .

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C) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5


D ) of Nkx2 - 1 T4 at E17 . 5 surface demonstrating T4 retention in Tubb1 - / - versus wt thyroids . data are of T4 or calcitonin ( CT ) surface versus total thyroid ( estimated from Nkx2 - 1 - stained surface ) . Scale bar : 50 μm . Student " s , * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 . Experiments were done on at least three tissues per stage for each

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D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen


A ) Serum TSH T4 in 3 - month - old Tubb1 - / - wild - type mice . Numbers of tested were 10 wild - type 14 Tubb1 - / - for TSH for 14 wild - type 22 Tubb1 - / - for T4 . Tubb1 - / - mice had hypothyroidism with elevated TSH decreased T4 versus wild - type mice . Student " s , * * p <

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A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p<


B ) Nkx2 - 1 ( in brown ) immunostaining of adult thyroid tissue . Note disorganisation of thyroid tissue in Tubb1 - / - versus wild - type

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B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type mice


C ) Ultrastructural alterations shown by electron microscopy in wild - type Tubb1 - / - thyroid tissues . In Tubb1 - / - tissues , note disorganisation of secretion vesicles ( asterisks ) rods of identical density to secretion vesicles ( arrow ) . ER is considerably dilated in Tubb1 - / - thyrocytes . Representative views . Scale bars at bottom left for each view . Co :

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C) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloi


D ) ER stress in Tubb1 - / - thyroid tissue . Top : Chop XBPs expression by quantitative PCR in adults , normalized to peptidylprolyl isomerase A . Experiments with at least three tissues per stage for each genotype ; wt in black Tubb1 - / - in grey . Bottom : Chop Actin expression by western blotting in three representative wild - type Tubb1 - / - mice . Chop band normalised for Actin confirms increased Chop expression demonstrated by quantitative PCR in Tubb1 - / - versus wild - type thyroids . lanes are from same blot , which was cut where indicated . Student " s , * p < 0 . 05 * * * p <

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D) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p<


E ) Co - immunostaining of endoplasmic reticulum ( ER ) marker ( KDEL , in red ) , thyroglobulin ( Tg in green ) , both merged ( from left to right ) in adult thyroid tissue . Note thyrocyte disorganisation in Tubb1 - / -

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O B-CELL_TYPE O O B-GENEPROD O O O

E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice


A ) Representative blood smears of with TUBB1 mutations ( P1 P3 homozygous for p . P160L , P4 heterozygous for p . Y106X , P6 P7 heterozygous for c . 35delG ) after May - Grünwald Giemsa . Variable platelet in was observed with three mutations with presence of macroplatelets ( arrows )

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A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows)


B ) Transmission electron microscopy ( ) of platelet ultrastructure in . results confirm abnormal platelet

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B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphology


C ) Representative of proplatelet formation in P1 P4 . Cultured control megakaryocytes ( MKs ) after thrombopoietin - induced differentiation were spread over a BSA - coated coverslip on day 10 . On day 13 or 14 , MKs were fixed ( 4 % paraformaldehyde ) proplatelet structure was observed after β - tubulin . Scale bars : 20 μm . Only P1 P4 could be studied . graphs show mean ± of shaft thickness of proplatelet extensions ( left ) diameter of coiled elements ( right ) of future proplatelets , both of which were significantly increased in , indicating that TUBB1 mutations ( homozygous p . P160L heterozygous p . Y106X ) affected proplatelet . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons ( * p < 0 . 05 , * * * p < 0 . 001 ; 20 - 60 MKs were analysed /

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C) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat


A ) Relative expression of α - tubulin β1 - tubulin in platelets from P1 P3 ( homozygous p . P160L ) , P4 ( heterozygous p . Y106X ) , P6 P7 ( heterozygous c . 35delG ) was quantified by Western blotting using specific antibodies against α - tubulin or β1 - tubulin . With both mutations , α - tubulin expression was normal but β1 - tubulin expression was significantly decreased . β1 - tubulin antibody recognizes C - terminal

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domain


B ) αIIbβ3 integrin activation was evaluated by flow cytometry ( % of positive platelets mean ) in whole blood from P1 P3 ( homozygous p . P160L ) from P6 P7 ( heterozygous c . 35delG ) ; P4 could not be studied . While no αIIbβ3 integrin activations were detected in P6 P7 ( family 3 ) , P1 P3 ( family 1 ) exhibited a significant increase of αIIbβ3 activation , indicating basal platelet

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B) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activation


C ) Aggregation of washed platelets induced by ADP ( 5 or 10 μM ) or collagen ( 0 . 6 , 0 . 8 or 2 μg / mL ) was evaluated in at least one member of each family . from families 1 2 had increased platelet aggregation in response to low doses of agonist versus controls , suggesting that TUBB1 affected by homozygous p . P160L heterozygous p . Y106X mutations affected platelet function . Platelet aggregation to both agonists was normal in both from family 3 ( heterozygous c . 35delG ) . Family F1 , n = 3 independent experiments ; Family F2 , n = 1 experiment ; Family F3 , n = 2 independent experiments . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , nd : statistical significance not determined

O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because


( C - C ' ' ' ) Terminal expressing reporters for ( C ) , ER ( C ' ) Golgi apparatus ( C ' ' ) . Red arrowheads : CD4 : : mIFP vesicles associated markers at tip of .

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(C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C''). Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


( D ) Mean 95 % confidence interval of tube branch during first hour of growth . Numbers represent rate of growth estimated from slope of curve + / - SD , p = 0 . 39 , Mann - Whitney , n = 6 .

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(D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6.


( E ) of tip of tube , leading ER Golgi accumulations from junction that defines base of . shading represent mean 95 % confidence interval , n = 4 .

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(E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4.


( F - I ) Terminal expressing general marker CD4 : : mIFP in combination with different or polarity markers : PH : : mCherry , a PIP2 sensor commonly used as apical plasma marker ( F ' ) , an endogenously GFP - tagged Crb ( G ' ) , polarity Par3 : : YFP ( H ' ) Par6 : : mCherry ( H ' ' ) . boxed region in ( H ' ' ' ) is shown at higher magnification in ( I ) over 4 time points . Red arrowheads : CD4 : : mIFP vesicles associated markers at tip of .

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(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.


tomograms from high - pressure frozen . ( A - A ' ) Low magnification ( 2 . 5 nm voxels ) tomogram 3D reconstruction from 10 serial 300 nm sections that cover one terminal . ( A ' ' - A ' ' ' ) 3D reconstructions derived from higher resolution tomograms showing different organelles in .

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TEM tomograms from high-pressure frozen embryos. (A-A') Low magnification (2.5 nm voxels) tomogram and 3D reconstruction from 10 serial 300 nm sections that cover one terminal cell. (A''-A''') 3D reconstructions derived from higher resolution tomograms showing different organelles in the cell.


( B ) Example of an high - resolution ( 0 . 78nm voxels ) tomograms used to generate 3D reconstructions .

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(B) Example of an individual high-resolution (0.78nm voxels) tomograms used to generate 3D reconstructions.


( C ) Low resolution tomogram of another ; dotted box is magnified in ( D ) . ( D ) High - resolution tomogram . ( D ' ) Model derived from 4 serial 3D reconstructions including one shown in ( D ) .

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(C) Low resolution tomogram of another cell; the dotted box is magnified in (D). (D) High-resolution tomogram. (D') Model derived from 4 serial 3D reconstructions including the one shown in (D).


( B ) Distribution of three commonly seen classes of vesicles , expressed as their density per of cytoplasm screened , in two regions of . Box plots represent median , interquartile range ( IQR ) , IQR * 1 . 5 below above IQR . n = 62 tomograms from two different . ( C ) Representative examples for each vesicle class are shown next to plots .

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(B) Distribution of three commonly seen classes of vesicles, expressed as their density per area of cytoplasm screened, in the two regions of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=62 tomograms from two different cells. (C) Representative examples for each vesicle class are shown next to the quantification plots.


( D ) Distribution of endocytic events at apical or basal compartment , expressed as density per surface screened . Left shows tomograms corresponding to branch , right shows tomograms corresponding to tip of . Box plots represent median , interquartile range ( IQR ) , IQR * 1 . 5 below above IQR . n = 48 tomograms from two different .

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(D) Distribution of endocytic events at the apical or basal membrane compartment, expressed as density per membrane surface screened. Left shows tomograms corresponding to the cell body and branch, and right shows tomograms corresponding to the tip of the cell. Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. n=48 tomograms from two different cells.