F . HopAO1 inhibits 3 - OH - C10 : 0 - induced disease resistance . Est : HopAO1 were pretreated with 1 μM 3 - OH - C10 : 0 ( C10 ) and100 μM estradiol , then were inoculated with 5 × 104 cfu / ml Pst DC3000 24 hrs later . titer was analyzed at 3 dpi . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 . 01

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F. HopAO1 inhibits 3-OH-C10:0-induced disease resistance. The Est:HopAO1 plants were pretreated with 1 μM 3-OH-C10:0 (C10) and100 μM estradiol, and then the plants were inoculated with 5×104 cfu/ml Pst DC3000 24 hrs later. The bacterial titer was analyzed at 3 dpi. Values are means ± SD (n=6; ANOVA; p < 0.01


F . HopAO1 inhibits 3 - OH - C10 : 0 - induced disease resistance . Est : HopAO1 were pretreated with 1 μM 3 - OH - C10 : 0 ( C10 ) and100 μM estradiol , then were inoculated with 5 × 104 cfu / ml Pst DC3000 24 hrs later . titer was analyzed at 3 dpi . Values are means ± SD ( n = 6 ; ANOVA ; p < 0 .

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Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 treated with drugs at indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with drugs at the indicated concentrations for six days.


Human spheroids established from BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) DLD1 , were incubated with 1 . 25 µM olaparib or 0 . 5 µM chlorambucil over indicated period of time .

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Human spheroids established from BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) DLD1 cells, were incubated with 1.25 µM olaparib or 0.5 µM chlorambucil over the indicated period of time.


Dose - dependent viability assays of BRCA1 - proficient ( + BRCA1 ) or - deficient ( - BRCA1 ) human RPE1 - hTERT treated with drugs at indicated concentrations for six days .

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Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) human RPE1-hTERT cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA1 - proficient ( + BRCA1 ) or - deficient ( - BRCA1 ) TP53 - deleted treated with drugs at indicated concentrations for six days .

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Dose-dependent viability assays of BRCA1-proficient (+BRCA1) or -deficient (-BRCA1) TP53-deleted cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of Brca1 + / + Brca1 - / - mouse mammary tumour - derived lines treated with drugs at indicated concentrations for six days .

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Dose-dependent viability assays of Brca1+/+ and Brca1-/- mouse mammary tumour-derived cell lines treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA2 - deficient ( Capan - 1 ) or - proficient ( MIA PaCa - 2 ) human pancreatic carcinoma - derived treated with drugs at indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-deficient (Capan-1) or -proficient (MIA PaCa-2) human pancreatic carcinoma-derived cells treated with drugs at the indicated concentrations for six days.


Dose - dependent viability assays of BRCA2 - deficient ( PEO1 ) or - proficient ( C4 - 2 ) human ovarian tumour - derived treated with drugs at indicated concentrations for six days .

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Dose-dependent viability assays of BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells treated with drugs at the indicated concentrations for six days.


BRCA2 - deficient ( PEO1 ) or - proficient ( C4 - 2 ) human ovarian tumour - derived were infected with lentiviruses expressing control or CHD4 shRNAs , followed by selection with puromycin for 72 hours . Dose - dependent viability assays were performed on treated with drugs at indicated concentrations for six days .

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BRCA2-deficient (PEO1) or -proficient (C4-2) human ovarian tumour-derived cells were infected with lentiviruses expressing control or CHD4 shRNAs, followed by selection with puromycin for 72 hours. Dose-dependent viability assays were performed on cells treated with drugs at the indicated concentrations for six days.


BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 were incubated with 1 µM chlorambucil ( Chl ) . Whole extracts prepared at indicated time points during treatment were immunoblotted as shown . GAPDH was used as a loading control .

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BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells were incubated with 1 µM chlorambucil (Chl). Whole cell extracts prepared at the indicated time points during treatment were immunoblotted as shown. GAPDH was used as a loading control.


of chromosome aberrations chromatid / chromosome break frequencies in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h . Data were obtained from three independent experiments normalised to untreated controls . A minimum of 60 Giemsa - stained metaphases were analysed for each sample . Cis , cisplatin ; Chl , chlorambucil .

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Quantification of chromosome aberrations and chromatid/chromosome break frequencies in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 72 h. Data were obtained from three independent experiments and normalised to untreated controls. A minimum of 60 Giemsa-stained metaphases were analysed for each sample. Cis, cisplatin; Chl, chlorambucil.


of G2 / M frequency relative to solvent control , using FACS analyses of incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours . Cis , cisplatin ; Chl , chlorambucil .

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Quantification of G2/M cell frequency relative to solvent control, using FACS analyses of cells incubated with 1 μM chlorambucil or 1 µM cisplatin for 48 hours. Cis, cisplatin; Chl, chlorambucil.


Human H1299 were treated with control ( CTR ) or indicated siRNAs two days before drugs were added to media for dose - dependent viability assays . extracts prepared at time of drug addition were immunoblotted as indicated . SMC1 was used as a loading control .

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Human H1299 cells were treated with control (CTR) or indicated siRNAs two days before drugs were added to the media for dose-dependent viability assays. Cell extracts prepared at the time of drug addition were immunoblotted as indicated. SMC1 was used as a loading control.


Nude mice ( nu / nu ) were injected subcutaneously with 5 x 106 human DLD1 , BRCA2 - proficient ( A ) or BRCA2 - deficient ( B ) . Tumour - bearing mice were treated with 3 mg / kg daily chlorambucil administered intraperitoneally ( i . p . ) for a total of ten days . Tumour was determined on indicated days after initiation of treatment .

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Nude mice (nu/nu) were injected subcutaneously with 5 x 106 human DLD1 cells, BRCA2-proficient (A) or BRCA2-deficient (B). Tumour-bearing mice were treated with 3 mg/kg daily chlorambucil administered intraperitoneally (i.p.) for a total of ten days. Tumour weight was determined on the indicated days after initiation of the treatment.


PDTCs derived from breast cancer samples as previously described ( Bruna et al , 2016 ) were treated with chlorambucil at indicated doses . survival is represented relative to DMSO control . AB521 , ER - negative tumour , no BRCA1 alteration ; STG201 , tumour with BRCA1 promoter methylation loss of BRCA1 expression ; VHIO179 , tumour with BRCA1 germline mutation MAD2L2 inactivating mutation ( olaparib - resistant )

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PDTCs derived from breast cancer samples as previously described (Bruna et al, 2016) were treated with chlorambucil at the indicated doses. Cell survival is represented relative to DMSO control. AB521, ER-negative tumour, no BRCA1 alteration; STG201, tumour with BRCA1 promoter methylation and loss of BRCA1 expression; VHIO179, tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant)


CB17 / SCID mice were injected intramuscularly with 5 x 106 human BRCA2 - deficient HCT116 . Tumour - bearing mice were treated on indicated days with chlorambucil or cisplatin administered intraperitoneally ( i . p . ) , or talazoparib administered orally ( o . s . ) Tumour volume was measured on indicated days after treatment initiation was expressed relative to tumour volume at beginning of treatment ( day 1 ) . Scale bar , 40 µm . Each experimental group included n = 5 mice . Error bars represent . Tumour sections were assessed at end of each treatment using immunohistochemistry of γH2AX .

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CB17/SCID mice were injected intramuscularly with 5 x 106 human BRCA2-deficient HCT116 cells. Tumour-bearing mice were treated on the indicated days with chlorambucil or cisplatin administered intraperitoneally (i.p.), or talazoparib administered orally (o.s.) Tumour volume was measured on the indicated days after treatment initiation and was expressed relative to tumour volume at the beginning of treatment (day 1). Scale bar, 40 µm. Each experimental group included n = 5 mice. Error bars represent SEM. Tumour sections were assessed at the end of each treatment using immunohistochemistry of γH2AX staining.


Wild type Balb / c mice were injected intra - peritoneally with solvent ( daily ) or 3 mg / kg chlorambucil ( daily for 5 days ) or 3 . 3 mg / kg cisplatin ( daily for 3 days ) . Uptake of apoptosis tracer 99mTc - Duramycin 2 h after intra - venous injection was quantified in selected organs using SPECT imaging in indicated organs . Representative maximum partial projections showing tracer distribution are shown .

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Wild type Balb/c mice were injected intra-peritoneally with solvent (daily) or 3 mg/kg chlorambucil (daily for 5 days) or 3.3 mg/kg cisplatin (daily for 3 days). Uptake of the apoptosis tracer 99mTc-Duramycin 2 h after intra-venous injection was quantified in selected organs using SPECT imaging in the indicated organs. Representative maximum intensity partial projections showing tracer distribution are shown.


Immunohistochemical analyses of γH2AX in organs from mice treated as in A . Scale bar , 25 µm .

Immunohistochemical analyses of γH2AX staining in organs from mice treated as in A. Scale bar, 25 µm.


( A - D ) of a representative wild - type adult eye ( A ) or adult eyes containing mtm mutant clones ( B C ) . of relative eye sizes ( n = 10 ) ( D ) .

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(A-D) Images of a representative wild-type adult eye (A) or adult eyes containing mtm mutant clones (B and C). Quantification of relative eye sizes (n=10) (D).


( F - G ) A pupal eye disc containing mtm mutant clones ( marked by absence of GFP ) was stained for Dlg . Note increased interommatidial in mtm mutant clone . of numbers of interommatidial per ommatidium ( n = 10 ) ( G ) .

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(F-G) A pupal eye disc containing mtm mutant clones (marked by the absence of GFP) was stained for Dlg. Note the increased interommatidial cells in the mtm mutant clone. Quantification of numbers of interommatidial cells per ommatidium (n=10) (G).


( K ) RNAi of Mtm reduces Yki phosphorylation . Kc167 were incubated with dsRNA of gfp or mtm for three days before western blot . Total lysates were probed with anti - phospho - Yki ( p - Yki ) antibody . Note that RNAi of mtm decreased Yki phosphorylation .

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(K) RNAi of Mtm reduces Yki phosphorylation. Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis. Total cell lysates were probed with anti-phospho-Yki (p-Yki) antibody. Note that RNAi of mtm decreased Yki phosphorylation.


PI ( 3 ) P expression detected by a 2xFYVE - GFP reporter in a third instar larval wing disc ( A ) eye disc ( B ) containing mtm mutant clones ( marked by absence of RFP ) . Ten different mtm clones surrounding regions in three biological replicates were used for . Note strong increases of PI ( 3 ) P in mtm mutant clones .

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PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


of mean FYVE - GFP of mtm mutant clones surrounding wild - type ( WT ) are shown ( C ) . Ten different mtm clones surrounding regions in three biological replicates were used for . Note strong increases of PI ( 3 ) P in mtm mutant clones .

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Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones.


( A ) Following transfection of given siRNAs synchronization in G2 - phase , Hek293 were DRB - or mock - treated then subjected to IFM using indicated antibodies . Lower panels display a 3 - fold magnification of boxed shown above . Scales bars correspond to 5 1 μm ,

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(A) Following transfection of given siRNAs and synchronization in G2-phase, Hek293 cells were DRB- or mock-treated and then subjected to IFM using the indicated antibodies. Lower panels display a 3-fold magnification of the boxed area shown above. Scales bars correspond to 5 and 1 μm, respectively


( B ) Hek293 were thymidine - arrested for 20h , mock - or OHT - treated to induce DSBs by accumulation of ER - AsiSI then subjected to ChIP - multiplex PCR

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(B) Hek293 cells were thymidine-arrested for 20h, mock- or OHT-treated to induce DSBs by nuclear accumulation of ER-AsiSI and then subjected to ChIP-multiplex PCR


( C ) ChIP samples from ( B ) were analysed by qPCR . Shown are averages ( bars ) of 3 independent experiments ( dots )

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(C) ChIP samples from (B) were analysed by qPCR. Shown are averages (bars) of 3 independent experiments (dots)


Separase interacts with γH2AX in DSB - containing G2 but not G1 . Transgenic Hek293 treated with Dox to induce expression of Myc - separase - WT with OHT to induce accumulation of ER - AsiSI infliction of DSBs were synchronized in G1 - or G2 - phase analysed by IP -

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Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed by IP-Western


Separase interacts with γH2AX in DSB - containing G2 but not G1 . Transgenic Hek293 treated with Dox to induce expression of Myc - separase - WT with OHT to induce accumulation of ER - AsiSI infliction of DSBs were synchronized in G1 - or G2 - phase analysed b IFM for γH2AX - Myc - separase - positive foc of IFM in ( E ) shows averages ( bars ) of 3 independent experiments ( dots ) counting ≥ 100

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Separase interacts with γH2AX in DSB-containing G2 but not G1 cells. Transgenic Hek293 cells treated with Dox to induce expression of Myc-separase-WT and with OHT to induce nuclear accumulation of ER-AsiSI and infliction of DSBs were synchronized in G1- or G2-phase and analysed b IFM for γH2AX- and Myc-separase-positive foc The quantification of the IFM in (E) shows averages (bars) of 3 independent experiments (dots) counting ≥ 100 cells each


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) U2OS EJ5 - GFP ( NHEJ reporter ) were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , then supplemented with OHT in G2 - phase to induce accumulation of homing endonuclease . Ethanol - supplemented samples served as negative controls . Two days later , were subjected to immunoblotting (

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected to immunoblotting (A


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) U2OS EJ5 - GFP ( NHEJ reporter ) were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , then supplemented with OHT in G2 - phase to induce accumulation of homing endonuclease . Ethanol - supplemented samples served as negative controls . Two days later , were subjected t flow cytometry to quantify of GFP positive ( B GFP in ( B ) displays averages ( bars ) of 3 independent experiments ( dots )

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls. Two days later, cells were subjected t flow cytometry to quantify the percentage of GFP positive cells (B The GFP quantification in (B) displays averages (bars) of 3 independent experiments (dots)


Separase is required for proper HDR but dispensable for NHEJ . U2OS DR - GFP ( HDR reporter ) U2OS EJ5 - GFP ( NHEJ reporter ) were separase - depleted by RNAi ( SEP - 1 ) or control - treated with GL2 siRNA , transfected to express HA - tagged ER - I - SceI , then supplemented with OHT in G2 - phase to induce accumulation of homing endonuclease . Ethanol - supplemented samples served as negative controls Two days later PI - stained ( C )

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Separase is required for proper HDR but dispensable for NHEJ. U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells were separase-depleted by RNAi (SEP-1) or control-treated with GL2 siRNA, transfected to express HA-tagged ER-I-SceI, and then supplemented with OHT in G2-phase to induce nuclear accumulation of the homing endonuclease. Ethanol-supplemented samples served as negative controls Two days later PI-stained cellular DNA (C)


Co - depletion of Wapl separase has a synergistic effect on HDR . U2OS DR - GFP were transfected with indicated siRNAs , treated as in ( A + B ) analysed by GFP - flow cytometry ( D Shown in ( D ) are averages ( bars ) of 2 - 3 independent experiments ( dots )

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Co-depletion of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed by GFP-flow cytometry (D Shown in (D) are averages (bars) of 2-3 independent experiments (dots)


Co - depletio of Wapl separase has a synergistic effect on HDR . U2OS DR - GFP were transfected with indicated siRNAs , treated as in ( A + B ) analysed b immunoblotting ( E )

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Co-depletio of Wapl and separase has a synergistic effect on HDR. U2OS DR-GFP cells were transfected with the indicated siRNAs, treated as in (A+B) and analysed b immunoblotting (E)


( A ) Transgenic Hek293 constitutively expressing a separase sensor ( cartoon below ) inducibly expressing Myc - separase were transiently transfected to express Flag - AsiSI - ER , Dox - / or OHT - treated in G2 - phase analysed by ( IP - )

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(A) Transgenic Hek293 cells constitutively expressing a separase sensor (cartoon below) and inducibly expressing Myc-separase were transiently transfected to express Flag-AsiSI-ER, Dox- and/or OHT-treated in G2-phase and analysed by (IP-)Western


( B ) from ( A ) were analysed by IP - Western for cleavage of endogenous Rad21 . As a control , in vitro - translated ( IVT ) Rad21 was incubated with hyperactive separase - SA or a protease - dead ( PD ) varian

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(B) Cells from (A) were analysed by IP-Western for cleavage of endogenous Rad21. As a control, in vitro-translated (IVT) Rad21 was incubated with hyperactive separase-SA or a protease-dead (PD) varian


( C ) Sensor expressing were treated in G2 - phase with DRB nocodazole for 6 2 h , respectively , prior to chromosome spreading IFM using Hec1 - γH2AX - antibodies . separase sensor was detected on autofluorescence of eGFP mCherry , while Hec1 - γH2AX - antibodies were detected with corresponding Cy5 - marina blue - labeled secondary antibodies , respectively . Note that sizes of spread chromosomes vary greatly with buffer conditions , which is why no scale bar is shown

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O 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) Sensor expressing cells were treated in G2-phase with DRB and nocodazole for 6 and 2 h, respectively, prior to chromosome spreading and IFM using Hec1- and γH2AX-antibodies. The separase sensor was detected based on autofluorescence of eGFP and mCherry, while Hec1- and γH2AX-antibodies were detected with corresponding Cy5- and marina blue-labeled secondary antibodies, respectively. Note that sizes of spread chromosomes vary greatly with buffer conditions, which is why no scale bar is shown


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 Myc - separase - WT or - S1660A expressing were subjected to ( IP - ) Western using , amongst others , a pan - specific antibody against phosphorylated serine ( A , pan -

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

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 Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst others, a pan-specific antibody against phosphorylated serine (A, pan-pS


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 Myc - separase - WT or - S1660A expressing were subjected to ( IP - ) Western using , amongst other a separase antibody specific for phosphorylated Ser1660 ( B , pS1660 )

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

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 Myc-separase-WT or -S1660A expressing cells were subjected to (IP-)Western using, amongst other a separase antibody specific for phosphorylated Ser1660 (B, pS1660)


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

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

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

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

(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

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

(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

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

(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 ,

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

(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

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

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

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

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

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

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

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

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

O O O O O B-CONTROLLED_VAR 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

(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

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

(G) 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

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

(A, B) 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 (

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

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

(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 ) .

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

(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 .

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

(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

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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 .

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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 .

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

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 .

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

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 .

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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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 ) .

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

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

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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 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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 B-MEASURED_VAR 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 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

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 .

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 .

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

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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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

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