Immunoprecipitation and biotinylation of SUMM2NB - FLAG ( A ) , RPP4NB - FLAG ( B ) , or CHS1NB - FLAG ( C ) by SNIPER1 H129Y - HATurboID in N . benthamiana . Two biological repeats were carried out with similar results .

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

Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.


Interaction of SNIPER1 H129Y - CLuc and SUMM2NB - NLuc ( D ) , RPP4NB - NLuc ( E ) or CHS1NB - NLuc ( F ) as tested by split - luciferase complementary assay in N . benthamiana . Three biological repeats with four technical repeats each were carried out with similar results .

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

Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.


Ubiquitination of the NB domains from SUMM2 ( A ) , RPP4 ( B ) and CHS1 ( C ) . Immunoblot of bacterial lysates form E . coil strains expressing AtUBA1 - S , MBP - NB - HA , AtUBC8 - S , HIS - FLAG - UBQ10 and SNIPER1 - myc , or strains without one of these components . Two biological repeats were carried out with similar results .

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

Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a),


( a , b ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP ( a ) . The high and low 20 % were sorted ( a ) , re - plated and treated with lysosomal protease inhibitors pepstatin and E - 64d for 1 h ; lysates were then immunoblotted for the indicated proteins ( b ) . ( c ) Densitometry of LC3 - II and p62 western blots ( normalized to actin and hour 0 , mean ± s . e . m . , n = 3 blots from 2 independent experiments , * P = 0 . 051 , * * P = 0 . 0091 ) .

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

(a,b) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP (a). The high and low 20% were sorted (a), re-plated and treated with lysosomal protease inhibitors pepstatin and E-64d for 1 h; lysates were then immunoblotted for the indicated proteins (b). (c) Densitometry of LC3-II and p62 western blots (normalized to actin and hour 0, mean ± s.e.m., n = 3 blots from 2 independent experiments, *P = 0.051, **P = 0.0091).


( d ) ; autophagic LC3 puncta were assessed by quantitative

(d); autophagic LC3 puncta were assessed by quantitative microscopy


( d ) ; autophagic LC3 puncta were assessed by quantitative microscopy ( e ; punctate area per cell , mean ± s . e . m . , n = 50 fields , * P = 0 . 010 , * * P = 0 . 053 ) .

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

(d); autophagic LC3 puncta were assessed by quantitative microscopy (e; punctate area per cell, mean ± s.e.m., n = 50 fields, *P = 0.010, **P = 0.053).


f ) Electron micrographs of HeLa mCherry - GFP - LC3 cells sorted for autophagic flux as in a . Yellow arrows denote autophagosomes ; red arrows indicate autolysosomes . ( g ) Quantification of autophagosomes and autolysomes from electron micrographs ( mean ± s . e . m . , n = 50 fields ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

f) Electron micrographs of HeLa mCherry-GFP-LC3 cells sorted for autophagic flux as in a. Yellow arrows denote autophagosomes; red arrows indicate autolysosomes. (g) Quantification of autophagosomes and autolysomes from electron micrographs (mean ± s.e.m., n = 50 fields). Uncropped images of blots are shown in Supplementary Fig. 6.


a - c ) BJAB lymphoma cells stably expressing mCherry - GFP - LC3 were serially cultured at log phase followed by fluorescence - activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry / GFP . Cells were then re - plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points ( median ratio of mCherry / GFP fluorescence ± s . e . m . , n = 3 wells ) . Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies ( b ) . Representative flow cytometry histograms for cells at 0 h and 24 h after sorting ( c ) .

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

a-c) BJAB lymphoma cells stably expressing mCherry-GFP-LC3 were serially cultured at log phase followed by fluorescence-activated cell sorting for cells with high and low autophagic flux using the ratio of mCherry/GFP. Cells were then re-plated in growth medium and autophagic flux was again measured by flow cytometry at the indicated time points (median ratio of mCherry/GFP fluorescence ± s.e.m., n = 3 wells). Lysates from cells collected at the indicated time points were immunoblotted with the indicated antibodies (b). Representative flow cytometry histograms for cells at 0 h and 24 h after sorting (c).


( d ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as in a ( top and bottom 20 % ) . Following treatment with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) , cell viability was determined by MTS assay ( a tetrazolium reduction - based metabolic measurement ) at 24 h ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 2 . 3 × 10 − 4 , * * P = 0 . 0036 ) .

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

(d) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as in a (top and bottom 20%). Following treatment with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1), cell viability was determined by MTS assay (a tetrazolium reduction-based metabolic measurement) at 24 h (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 2.3×10−4, **P = 0.0036).


( e ) Long - term growth of BJAB mCherry - GFP - LC3 cells sorted for autophagic flux followed by treatment with Fas ligand ( 4 ng ml − 1 ) or TRAIL ( 15 ng ml − 1 ) for 24 h . Cells were then re - plated and allowed to recover for 5 ( Fas ligand ) or 6 ( TRAIL ) days then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 012 ) .

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

(e) Long-term growth of BJAB mCherry-GFP-LC3 cells sorted for autophagic flux followed by treatment with Fas ligand (4 ng ml−1) or TRAIL (15 ng ml−1) for 24 h. Cells were then re-plated and allowed to recover for 5 (Fas ligand) or 6 (TRAIL) days then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.012).


( f ) BJAB mCherry - GFP - LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI ( mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB mCherry-GFP-LC3 cells were sorted for autophagic flux as above and apoptosis was measured at 1 h by flow cytometry using AnnexinV and DAPI (mean ± s.e.m., n = 3 wells, *P = 0.0046).


( g ) Cells sorted as in a , were re - plated and , starting at the indicated times following sorting , treated with Fas ligand ( 4 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( percentage of untreated control , mean ± s . e . m . , n = 3 wells ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells sorted as in a, were re-plated and, starting at the indicated times following sorting, treated with Fas ligand (4 ng ml−1) for 24 h; cell viability was then determined by MTS (percentage of untreated control, mean ± s.e.m., n = 3 wells). Uncropped images of blots are shown in Supplementary Fig. 6.


( a ) BJAB and Jurkat cells were treated with vehicle or chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( 1 . 5 ng ml − 1 ) . Cell viability was determined by MTS 24 h later ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 0 . 0058 ) .

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

(a) BJAB and Jurkat cells were treated with vehicle or chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (1.5 ng ml−1). Cell viability was determined by MTS 24 h later (percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 0.0058).


( b ) Immunoblots of cells in a , c probed for the indicated antibodies . CQ ,

(b) Immunoblots of cells in a,c probed for the indicated antibodies. CQ, chloroquine


( c ) BJAB and Jurkat cells were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by treatment with Fas ligand ( 4 ng ml − 1 ) for 24 h ( same technical replicate as a ) . Cells were then re - plated at low density in growth media and allowed to recover for 5 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0024 ) .

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

(c) BJAB and Jurkat cells were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by treatment with Fas ligand (4 ng ml−1) for 24 h (same technical replicate as a). Cells were then re-plated at low density in growth media and allowed to recover for 5 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0024).


( d ) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs , followed by 3 days of puromycin selection . Selected cells were plated and treated with Fas ligand ( 1 . 5 ng ml − 1 ) or TRAIL ( 4 ng ml − 1 ) for 24 h and viability was assessed by MTS ( percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 , * P = 1 . 7 × 10 − 4 , * * P = 0 . 024 ) .

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

(d) BJAB and Jurkat cells were transduced with the indicated lentiviral shRNA constructs, followed by 3 days of puromycin selection. Selected cells were plated and treated with Fas ligand (1.5 ng ml−1) or TRAIL (4 ng ml−1) for 24 h and viability was assessed by MTS (percentage of control (no ligand), mean ± s.e.m., n = 3, *P = 1.7×10−4, **P = 0.024).


( e ) Immunoblots for Atg5 , Atg7 and p62 confirm protein depletion and autophagy inhibition in d , f .

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

(e) Immunoblots for Atg5, Atg7 and p62 confirm protein depletion and autophagy inhibition in d,f.


( f ) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand ( 15 ng ml − 1 ) or TRAIL ( 12 . 5 ng ml − 1 ) for 24 h ( same technical replicate as d ) . Cells were then re - plated at low density and allowed to recover for 5 - 6 days , and then assayed for viability ( percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0089 ) . Atg7 knockdown data were not included for long - term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment . Uncropped images of blots are shown in Supplementary Fig . 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BJAB and Jurkat cells expressing control or Atg5 shRNA were treated with Fas ligand (15 ng ml−1) or TRAIL (12.5 ng ml−1) for 24 h (same technical replicate as d). Cells were then re-plated at low density and allowed to recover for 5-6 days, and then assayed for viability (percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0089). Atg7 knockdown data were not included for long-term viability owing to the growth suppressive effect of Atg7 knockdown in the absence of drug treatment. Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells stably expressing mCherry - GFP - LC3 were flow sorted for high and low autophagic flux , treated with Fas ligand ( 1 . 5 ng ml − 1 ) for 24 h and viability was assessed by MTS ( a ; percentage of no ligand control , mean ± s . e . m . , n = 3 wells , * P = 0 . 0046 ) . ( b ) Immunoblots following sorting of the samples in a .

(a,b) BJAB and Jurkat cells stably expressing mCherry-GFP-LC3 were flow sorted for high and low autophagic flux, treated with Fas ligand (1.5 ng ml−1) for 24 h and viability was assessed by MTS (a; percentage of no ligand control, mean ± s.e.m., n = 3 wells, *P = 0.0046). (b) Immunoblots following sorting of the samples in a.


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c).


( c , d ) The indicated cell lines were treated with chloroquine ( BJAB , CEM 20 μM ; SKW6 . 4 , 25 μM ; Jurkat , 10 μM ) for 16 h , followed by Fas ligand ( BJAB , 12 . 5 ng ml − 1 ; SKW6 . 4 , 50 ng ml − 1 ; Jurkat , 0 . 4 ng ml − 1 ; CEM , 40 ng ml − 1 ) . Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies ( c ) . Cell viability was assessed by MTS 24 h following Fas ligand treatment ( d ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 1 . 6 × 10 − 4 ) .

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

(c,d) The indicated cell lines were treated with chloroquine (BJAB, CEM 20 μM; SKW6.4, 25 μM; Jurkat, 10 μM) for 16 h, followed by Fas ligand (BJAB, 12.5 ng ml−1; SKW6.4, 50 ng ml−1; Jurkat, 0.4 ng ml−1; CEM, 40 ng ml−1). Lysates from cells collected after chloroquine treatment were immunoblotted with the indicated antibodies (c). Cell viability was assessed by MTS 24 h following Fas ligand treatment (d; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 1.6×10−4).


( e , f ) BJAB and Jurkat cells were transduced with control , Atg5 , Atg7 or Vps34 shRNA lentiviruses , followed by three days of puromycin selection . Cells were then treated with Fas ligand ( 1 . 25 ng ml − 1 ) or TRAIL ( 1 . 25 ng ml − 1 ) for 24 h and viability was assessed by MTS ( e ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 1 . 4 × 10 − 6 , * * P = 2 . 3 × 10 − 5 , * * * P = 3 . 9 × 10 − 5 , § P = 9 . 2 × 10 − 6 , § § P = 3 . 4 × 10 − 6 , § § § P = 1 . 7 × 10 − 5 ) . Immunoblots demonstrate Atg5 , Atg7 and Vps34 knockdown , autophagy inhibition and altered Fap - 1 levels . The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other ; the Fap - 1 blot is not separated to demonstrate the lack of Fap - 1 protein in Type II Jurkat cells ( f ) . Fap - 1 blots ( b , c , f ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(e,f) BJAB and Jurkat cells were transduced with control, Atg5, Atg7 or Vps34 shRNA lentiviruses, followed by three days of puromycin selection. Cells were then treated with Fas ligand (1.25 ng ml−1) or TRAIL (1.25 ng ml−1) for 24 h and viability was assessed by MTS (e; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 1.4×10−6, **P = 2.3×10−5, ***P = 3.9×10−5, §P = 9.2×10−6, §§P = 3.4×10−6, §§§P = 1.7×10−5). Immunoblots demonstrate Atg5, Atg7 and Vps34 knockdown, autophagy inhibition and altered Fap-1 levels. The blots are separated because different exposures were required to detect the proteins in one cell line without overexposing the lanes for the other; the Fap-1 blot is not separated to demonstrate the lack of Fap-1 protein in Type II Jurkat cells (f). Fap-1 blots (b,c,f) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


( a , b ) BJAB and Jurkat cells expressing the indicated Fap - 1 wild - type ( WT ) and catalytically inactive ( ΔCD ) constructs were treated with chloroquine ( BJAB , 20 μM ; Jurkat , 10 μM ) for 16 h followed by Fas ligand ( BJAB , 1 . 5 ng ml − 1 ; Jurkat 15 ng ml − 1 ) for 24 h . Cell viability was determined by MTS ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 2 . 7 × 10 − 4 , * * P = 2 . 4 × 10 − 5 , * * * P = 0 . 0024 ) . Cell lysates were blotted and probed with the indicated antibodies ( b ) .

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

(a,b) BJAB and Jurkat cells expressing the indicated Fap-1 wild-type (WT) and catalytically inactive (ΔCD) constructs were treated with chloroquine (BJAB, 20 μM; Jurkat, 10 μM) for 16 h followed by Fas ligand (BJAB, 1.5 ng ml−1; Jurkat 15 ng ml−1) for 24 h. Cell viability was determined by MTS (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 2.7×10−4, **P = 2.4×10−5, ***P = 0.0024). Cell lysates were blotted and probed with the indicated antibodies (b).


c - e ) BJAB cells expressing control or Fap - 1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand ( 12 . 5 ng ml − 1 ) or TRAIL ( 25 ng ml − 1 ) for 24 h ; cell viability was then determined by MTS ( c ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 4 . 8 × 10 − 6 , * * P = 8 . 1 × 10 − 5 , * * * P = 8 . 0 × 10 − 4 , § P = 0 . 013 , NS P > 0 . 05 ) .

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

c-e) BJAB cells expressing control or Fap-1 shRNAs were treated with 20 μM chloroquine for 16 h followed by Fas ligand (12.5 ng ml−1) or TRAIL (25 ng ml−1) for 24 h; cell viability was then determined by MTS (c; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 4.8×10−6, **P = 8.1×10−5, ***P = 8.0×10−4, §P = 0.013, NS P>0.05).


( d ) Immunoblots of lysates collected following chloroquine treatment . Long - term growth was assessed in cells from c by re - plating them at low density and allowing them to recover for 5 days , followed by viability assay ( percentage of untreated control , mean ± s . e . m . , n = 3 wells , * P = 0 . 043 ; e ) . Fap - 1 blots ( b , d ) were run on separate gels owing to the quantity of protein required for detection ( see Methods ) . Uncropped images of blots are shown in Supplementary Fig . 6 .

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

(d) Immunoblots of lysates collected following chloroquine treatment. Long-term growth was assessed in cells from c by re-plating them at low density and allowing them to recover for 5 days, followed by viability assay (percentage of untreated control, mean ± s.e.m., n = 3 wells, *P = 0.043; e). Fap-1 blots (b,d) were run on separate gels owing to the quantity of protein required for detection (see Methods). Uncropped images of blots are shown in Supplementary Fig. 6.


a , b ) Cell viability was determined by MTS assay following Fas ligand ( 4 ng ml − 1 ) treatment in BJAB cells expressing control or p62 lentiviral shRNAs ( a ; percentage of control ( no ligand ) , mean ± s . e . m . , n = 3 wells , * P = 3 . 2 × 10 − 5 , * * P = 1 . 8 × 10 − 6 ) . Immunoblots confirm p62 depletion and Fap - 1 levels ( b ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell viability was determined by MTS assay following Fas ligand (4 ng ml−1) treatment in BJAB cells expressing control or p62 lentiviral shRNAs (a; percentage of control (no ligand), mean ± s.e.m., n = 3 wells, *P = 3.2×10−5, **P = 1.8×10−6). Immunoblots confirm p62 depletion and Fap-1 levels (b).


( c ) Co - immunoprecipitation of endogenous p62 and Fap - 1 in BJAB cells treated with 20 μM chloroquine for 16 h , followed by treatment with Fas ligand ( 50 ng ml − 1 ) for 2 h at 4 ° C .

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

(c) Co-immunoprecipitation of endogenous p62 and Fap-1 in BJAB cells treated with 20 μM chloroquine for 16 h, followed by treatment with Fas ligand (50 ng ml−1) for 2 h at 4°C.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) the TAK1 inhibitor NG25 ( 2 μM ) , the IKKβ inhibitor BI605906 ( 5 μM ) or the IKKβ inhibitor PS1145 ( 10 μM ) . The cells were then co - stimulated for 30 min with 100 ng / ml LPS and 4 mM ATP . Cell lysates ( 10 μg protein ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . ( B ) As in A , except that the cells were co - stimulated for 1 h with 100 ng / ml LPS and 5 μM nigericin . Similar results were obtained in three independent experiments in A and B .

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

(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25 (2 μM), the IKKβ inhibitor BI605906 (5 μM) or the IKKβ inhibitor PS1145 (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. (B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B.


( C , D ) As in A , B , except that primary human monocyte - derived macrophages were used instead of mouse BMDM . Similar results were obtained in two 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

(C, D) As in A, B, except that primary human monocyte-derived macrophages were used instead of mouse BMDM. Similar results were obtained in two independent experiments.


( A , B ) The Mouse macrophage J774A . 1 cell line was transfected with control siRNA or siRNA against IKKβ ( A ) or IKKα ( B ) . 72 h post - transfection , the cells were co - stimulated for 60 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 5 μM nigericin . Cell lysates ( 10 μg ) were denatured in SDS , subjected to SDS - PAGE and immunoblotted with the antibodies indicated . Similar results were obtained in three ( A ) or two ( B ) 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 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 O O 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) The Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or IKKα (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.


( C , D ) As in ( A , B ) except that BMDM from IKKβ - LysM - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT control mice were co - stimulated without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( C ) or with 100 ng / ml LPS and / or 5 μM nigericin ( D ) . Similar results were obtained in two 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 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(C, D) As in (A, B) except that BMDM from IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (C) or with 100 ng/ml LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.


( A , B ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) , the IKKβ inhibitors BI605906 ( 5 μM ) , TPCA - 1 ( 5 μM ) or PS1145 ( 10 μM ) , the TAK1 inhibitor NG25 ( 2 μM ) or the NLRP3 inhibitor MCC950 . The cells were then co - stimulated for 30 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( A ) and / or 5 μM nigericin ( B ) . The cell culture medium was removed and the cells lysed . Protein in the culture medium was precipitated ( see Methods ) , dissolved in SDS . and subjected to SDS - PAGE , along with cell lysates . After transfer to PVDF membranes , immunoblotting was performed with antibodies recognizing both full length ( FL ) and cleaved ( CL ) gasdermin D ( GSDMD ) , the p20 and p10 fragments of caspase - 1 and IL - 18 . Similar results were obtained in two independent experiments . ( C ) As in A , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) and control WT cells were used . Similar results were obtained in 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) 5 μM BI605906 , then stimulated for 1 h with ( + ) 100 ng / ml LPS and / or 5 μM nigericin or left unstimulated . The cells were lysed in buffer containing 1 % ( v / v ) Triton - X - 100 , and the Triton - X - 100 - soluble and Triton - X - 100 - insoluble fractions were prepared as in Methods . The Triton - X - 100 - insoluble fraction was dissolved SDS subjected to SDS - PAGE and immunoblotted with the antibodies indicated . The nuclear protein Histone H3B ( H3B ) was used as a loading control in the Tritin X - 100 insoluble fraction . Similar results were obtained in three independent experiments . ( B ) As in A , except that the Triton - X - 100 - insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2 . 0 mM DSS .

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

(A) WT BMDM were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v) Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS subjected to SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments. (B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS.


( C , As in A , B except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) mice ( IKKβ ( fl / fl ) ) were used . The U2 Small Nuclear RNA Auxiliary Factor 1 ( U2AF1 ) was used as a loading control . Similar results were obtained in two 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 O O O O O O O O O O O O O O O O O O O O O O

(C, As in A, B except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) mice (IKKβ (fl/fl)) were used. The U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) was used as a loading control. Similar results were obtained in two independent experiments.


( E , F ) As in A , B except that BMDM from caspase - 1 KO mice were used . Similar results were obtained in two 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

(E, F) As in A, B except that BMDM from caspase-1 KO mice were used. Similar results were obtained in two independent experiments.


( A - H ) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA - 1 ( 5 μM ) ( C , G ) or BI605906 ( 5 μM ) ( D , H ) . The cells were then stimulated for 1 h with 100 ng / ml LPS ( E ) or 5 μM nigericin ( F , G , H ) , or co - stimulated with 100 ng / ml LPS and 5 μM nigericin ( B , C , D ) or left unstimulated ( control ) ( A ) . The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3 . A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity . ( A - H ) the panel images show the PLA signal ( red ) and DAPI staining for nuclei ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were obtained in three independent experiments and representative images are shown . Scale bar = 50 μm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA-1 (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml LPS (E) or 5 μM nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the PLA signal (red) and DAPI staining for nuclei (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.


( I - L ) As in A - H , except that IKKβ inhibitors were omitted and iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated ( control ) . Scale bar = 50 μm .

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

(I-L) As in A-H, except that IKKβ inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.


( A - H ) The experiments were performed as in Figs 5A - 5H , except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody , which was visualized using a secondary antibody ( red ) . Nuclei were counterstained with DAPI ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were observed in many fields and obtained in three independent experiments . Representative images are shown Data information : In all panels , scale bar = 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm.


( I - L ) As in A - H , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated . Similar results were obtained in two independent experiments . Data information : In all panels , scale bar = 50 μm .

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

(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm.


( A ) Western blotting of Mcrs1 at the GV ( 0 h ) , GVBD ( 4 h ) , MI ( 8 h ) , and MII ( 12 h ) stages of mouse oocyte meiosis . The blots were probed with anti - Mcrs1 and anti - actin antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting of Mcrs1 at the GV (0 h), GVBD (4 h), MI (8 h), and MII (12 h) stages of mouse oocyte meiosis. The blots were probed with anti-Mcrs1 and anti-actin antibodies.


( G ) Percentages of control ( n = 210 ) , Mcrs1 - KD ( n = 182 ) , and Mcrs1 - rescue ( n = 201 ) oocytes that underwent GVBD and PBE . The bars are representing the mean ± SEM . The P - values were calculated using Student ' s t - test . GVBD % : * * * P = 0 . 0003 ; * P = 0 . 01 , PB1 % : * P = 0 . 03 ; * P = 0 . 02 .

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(G) Percentages of control (n = 210), Mcrs1-KD (n = 182), and Mcrs1-rescue (n = 201) oocytes that underwent GVBD and PBE. The bars are representing the mean ± SEM. The P-values were calculated using Student's t-test. GVBD%: ***P = 0.0003; *P = 0.01, PB1%: *P = 0.03; *P = 0.02.


( A ) Representative images of the occurrence of GVBD in control , Mcrs1 - KD , and Mcrs1 - rescue ( Mcrs1 siRNA + Mcrs1 mRNA ) oocytes at 3 h following release from IBMX . Scale bar , 100 μm . ( B ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 155 ) , Mcrs1 - KD ( n = 157 ) , and Mcrs1 - rescue ( n = 150 ) oocytes .

O O O O O O O O O O O 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 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

(A) Representative images of the occurrence of GVBD in control, Mcrs1-KD, and Mcrs1-rescue (Mcrs1 siRNA + Mcrs1 mRNA) oocytes at 3 h following release from IBMX. Scale bar, 100 μm. (B) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 155), Mcrs1-KD (n = 157), and Mcrs1-rescue (n = 150) oocytes.


( D ) Expression levels of CDK1 in Mcrs1 - KD and control oocytes . The bars represent the mean ( ± SEM ) of 5 biological replicates . The P - values were calculated using Student ' s t - test . * P = 0 . 01 . ( E ) Expression levels of p - CDK1 ( Y15 ) in Mcrs1 - KD and control oocytes at 1 h following release from IBMX ( 200 oocytes per sample ) . * * P = 0 . 006 . ( F ) Expression levels of cyclin B1 in control and Mcrs1 - KD oocytes at 1 h following release from IBMX ( 200 oocytes per sample ) . The bars represent the mean ( ± SEM ) of 3 biological replicates . The P - values were calculated using Student ' s t - test . * * P = 0 . 0042 .

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

(D) Expression levels of CDK1 in Mcrs1-KD and control oocytes. The bars represent the mean (± SEM) of 5 biological replicates. The P-values were calculated using Student's t-test. *P = 0.01. (E) Expression levels of p-CDK1(Y15) in Mcrs1-KD and control oocytes at 1 h following release from IBMX (200 oocytes per sample). **P = 0.006. (F) Expression levels of cyclin B1 in control and Mcrs1-KD oocytes at 1 h following release from IBMX (200 oocytes per sample). The bars represent the mean (± SEM) of 3 biological replicates. The P-values were calculated using Student's t-test. **P = 0.0042.


( I ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control oocytes ( n = 112 ) , Mcrs1 - KD oocytes ( n = 98 ) , and Mcrs1 - KD oocytes treated with 5 mM WEE1 - 1N - 4 ( n = 102 ) . ( J ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 123 ) , Mcrs1 - KD ( n = 134 ) , and Mcrs1 siRNA + cyclin B1 - GFP ( n = 95 ) oocytes . ( K ) The incidence of GVBD at 1 , 2 , and 3 h post - IBMX release in control ( n = 92 ) , Mcrs1 - KD ( n = 97 ) , and Mcrs1 siRNA + cyclin B1 - GFP oocytes treated with 5 mM WEE1 - 1N - 4 ( n = 106 ) .

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

(I) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control oocytes (n = 112), Mcrs1-KD oocytes (n = 98), and Mcrs1-KD oocytes treated with 5 mM WEE1-1N-4 (n = 102). (J) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 123), Mcrs1-KD (n = 134), and Mcrs1 siRNA + cyclin B1-GFP (n = 95) oocytes. (K) The incidence of GVBD at 1, 2, and 3 h post-IBMX release in control (n = 92), Mcrs1-KD (n = 97), and Mcrs1 siRNA + cyclin B1-GFP oocytes treated with 5 mM WEE1-1N-4 (n = 106).


( B ) Detection of newly synthesized RNAs by EU incorporation in control , Mcrs1 - KD , and Mcrs1 - rescue GV oocytes . The scattergram shows the relative fluorescence intensity of EU in control ( n = 43 ) , Mcrs1 - KD ( n = 52 ) , and Mcrs1 - rescue ( n = 30 ) GV oocytes . Scale bar , 10 μm . * * * P = 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Detection of newly synthesized RNAs by EU incorporation in control, Mcrs1-KD, and Mcrs1-rescue GV oocytes. The scattergram shows the relative fluorescence intensity of EU in control (n = 43), Mcrs1-KD (n = 52), and Mcrs1-rescue (n = 30) GV oocytes. Scale bar, 10 μm. ***P = 0.0001.


( C ) Immunofluorescence analysis of H3K4me2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H3K4me2 signals in control ( n = 26 ) and Mcrs1 - KD ( n = 30 ) GV oocytes . * * * P = 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of H3K4me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K4me2 signals in control (n = 26) and Mcrs1-KD (n = 30) GV oocytes. ***P = 0.0001.


( E ) Immunofluorescence analysis of H3K9me2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H3K9me2 signals in control ( n = 29 ) and Mcrs1 - KD ( n = 22 ) GV oocytes . * * P = 0 . 0035 . ( F ) Immunofluorescence analysis of H4K16ac in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of H4K16ac signals in control ( n = 30 ) and Mcrs1 - KD ( n = 28 ) GV oocytes . * P = 0 . 022 .

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

(E) Immunofluorescence analysis of H3K9me2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H3K9me2 signals in control (n = 29) and Mcrs1-KD (n = 22) GV oocytes. **P = 0.0035. (F) Immunofluorescence analysis of H4K16ac in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of H4K16ac signals in control (n = 30) and Mcrs1-KD (n = 28) GV oocytes. *P = 0.022.


( G ) Immunofluorescence analysis of HDAC2 in control and Mcrs1 - KD oocytes . Scale bar , 10 μm . Relative fluorescence intensity of HDAC2 signals in control ( n = 27 ) and Mcrs1 - KD ( n = 24 ) GV oocytes . * * * P = 0 . 0005 .

(G) Immunofluorescence analysis of HDAC2 in control and Mcrs1-KD oocytes. Scale bar, 10 μm. Relative fluorescence intensity of HDAC2 signals in control (n = 27) and Mcrs1-KD (n = 24) GV oocytes. ***P = 0.0005.


( A ) Representative images of spindle assembly at 3 h post - release from IBMX in control and Mcrs1 - KD oocytes . Scale bar , 20 μm .

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(A) Representative images of spindle assembly at 3 h post-release from IBMX in control and Mcrs1-KD oocytes. Scale bar, 20 μm.


( G ) Immunofluorescence analysis of H3S10ph and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of H3S10ph signals in control ( n = 21 ) and Mcrs1 - KD ( n = 23 ) oocytes . * P = 0 . 017 .

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(G) Immunofluorescence analysis of H3S10ph and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of H3S10ph signals in control (n = 21) and Mcrs1-KD (n = 23) oocytes. *P = 0.017.


( H ) Immunofluorescence analysis of γ - tubulin and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The histogram shows the percentages of control ( n = 55 ) and Mcrs1 - KD ( n = 63 ) oocytes with abnormal γ - tubulin . * P = 0 . 014 . ( I ) Immunofluorescence analysis of acetylated tubulin and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control ( n = 83 ) and Mcrs1 - KD ( n = 66 ) oocytes . * * P = 0 . 0046 .

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(H) Immunofluorescence analysis of γ-tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 55) and Mcrs1-KD (n = 63) oocytes with abnormal γ-tubulin. *P = 0.014. (I) Immunofluorescence analysis of acetylated tubulin and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of acetylated tubulin signals in control (n = 83) and Mcrs1-KD (n = 66) oocytes. **P = 0.0046.


( C ) Levels of p - Aurka and p - Aurkc in control and Mcrs1 - KD MI oocytes . The blots were probed with anti - p - Aurora A / B / C and anti - actin antibodies . ( D ) Co - IP was performed with an anti - Mcrs1 antibody . The immunoblots of protein precipitates were probed with an anti - Aurka antibody .

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(C) Levels of p-Aurka and p-Aurkc in control and Mcrs1-KD MI oocytes. The blots were probed with anti-p-Aurora A/B/C and anti-actin antibodies. (D) Co-IP was performed with an anti-Mcrs1 antibody. The immunoblots of protein precipitates were probed with an anti-Aurka antibody.


( G ) Percentages of control ( n = 92 ) , Mcrs1 - KD ( n = 73 ) , and Aurka - rescue ( n = 67 ) oocytes that underwent PBE . * P = 0 . 033 ; * P = 0 . 018 .

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(G) Percentages of control (n = 92), Mcrs1-KD (n = 73), and Aurka-rescue (n = 67) oocytes that underwent PBE. *P = 0.033; *P = 0.018.


( H ) Percentages of control ( n = 69 ) , Mcrs1 - KD ( n = 76 ) , and Aurka - rescue ( n = 43 ) oocytes with abnormal spindles . * P = 0 . 018 ; * P = 0 . 024 .

(H) Percentages of control (n = 69), Mcrs1-KD (n = 76), and Aurka-rescue (n = 43) oocytes with abnormal spindles. *P = 0.018; *P = 0.024.


( K ) Immunofluorescence analysis of Kif2A in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The histogram shows the percentages of control ( n = 50 ) and Mcrs1 - KD ( n = 61 ) oocytes with abnormal Kif2A . * * P = 0 . 003 .

(K) Immunofluorescence analysis of Kif2A in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The histogram shows the percentages of control (n = 50) and Mcrs1-KD (n = 61) oocytes with abnormal Kif2A. **P = 0.003.


( L ) Immunofluorescence analysis of TPX2 and α - tubulin in control and Mcrs1 - KD oocytes . Scale bar , 20 μm . The scattergram shows the relative fluorescence intensity of TPX2 signals in control ( n = 48 ) and Mcrs1 - KD ( n = 52 ) oocytes .

(L) Immunofluorescence analysis of TPX2 and α-tubulin in control and Mcrs1-KD oocytes. Scale bar, 20 μm. The scattergram shows the relative fluorescence intensity of TPX2 signals in control (n = 48) and Mcrs1-KD (n = 52) oocytes.


( B ) The active fragment of mTOR or active Akt2 was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC - 0068 .

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(B) The active fragment of mTOR or active Akt2 was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Akt inhibitor GDC-0068.


( C ) HEK - 293E Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls .

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(C) HEK-293E Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.


( D ) HEK - 293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time . Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways . Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls .

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(D) HEK-293E cells were deprived of total amino acids for 1hr followed by addition of amino acids for the indicated time. Lysates were analysed by Western blot for phosphorylation of RagC and proteins known to belong to the Akt and mTOR pathways. Graph shows mean of quantitative analyses of the Western blots after normalization for total protein or the loading controls.


( E ) Control or iRapKO MEFs were treated with or without 2 μM 4 - Hydroxytamoxifen ( 4 - OHT ) for 4 days to induce Raptor knockout . Treated cells were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot .

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(E) Control or iRapKO MEFs were treated with or without 2 μM 4-Hydroxytamoxifen (4-OHT) for 4 days to induce Raptor knockout. Treated cells were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.


( F ) SIN1 WT was expressed in SIN1 KO MEFs , and cells were selected by FACS . Cell lines were deprived of serum for 2 hr , followed by insulin ( 100 nM ) stimulation for 10 min , and samples were analysed by Western blot .

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(F) SIN1 WT was expressed in SIN1 KO MEFs, and cells were selected by FACS. Cell lines were deprived of serum for 2 hr, followed by insulin (100 nM) stimulation for 10 min, and samples were analysed by Western blot.


The active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Mass Spectrum for pRagC

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

The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Mass Spectrum for pRagC T394


The active fragment of mTOR was incubated with Flag - RagC WT purified from HEK - 293E cells . The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin . Samples were analysed by Western blot for pRagC

The active fragment of mTOR was incubated with Flag-RagC WT purified from HEK-293E cells. The kinase reaction was performed in the presence or absence of mTOR inhibitor Torin or Rapamycin. Samples were analysed by Western blot for pRagC S21


( A ) The HA - RagC HeLa Flp - In T - Rex stable cell lines were created ( See methods for details ) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight . Cells were deprived of serum for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , # no significant difference , two tailed Student ' s t test , n = 3 ) . ( B ) Expression of RagC WT , 3A or 3E was induced by addition of tetracycline overnight . Cells were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for 20 min . Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , # no significant difference , two tailed Student ' s t test , n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The HA-RagC HeLa Flp-In T-Rex stable cell lines were created (See methods for details) and the expression of RagC WT or mutants was induced by addition of tetracycline overnight. Cells were deprived of serum for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3). (B) Expression of RagC WT, 3A or 3E was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for 20 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, # no significant difference, two tailed Student's t test, n=3)


( C ) Expression of RagC WT and 3A was induced by addition of tetracycline overnight . Cells were serum starved for 2 hr followed by insulin ( 100nM ) stimulation for indicated time . Left : Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Right : Graph shows mean ± S . E . M . of quantitative analyses of the Western blots after normalization for total protein , n = 3

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

(C) Expression of RagC WT and 3A was induced by addition of tetracycline overnight. Cells were serum starved for 2 hr followed by insulin (100nM) stimulation for indicated time. Left: Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Right: Graph shows mean ± S.E.M. of quantitative analyses of the Western blots after normalization for total protein, n=3


( D ) HEK - 293E cells stably expressing RagC WT , 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min . Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1 . Graphs indicate the mean ± S . E . M . of quantitative analyses of the western blots after normalization for total protein ( * p < 0 . 05 , * * p < 0 . 01 , two tailed Student ' s t test , n = 3 )

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

(D) HEK-293E cells stably expressing RagC WT, 3A or 3E were deprived of total amino acids for 1hr followed by addition of amino acids for 10 min. Lysates were analyzed by Western blot for the levels of the specified proteins and the phosphorylation state of S6K1. Graphs indicate the mean ± S.E.M. of quantitative analyses of the western blots after normalization for total protein (* p<0.05, ** p<0.01, two tailed Student's t test, n=3)


( A ) Expression of RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 4 )

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(A) Expression of RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=4)


( B ) Phospho - RagC positively regulates wing compartment size in Drosophila . Over - expression ( OE ) of dRagC WT , 3A , or 3E in wing posterior compartment using en - Gal4 driver and the posterior compartment sizes were measured in each genotype ( ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Bonferroni multiple comparisons , n ≥ 63 flies per condition )

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(B) Phospho-RagC positively regulates wing compartment size in Drosophila. Over-expression (OE) of dRagC WT, 3A, or 3E in wing posterior compartment using en-Gal4 driver and the posterior compartment sizes were measured in each genotype ((Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. ** p<0.01, *** p<0.001, **** p<0.0001, One-way ANOVA with Bonferroni multiple comparisons, n ≥ 63 flies per condition)


( C ) Empty vector ( EV ) , RagC WT , 3A or 3E were transiently expressed in stable RagC knockdown HEK - 293E cells followed by cell size analysis using a particle size counter ( Horizontal lines , box limits and whiskers represent the median , the 25th percentile ot the 75th percentile and the minimum to the maximum respectively . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , # no significant difference , One - way ANOVA with Bonferroni multiple comparisons , n = 2 for C2 and n = 4 for other conditions )

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(C) Empty vector (EV), RagC WT, 3A or 3E were transiently expressed in stable RagC knockdown HEK-293E cells followed by cell size analysis using a particle size counter (Horizontal lines, box limits and whiskers represent the median, the 25th percentile ot the 75th percentile and the minimum to the maximum respectively. * p<0.05, ** p<0.01, **** p<0.0001, # no significant difference, One-way ANOVA with Bonferroni multiple comparisons, n=2 for C2 and n=4 for other conditions)


( D ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Lysates were analyzed by Western blot for phosphorylation of ULK1 and total

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

(D) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Lysates were analyzed by Western blot for phosphorylation of ULK1 and total ULK1


( E ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were expressed in HEK - 293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . Lysates were analyzed by Western blot for levels of LC3 - II and 14 - 3 - 3 . Graphs show mean ± S . E . M . of quantitative analyses of LC3 - II blotting after normalization for 14 - 3 - 3 . * p < 0 . 05 , two tailed Student ' s t test , n = 4

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(E) Empty vector (EV), HA-RagC WT, 3A or 3E were expressed in HEK-293E cells followed 48 hr later by incubation with DMEM in the presence or absence of serum for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. Lysates were analyzed by Western blot for levels of LC3-II and 14-3-3.Graphs show mean ± S.E.M. of quantitative analyses of LC3-II blotting after normalization for 14-3-3. * p<0.05, two tailed Student's t test, n=4


( F ) GFP - LC3 was co - expressed with empty vector , RagC WT , 3A or 3E in HEK - 293E cells followed 48 hr later by serum deprivation for 4 hr . Chloroquine ( 100μM ) was added to media 30 min prior to harvest . LC3 and RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm

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(F) GFP-LC3 was co-expressed with empty vector, RagC WT, 3A or 3E in HEK-293E cells followed 48 hr later by serum deprivation for 4 hr. Chloroquine (100μM) was added to media 30 min prior to harvest. LC3 and RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( G ) Quantitative analysis of GFP puncta ( F ) ( * * * * p < 0 . 0001 , two tailed Student ' s t test ; n ≥ 21 ) . Mean value is shown as a horizontal bar

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(G) Quantitative analysis of GFP puncta (F) (**** p<0.0001, two tailed Student's t test; n ≥ 21). Mean value is shown as a horizontal bar


( A ) Empty vector ( EV ) , HA - RagC WT , 3A or 3E were transiently expressed in HEK - 293E cells . RagC and lysosome maker , Lamp1 , were monitored by confocal fluorescence microscopy . Scale bar , 10 μm

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(A) Empty vector (EV), HA-RagC WT, 3A or 3E were transiently expressed in HEK-293E cells. RagC and lysosome maker, Lamp1, were monitored by confocal fluorescence microscopy. Scale bar, 10 μm


( B ) Empty vector ( EV ) , HA - RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK - 293E cells . mTOR and HA - RagC were monitored by confocal fluorescence microscopy . Scale bar , 10 µm

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(B) Empty vector (EV), HA-RagC WT or 3A were transiently expressed with scramble or RagC & RagD shRNA in HEK-293E cells. mTOR and HA-RagC were monitored by confocal fluorescence microscopy. Scale bar, 10 µm


( C ) Empty vector ( EV ) , Flag - RagC WT , 3A or 3E were transiently expressed with HA - RagA in HEK - 293E cells . Total cell lysates and amounts of mTOR , Raptor and HA - RagA in the Flag - RagC immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * p < 0 . 05 , two tailed Student ' s t test , n ≥ 3 )

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(C) Empty vector (EV), Flag-RagC WT, 3A or 3E were transiently expressed with HA-RagA in HEK-293E cells. Total cell lysates and amounts of mTOR, Raptor and HA-RagA in the Flag-RagC immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (* p<0.05, two tailed Student's t test, n ≥ 3)


( D ) Expression of HA - RagC WT , 3A , or 3E was induced by overnight addition of tetracycline in HeLa cells . Total cell lysates ( TCL ) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot . Graphs show mean ± S . E . M . of quantitative analyses of Western blots ( * * * p < 0 . 001 , Student ' s t test , n = 3 )

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(D) Expression of HA-RagC WT, 3A, or 3E was induced by overnight addition of tetracycline in HeLa cells. Total cell lysates (TCL) and amount of mTOR in the Raptor immunoprecipitates were analyzed by Western blot. Graphs show mean ± S.E.M. of quantitative analyses of Western blots (*** p<0.001, Student's t test, n=3)


A , Rb1 protein ( RB ) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice . Scale bar = 100μm ; arrowhead , embryo . B , Phosphorylated RB ( pRB ) protein , the inactive form of RB , was expressed in the luminal epithelium on days 2 and 3 of pregnancy . Scale bar = 100μm .

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A, Rb1 protein (RB) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice. Scale bar = 100μm; arrowhead, embryo. B, Phosphorylated RB (pRB) protein, the inactive form of RB, was expressed in the luminal epithelium on days 2 and 3 of pregnancy. Scale bar = 100μm.


C , RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 ( Rb1d / d mice ) on day 4 of pregnancy . Scale bar = 100μm . D , RB expression was normal in the ovary of Rb1d / d mice on day 4 of pregnancy . Scale bar = 100μm .

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C, RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 (Rb1d/d mice) on day 4 of pregnancy. Scale bar = 100μm. D, RB expression was normal in the ovary of Rb1d/d mice on day 4 of pregnancy. Scale bar = 100μm.


E , The number of new - born pups was decreased in Rb1d / d dams ( n = 37 different dams ) compared to their littermate controls ( Rb1f / f mice , n = 20 different dams ) ( mean ± SEM , Student ' s t test ) .

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E, The number of new-born pups was decreased in Rb1d/d dams (n=37 different dams) compared to their littermate controls (Rb1f/f mice, n=20 different dams) (mean ± SEM, Student's t test).


A - C , Ovulation , fertilization and development of preimplantation embryos were normal in Rb1d / d mice ( A & C , mean ± SEM , Student ' s t test ; n = 5 mice for each group ; B , Fisher ' s exact test ; n = 5 mice for each group ) .

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A-C, Ovulation, fertilization and development of preimplantation embryos were normal in Rb1d/d mice (A&C, mean ± SEM, Student's t test; n=5 mice for each group; B, Fisher's exact test; n=5 mice for each group).


Number of Ki67 - positive cells on day 4 in uterine epithelium of Rb1d / d mice was higher than that of Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

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Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.


F - G , Embryo attachment occurred normally in Rb1d / d mice at 0900h on day 5 ( mean ± SEM , Student ' s t test ; n = 5 mice for each group ) . Arrow , implantation site ; scale bar = 1 cm .

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F-G, Embryo attachment occurred normally in Rb1d/d mice at 0900h on day 5 (mean ± SEM, Student's t test; n=5 mice for each group). Arrow, implantation site; scale bar = 1 cm.


H - I , Rb1d / d mice showed higher rate of embryo resorption on day 8 ( * P < 0 . 05 ; Fisher ' s exact test ; n = 45 implantation sites for Rb1f / f mice and n = 46 implantation sites for Rb1d / d mice ) . Arrow , implantation site ; scale bar = 1 cm .

H-I, Rb1d/d mice showed higher rate of embryo resorption on day 8 (*P<0.05; Fisher's exact test; n=45 implantation sites for Rb1f/f mice and n=46 implantation sites for Rb1d/d mice). Arrow, implantation site; scale bar = 1 cm.


J , H & E staining showed embryo resorption in Rb1d / d mice on day 8 of pregnancy . Scale bar = 200 μm ; arrowhead , embryo ; arrow , broken embryo with blood cell infiltration .

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J, H&E staining showed embryo resorption in Rb1d/d mice on day 8 of pregnancy. Scale bar = 200 μm; arrowhead, embryo; arrow, broken embryo with blood cell infiltration.


K , Weight of implantation site was comparable between Rb1f / f and Rb1d / d mice on day 6 , but reduced on day 8 in Rb1d / d mice compared to Rb1f / f mice ( mean ± SEM , Student ' s t test ; n = 6 mice for each group ) .

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K, Weight of implantation site was comparable between Rb1f/f and Rb1d/d mice on day 6, but reduced on day 8 in Rb1d/d mice compared to Rb1f/f mice (mean ± SEM, Student's t test; n=6 mice for each group).


L - M , Expression of Cox2 and Bmp2 , markers of decidualization , was comparable in between Rb1d / d and Rb1f / f mice at 0900h on day 6 in implantation sites ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

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L-M, Expression of Cox2 and Bmp2, markers of decidualization, was comparable in between Rb1d/d and Rb1f/f mice at 0900h on day 6 in implantation sites (mean ± SEM, Student's t test; n=4 mice for each group).


A , Immunostaining of cytokeratin 8 ( CK8 ) , a marker of epithelial cell lineage , showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f / f mice , while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d / d mice . Scale bar = 200μm ; arrowhead , an embryo .

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A, Immunostaining of cytokeratin 8 (CK8), a marker of epithelial cell lineage, showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f/f mice, while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d/d mice. Scale bar = 200μm; arrowhead, an embryo.


B , Transmission electron microscopic analyses ( TEM ) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f / f mice , while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d / d mice . Scale bar = 10µm ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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B, Transmission electron microscopic analyses (TEM) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f/f mice, while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d/d mice. Scale bar = 10µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


C , TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice ( Rb1f / f mice ) . Scale bar = 2µm . Arrowhead , cytoplasmic fragments engulfed by trophoblast ; yellow dotted line circle , cytoplasmic lipid droplets in the luminal epithelium ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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C, TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice (Rb1f/f mice). Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; yellow dotted line circle, cytoplasmic lipid droplets in the luminal epithelium; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


D , TEM showed that the microvilli , markers of uterine receptivity , were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice ( Rb1f / f mice ) . Scale bar = 2µm ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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D, TEM showed that the microvilli, markers of uterine receptivity, were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice (Rb1f/f mice). Scale bar = 2µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


E , Immunofluorescence of MFG - E8 , a marker of programmed cell death , showed that MFG - E8 is not expressed at the implantation sites of Rb1f / f mice at 0900h on day 5 and Rb1d / d mice at 0900h and 2000h on day 5 , but is expressed at the implantation sites of Rb1f / f mice at 2000h on day 5 . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; yellow signal , MFG - E8 ; arrowhead , embryo .

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E, Immunofluorescence of MFG-E8, a marker of programmed cell death, showed that MFG-E8 is not expressed at the implantation sites of Rb1f/f mice at 0900h on day 5 and Rb1d/d mice at 0900h and 2000h on day 5, but is expressed at the implantation sites of Rb1f/f mice at 2000h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; arrowhead, embryo.


A , Serum estradiol - 17β ( E2 ) levels on days 4 and 6 of pregnancy were comparable between Rb1f / f and Rb1d / d mice ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

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A, Serum estradiol-17β (E2) levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice (mean ± SEM, Student's t test; n=4 mice for each group).


B , Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f / f and Rb1d / d mice . In Rb1d / d mice , pre - implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 ( mean ± SEM , Student ' s t test ; n = 4 mice for each group ) .

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B, Serum P4 levels on days 4 and 6 of pregnancy were comparable between Rb1f/f and Rb1d/d mice. In Rb1d/d mice, pre-implantation P4 treatment on days 2 and 3 increased serum P4 levels on day 4 but did not affect those on day 6 (mean ± SEM, Student's t test; n=4 mice for each group).


D , Persistent Ki67 expression in the epithelium of Rb1d / d mice on day 4 was recovered by pre - implantation P4 treatment on days 2 and 3 . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . E , Ratio of Ki67 - positive epithelial cells in Rb1d / d mice was completely suppressed by pre - implantation P4 supplementation on days 2 and 3 . The same data of Rb1f / f and Rb1d / d mice without P4 treatment in Fig 2D were used ( mean ± SEM , Student ' s t test ; n = 5 different mice for each group ) . Three different high - powered fields per mouse were analyzed and each of the Ki67 - posivie ratios was demonstrated .

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D, Persistent Ki67 expression in the epithelium of Rb1d/d mice on day 4 was recovered by pre-implantation P4 treatment on days 2 and 3. Scale bar = 100μm; le, luminal epithelium; s, stroma. E, Ratio of Ki67-positive epithelial cells in Rb1d/d mice was completely suppressed by pre-implantation P4 supplementation on days 2 and 3. The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2D were used (mean ± SEM, Student's t test; n=5 different mice for each group). Three different high-powered fields per mouse were analyzed and each of the Ki67-posivie ratios was demonstrated.


G , Resorption rate was reduced on day 8 in Groups 1 ( n = 41 different implantation sites ) and 2 ( n = 30 different implantation sites ) , but not in Group 3 ( n = 40 different implantation sites ) . The same data of Rb1f / f and Rb1d / d mice without P4 treatment in Fig 2I were used . H & E staining was performed using all sections that shows the implantation sites , and resorption rate was evaluated . * P < 0 . 05 vs Rb1f / f mice ; * * P < 0 . 05 vs Rb1d / d mice ; Fisher ' s exact test .

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G, Resorption rate was reduced on day 8 in Groups 1 (n=41 different implantation sites) and 2 (n=30 different implantation sites), but not in Group 3 (n=40 different implantation sites). The same data of Rb1f/f and Rb1d/d mice without P4 treatment in Fig 2I were used. H&E staining was performed using all sections that shows the implantation sites, and resorption rate was evaluated. *P<0.05 vs Rb1f/f mice; **P<0.05 vs Rb1d/d mice; Fisher's exact test.


H , Number of pups delivered by Rb1d / d dams was normalized by pre - implantation P4 treatment on days 2 and 3 ( Group 2 ) ( mean ± SEM , Student ' s t test ) . The same data of Rb1f / f and Rb1d / d dams without P4 treatment in Fig 1E were used . The numbers of pups in 20 Rb1f / f and 37 Rb1d / d dams without P4 treatment and 7 Rb1d / d dams with P4 treatment were evaluated .

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H, Number of pups delivered by Rb1d/d dams was normalized by pre-implantation P4 treatment on days 2 and 3 (Group 2)(mean ± SEM, Student's t test). The same data of Rb1f/f and Rb1d/d dams without P4 treatment in Fig 1E were used. The numbers of pups in 20 Rb1f/f and 37 Rb1d/d dams without P4 treatment and 7 Rb1d/d dams with P4 treatment were evaluated.


A , pRIP3 , a central mediator of necroptosis , was not expressed at the implantation sites of Rb1d / d mice but was expressed at those of Rb1f / f mice at 1500h on day 5 . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; purple signal , pRIP3 ; le , luminal epithelium ; s , stroma ; arrowhead , embryo ; green dotted line , luminal epithelium ; red dotted line , stroma .

A, pRIP3, a central mediator of necroptosis, was not expressed at the implantation sites of Rb1d/d mice but was expressed at those of Rb1f/f mice at 1500h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo; green dotted line, luminal epithelium; red dotted line, stroma.


B , TNFα stimulated the expression of pMLKL , a critical mediator of necroptosis , in the primary mouse uterine epithelial cells . Scale bar = 50μm ; blue signal , nuclei stained by DAPI ; red signal , pMLKL .

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B, TNFα stimulated the expression of pMLKL, a critical mediator of necroptosis, in the primary mouse uterine epithelial cells. Scale bar = 50μm; blue signal, nuclei stained by DAPI; red signal, pMLKL.


C , Uterine expression of Tnfα was elevated in the control mice ( Rb1f / f mice ) on days 5 and 6 compared to on day 4 ( mean ± SEM , Student ' s t test ; n = 3 mice for each group ) . As for the uterine samples on days 5 and 6 , those with implantation sites were examined .

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C, Uterine expression of Tnfα was elevated in the control mice (Rb1f/f mice) on days 5 and 6 compared to on day 4 (mean ± SEM, Student's t test; n=3 mice for each group). As for the uterine samples on days 5 and 6, those with implantation sites were examined.


D , The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f / f and Rb1d / d mice . Immunostaining of TNFα at the implantation sites of Rb1f / f and Rb1d / d mice were performed . Scale bar = 100μm ; arrowhead , embryo .

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D, The implanting embryo and the uterus produced TNFα at the implantation site of both Rb1f/f and Rb1d/d mice. Immunostaining of TNFα at the implantation sites of Rb1f/f and Rb1d/d mice were performed. Scale bar = 100μm; arrowhead, embryo.


A - B , In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V , a marker of PS presentation at the outer membrane of Rb1f / f epithelial cells but does not in Rb1d / d ones , and P4 restores TNFα - primed annexin V expression in Rb1d / d uterine epithelial cells ( mean ± SEM , Student ' s t test ) . Using three lines of primary mouse epithelial cells obtained from both Rb1f / fand Rb1d / d mice , the in vivo experiments were performed three times . Each of the annexin V - positive ratios was demonstrated . Scale bar = 100μm ; green signal , annexin V .

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A-B, In vitro analyses using primary mouse uterine epithelial cells demonstrated that TNFα induces the expression of annexin V, a marker of PS presentation at the outer membrane of Rb1f/f epithelial cells but does not in Rb1d/d ones, and P4 restores TNFα-primed annexin V expression in Rb1d/d uterine epithelial cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.


C - D , Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth - arrested mouse epithelial cells with thymidine treatment , but does not in the control cells ( mean ± SEM , Student ' s t test ) . Using three lines of primary mouse epithelial cells obtained from both Rb1f / fand Rb1d / d mice , the in vivo experiments were performed three times . Each of the annexin V - positive ratios was demonstrated . Scale bar = 100μm ; green signal , annexin V .

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C-D, Annexin V assay using primary mouse uterine epithelial cells showed that TNFα administration increases the expression of annexin V in growth-arrested mouse epithelial cells with thymidine treatment, but does not in the control cells (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d mice, the in vivo experiments were performed three times. Each of the annexin V-positive ratios was demonstrated. Scale bar = 100μm; green signal, annexin V.


E , The expression of TNF receptor type 2 ( TNFR2 ) was upregulated in growth - arrested epithelial cells with thymidine and P4 supplementation ( mean ± SEM , Student ' s t test ) . Using three lines of primary mouse epithelial cells obtained from both Rb1f / fand Rb1d / d groups , qPCR was performed in duplicate .

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E, The expression of TNF receptor type 2 (TNFR2) was upregulated in growth-arrested epithelial cells with thymidine and P4 supplementation (mean ± SEM, Student's t test). Using three lines of primary mouse epithelial cells obtained from both Rb1f/fand Rb1d/d groups, qPCR was performed in duplicate.


A , In Rb1d / d mice with pre - implantation P4 treatment , the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells . Scale bar = 2µm . Arrowhead , cytoplasmic fragments engulfed by trophoblast ; dotted line circle , lipid droplets in the cytoplasm ; tr , trophoblast ; le , luminal epithelium ; s , stroma ; red dotted line , stroma ; green dotted line , luminal epithelium ; blue dotted line , trophoblast .

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A, In Rb1d/d mice with pre-implantation P4 treatment, the fragmented uterine epithelial cells with the cytoplasmic lipid droplets were engulfed by the trophoblast cells. Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; dotted line circle, lipid droplets in the cytoplasm; tr, trophoblast; le, luminal epithelium; s, stroma; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.


B , Pre - implantation P4 treatment rescued the expression of MFG - E8 on day 5 of pregnancy in Rb1d / d uteri . Scale bar = 100μm . Blue signal , nuclei stained by DAPI ; yellow signal , MFG - E8 ; le , luminal epithelium ; s , stroma ; arrowhead , embryo . C , Pre - implantation P4 treatment recovered the expression of pRIP3 , a mediator of necroptosis , on day 5 of pregnancy in Rb1d / d uteri . Scale bar = 100μm ; blue signal , nuclei stained by DAPI ; purple signal , pRIP3 ; le , luminal epithelium ; s , stroma ; arrowhead , embryo .

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B, Pre-implantation P4 treatment rescued the expression of MFG-E8 on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm. Blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; le, luminal epithelium; s, stroma; arrowhead, embryo. C, Pre-implantation P4 treatment recovered the expression of pRIP3, a mediator of necroptosis, on day 5 of pregnancy in Rb1d/d uteri. Scale bar = 100μm; blue signal, nuclei stained by DAPI; purple signal, pRIP3; le, luminal epithelium; s, stroma; arrowhead, embryo.


A , RB was efficiently deleted in the uterine epithelium of uterine epithelium - specific conditional knockout mice ( Rb1 eKO mice ) . Scale bar = 100μm . Rb1 eKO mice , Rb1loxP / loxPLtfCre / + mice ; Rb1 eControl mice ( littermate controls of Rb1 eKO mice ) , Rb1loxP / loxP mice ; le , luminal epithelium ; s , stroma .

A, RB was efficiently deleted in the uterine epithelium of uterine epithelium-specific conditional knockout mice (Rb1 eKO mice). Scale bar = 100μm. Rb1 eKO mice, Rb1loxP/loxPLtfCre/+ mice; Rb1 eControl mice (littermate controls of Rb1 eKO mice), Rb1loxP/loxP mice; le, luminal epithelium; s, stroma.


B , Number of new - born pups was comparable between Rb1 eKO and eControl dams ( mean ± SEM , Student ' s t test ; n = 10 different dams for each group ) .

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B, Number of new-born pups was comparable between Rb1 eKO and eControl dams (mean ± SEM, Student's t test; n=10 different dams for each group).


C , The expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO and Rb1 eControl mice . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

C, The expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO and Rb1 eControl mice. Scale bar, 100μm; le, luminal epithelium; s, stroma.


A , Representative pictures of Ki67 staining in the human endometria obtained from women at the time of embryo implantation . Samples were divided into two groups according to the outcome of clinical pregnancy following endometrial biopsy . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . B , Ratio of Ki67 - positive cells in the human uterine luminal epithelium during the implantation period was lower in the group with pregnancy than the group without pregnancy ( mean ± SEM , Student ' s t test ; n = 10 different individuals for each group ) . Three different high - powered fields per sample were analyzed and each of the Ki67 - posivie ratios was demonstrated .

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A, Representative pictures of Ki67 staining in the human endometria obtained from women at the time of embryo implantation. Samples were divided into two groups according to the outcome of clinical pregnancy following endometrial biopsy. Scale bar = 100μm; le, luminal epithelium; s, stroma. B, Ratio of Ki67-positive cells in the human uterine luminal epithelium during the implantation period was lower in the group with pregnancy than the group without pregnancy (mean ± SEM, Student's t test; n=10 different individuals for each group). Three different high-powered fields per sample were analyzed and each of the Ki67-posivie ratios was demonstrated.


A . Live - cell imaging assay to detect mitotic perturbations . HeLa cells stably expressing a chromatin marker ( H2B - mCherry ; red ) and a nuclear import substrate ( IBB - eGFP ; green ) were imaged by automated live - cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown ( mitotic entry ) until nuclear reassembly ( mitotic exit ) ( Schmitz et al . , 2010 ) .

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A. Live-cell imaging assay to detect mitotic perturbations. HeLa cells stably expressing a chromatin marker (H2B-mCherry; red) and a nuclear import substrate (IBB-eGFP; green) were imaged by automated live-cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown (mitotic entry) until nuclear reassembly (mitotic exit) (Schmitz et al., 2010).