( a ) MDCK / pGFP - LC3 infected with a wild - type or ΔactA2 of L . monocytogenes were stained with an anti - Listeria antibody ( red ) rhodamine phalloidin ( stains F - actin ; blue ) . Scale bars , 10 μm . Arrowheads indicate GFP - LC3 - positive . ( b ) High magnification of framed in a , graphs show of fluoresence signal along arrow . ( c ) of number of GFP - LC3 - positive shown in a . Data are mean ± s . e . m . At least 500 were counted in each experiment ( n = 7 6 experiments at 2 4 h , respectively ) .

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(a) MDCK/pGFP-LC3 cells infected with a wild-type or ΔactA2 strain of L. monocytogenes were stained with an anti-Listeria antibody (red) and rhodamine phalloidin (stains F-actin; light blue). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive bacteria. (b) High magnification of framed images in a, and graphs show intensity of the fluoresence signal along the arrow. (c) Quantification of the number of GFP-LC3-positive bacteria shown in a. Data are mean ± s.e.m. At least 500 bacteria were counted in each experiment (n = 7 and 6 experiments at 2 and 4 h, respectively).


( d ) LC3 in infected MDCK . MDCK were infected with wild - type or ΔactA2 for indicated times . EBSS ( Earle ' s balanced salt solution , a starvation medium ) rapamycin ( 100 μg ml − 1 ) were used as controls .

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(d) LC3 levels in infected MDCK cells. MDCK cells were infected with wild-type or ΔactA2 bacteria for the indicated times. EBSS (Earle's balanced salt solution, a starvation medium) and rapamycin (100 μg ml−1) were used as controls.


( e - f ) Intracellular survival of wild - type or ΔactA2 in MDCK ( e ; n = 8 ) , MDCK treated with 10 mM 3 - MA ( f ; n = 4 ) ,

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(e-f) Intracellular survival of wild-type or ΔactA2 bacteria in MDCK cells (e; n = 8), MDCK cells treated with 10 mM 3-MA (f; n = 4),


( e - h ) Intracellular survival of wild - type or ΔactA2 in atg5 + / + MEF ( g ; n = 8 7 for wild - type ΔactA2 , respectively ) or atg5 − / − MEF ( h ; n = 4 3 for wild - type ΔactA2 , respectively ) . Data are presented as number of colony - forming units ( CFU ) relative to that at 30 min after infection , are mean ± s . e . m . WT , wild type .

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(e-h) Intracellular survival of wild-type or ΔactA2 bacteria in atg5+/+ MEF cells (g; n = 8 and 7 for wild-type and ΔactA2, respectively) or atg5−/− MEF cells (h; n = 4 and 3 for wild-type and ΔactA2, respectively). Data are presented as the number of colony-forming units (CFU) relative to that at 30 min after infection, and are mean ± s.e.m. WT, wild type.


( b , c ) of number of GFP - LC3 - positive ( b ) or actin - accumulating ( c ) . Data are mean ± s . e . m . , at least 500 were counted in each experiment ( n = 3 experiments ) .

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(b, c) Quantification of the number of GFP-LC3-positive bacteria (b) or actin-accumulating bacteria (c). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments).


( d ) Colocalization of ubiquitin with ΔactA2 . MDCK / pGFP - LC3 infected with ΔactA2 were stained with an anti - Listeria antibody ( blue ) anti - ubiquitin antibody ( red ) . Scale bars , 10 μm . Arrowheads indicate GFP - LC3 - positive or ubiquitin - positive . ( e ) High magnification of framed image in d , graph shows of signal along arrow . ( f ) of number of GFP - LC3 - positive , ubiquitin - positive or GFP - LC3 - ubiquitin - positive . Data are mean ± s . e . m . , at least 500 were counted in each experiment ( n = 3 experiments ) .

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(d) Colocalization of ubiquitin with ΔactA2. MDCK/pGFP-LC3 cells infected with ΔactA2 bacteria were stained with an anti-Listeria antibody (blue) and anti-ubiquitin antibody (red). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive or ubiquitin-positive bacteria. (e) High magnification of framed image in d, and the graph shows the intensity of the fluorescence signal along the arrow. (f) Quantification of the number of GFP-LC3-positive, ubiquitin-positive or GFP-LC3- and ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments).


( g ) Immunogold electron micrograph of MDCK / pGFP - LC3 infected with ΔactA2 . Gold particles of 5 nm ( arrowheads ) indicate GFP - LC3 , gold particles of 10 nm ( arrows ) indicate ubiquitin . Scale bars , 0 . 5 μm .

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(g) Immunogold electron micrograph of MDCK/pGFP-LC3 cells infected with ΔactA2. Gold particles of 5 nm (arrowheads) indicate GFP-LC3, and gold particles of 10 nm (arrows) indicate ubiquitin. Scale bars, 0.5 μm.


( h ) Kinetics of generation of GFP - LC3 - positive or ubiquitin - positive . Data are mean ± s . e . m . , at least 500 were counted in each experiment ( n = 3 experiments ) . * P 0 . 001 . ( i ) of number of ubiquitin - positive or GFP - LC3 - positive . MDCK / pGFP - LC3 infected with wild - type or ΔactA2 were untreated or treated with 3 - MA ( 10 mM ) . Data are mean ± s . e . m . , at least 500 were counted in each experiment ( n ≥ 3 experiments ) . Ub , ubiquitin . WT , wild type .

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(h) Kinetics of the generation of GFP-LC3-positive or ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). *P 0.001. (i) Quantification of the number of ubiquitin-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3 cells infected with wild-type or ΔactA2 bacteria were untreated or treated with 3-MA (10 mM). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n ≥ 3 experiments). Ub, ubiquitin. WT, wild type.


( a ) of number of ubiquitin - positive , p62 - positive or GFP - LC3 - positive . MDCK / pGFP - LC3 / pp62 - 3 × Myc were infected with a wild - type or ΔactA2 of L . monocytogenes for 2 h . Data are mean ± s . e . m . , at least 500 were counted in each experiment ( n = 6 experiments ) .

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(a) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3/pp62-3×Myc cells were infected with a wild-type or ΔactA2 strain of L. monocytogenes for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 experiments).


( b ) Colocalization of p62 with ΔactA2 . MDCK / pGFP - LC3 / pp62 - 3 × Myc were stained with an anti - ubiquitin antibody ( blue ) , anti - Myc antibody ( red ) , DAPI ( ) anti - GFP - LC3 ( green ) . Scale bar , 10 μm . Arrowheads indicate GFP - LC3 - , ubiquitin - or p62 - positive .

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(b) Colocalization of p62 with ΔactA2. MDCK/pGFP-LC3/pp62-3×Myc cells were stained with an anti-ubiquitin antibody (light blue), anti-Myc antibody (red), DAPI (white) and anti-GFP-LC3 (green). Scale bar, 10 μm. Arrowheads indicate GFP-LC3-, ubiquitin- or p62-positive bacteria.


( c ) of number of ubiquitin - positive , p62 - positive or GFP - LC3 - positive . p62 − / − / p62 were infected with ΔactA2 for 2 h . Data are mean ± s . e . m . , at least 500 were counted in each experiment ( n = 6 3 experiments for p62 − / − p62 − / − / p62 , respectively )

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(c) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. p62−/−/p62 cells were infected with ΔactA2 bacteria for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 and 3 experiments for p62−/− and p62−/−/p62, respectively)


( d , e ) Intracellular survival of wild - type or ΔactA2 in p62 − / − ( d ) or p62 − / − / p62 ( e ; colony - forming units , CFU , relative to CFU 30 min after infection ) . Data are presented as mean ± s . e . m . ( n ≥ 4 experiments ) .

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(d, e) Intracellular survival of wild-type or ΔactA2 bacteria in p62−/− cells (d) or p62−/−/p62 cells (e; colony-forming units, CFU, relative to CFU 30 min after infection). Data are presented as mean ± s.e.m. (n ≥ 4 experiments).


( g ) Generation of p62 - 3 × Myc mutants in p62 − / − .

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(g) Generation of p62-3×Myc mutants in p62−/− cells.


( h ) of number of GFP - positive or GFP - LC3 - positive . p62 − / − / p62 ( wild type , ΔLIR , ΔUBA , ΔLIR - ΔUBA or K7AD69A ) were infected with ΔactA2 stained with anti - anti - Myc antibodies ( Supplementary Information , Fig . S4e ) . Data are mean ± s . e . m . , at least 500 LC3 were counted in each experiment ( n = 3 experiments ) . P 0 . 001 versus p62 - positive LC3 in p62 − / − / p62 wild - type , P 0 . 001 versus GFP - p62 - positive in p62 − / − / ubiquitinwild - type . Ub , ubiquitin . WT , wild type . A full scan of g is shown in Supplementary Information , Fig . S7 .

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

(h) Quantification of the number of GFP-positive or GFP-LC3-positive bacteria. p62−/−/p62 (wild type, ΔLIR, ΔUBA, ΔLIR-ΔUBA or K7AD69A) cells were infected with ΔactA2 bacteria and stained with anti-bacteria and anti-Myc antibodies (Supplementary Information, Fig. S4e). Data are mean ± s.e.m., at least 500 LC3 were counted in each experiment (n = 3 experiments). P 0.001 versus p62-positive LC3 in p62−/−/p62 wild-type cells, P 0.001 versus GFP-p62-positive bacteria in p62−/−/ubiquitinwild-type cells. Ub, ubiquitin. WT, wild type. A full scan of g is shown in Supplementary Information, Fig. S7.


( b - e ) series of pEGFP - ActA - Q79C constructs was transfected into COS - 7 . were fixed with 4 % Paraformaldehyde after 18 h stained with an anti - Arp2 antibody ( b ) , an anti - VASP antibody ( c ) , an anti - ubiquitin antibody ( d ) or an anti - p62 antibody ( e ; red ) . Scale bars , 10 μm . Arrowheads indicate Arp2 - , VASP - , ubiquitin - or p62 - positive Q79C . Ub , ubiquitin .

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

(b-e) The series of pEGFP-ActA-Q79C constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 18 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive Q79C. Ub, ubiquitin.


( b - e ) series of pEGFP - ActA - 170 constructs was transfected into COS - 7 . were fixed with 4 % Paraformaldehyde after 14 h stained with an anti - Arp2 antibody ( b ) , an anti - VASP antibody ( c ) , an anti - ubiquitin antibody ( d ) or an anti - p62 antibody ( e ; red ) . Scale bars , 10 μm . Arrowheads indicate Arp2 - , VASP - , ubiquitin - or p62 - positive 170 . Ub , ubiquitin .

(b-e) The series of pEGFP-ActA-170 constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 14 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive 170. Ub, ubiquitin.


( b ) PC12 transfected with either 60Q or 60Q - HSC70bm . presence of two HSC70bm motifs in 60Q - HSC70bm reduced number of inclusions as compared to 60Q ( scale bar , 20 μm ) . ( c ) of PC12 with inclusions 24 h after transfection with either 60Q or 60Q - HSC70bm . 10 mM 3 - methyladenine ( 3 - MA ) , 10 μM pepstatin A 10 μM leupeptin were added to inhibit macroautophagy , proteases , cathepsins D E , cathepsin A , respectively . Bars in c represent relative mean values ± s . e . m . from four independent experiments , with of aggregation observed for 60Q normalized to a value of 1 . Quantifications were performed by ArrayScan .

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(b) PC12 cells transfected with either 60Q or 60Q-HSC70bm. The presence of two HSC70bm motifs in 60Q-HSC70bm reduced the number of inclusions as compared to 60Q (scale bar, 20 μm). (c) Quantification of the PC12 cells with inclusions 24 h after transfection with either 60Q or 60Q-HSC70bm. 10 mM 3-methyladenine (3-MA), 10 μM pepstatin A and 10 μM leupeptin were added to inhibit macroautophagy, the proteases, cathepsins D and E, and cathepsin A, respectively. Bars in c represent relative mean values ± s.e.m. from four independent experiments, with levels of aggregation observed for 60Q normalized to a value of 1. Quantifications were performed by ArrayScan.


( b ) Confocal of 150Q Neuro2a transfected with R , RH , RS , RHS , RQ RHQ . Molecules containing QBP1 co - localized with tNHTT - 150Q - EGFP inclusions ( bar , 5 μm ) . with inclusions were chosen to illustrate co - localization of QBP1 - containing molecules with tNHTT - 150Q - EGFP .

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(b) Confocal images of the 150Q Neuro2a cells transfected with R, RH, RS, RHS, RQ and RHQ. Molecules containing QBP1 co-localized with the tNHTT-150Q-EGFP inclusions (bar, 5 μm). Cells with inclusions were chosen to illustrate the co-localization of QBP1-containing molecules with tNHTT-150Q-EGFP.


( c ) expression of RQ RHQ in 150Q Neuro2a decreased accumulation of insoluble tNHTT - 150Q - EGFP on gel top . Note shift of tNHTT - 150Q - EGFP from an insoluble to a soluble form when RQ was co - expressed . of tNHTT - 16Q - EGFP were not affected . Full - western blots are presented in Supplementary Figure 13 .

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(c) The expression of RQ and RHQ in 150Q Neuro2a cells decreased the accumulation of insoluble tNHTT-150Q-EGFP on the gel top. Note the shift of tNHTT-150Q-EGFP from an insoluble to a soluble form when RQ was co-expressed. The levels of tNHTT-16Q-EGFP were not affected. Full-length western blots are presented in Supplementary Figure 13.


( d ) RHQ reduced of aggregated soluble forms of tNHTT - 150Q - EGFP by 81 . 8 % 33 . 6 % , respectively . WB , western blot . ( e ) of tNHTT - 16Q - EGFP .

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(d) RHQ reduced the levels of aggregated and soluble forms of tNHTT-150Q-EGFP by 81.8% and 33.6%, respectively. WB, western blot. (e) Quantification of tNHTT-16Q-EGFP protein levels.


( f ) ArrayScan revealed 28 % 56 % reductions in inclusion formation in 150Q Neuro2a by RQ RHQ , respectively , after 24 h of differentiation induction . ( g ) In 150Q - nls Neuro2a , expression of RQ RHQ reduced number of tNHTT - 150Q - nls - EGFP inclusion by 23 % ( RQ ) 53 % ( RHQ ) after 48 h of differentiation induction compared to RS RHS , respectively . Bars in d e represent mean values ± s . e . m . from four independent experiments . Quantifications were performed by ArrayScan .

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(f) ArrayScan analysis revealed 28% and 56% reductions in inclusion formation in 150Q Neuro2a cells by RQ and RHQ, respectively, after 24 h of differentiation and induction. (g) In 150Q-nls Neuro2a cells, expression of RQ and RHQ reduced the number of tNHTT-150Q-nls-EGFP inclusion by 23% (RQ) and 53% (RHQ) after 48 h of differentiation and induction compared to RS and RHS, respectively. Bars in d and e represent mean values ± s.e.m. from four independent experiments. Quantifications were performed by ArrayScan.


( a ) Western blot of chase experiment in 60Q Neuro2a . Full - western blots are presented in Supplementary Figure 13 . ( b ) RHQ enhanced tNHTT - 60Q - EGFP degradation during chase period . 5 μM MG132 , 10 mM 3 - methyladenine 15 mM ammonium chloride ( NH4Cl ) were used to inhibit proteasome , macroautophagy lysosomal degradation , respectively . NH4Cl had no effect on proteasome activity did not affect viability ( Supplementary Fig . 4a , b ) . Bars in b represent relative mean values ± s . e . m . from three independent experiments . at time 0 represent control condition ( arbitrarily set as 1 ) .

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(a) Western blot analysis of the chase experiment in 60Q Neuro2a cells. Full-length western blots are presented in Supplementary Figure 13. (b) RHQ enhanced the tNHTT-60Q-EGFP degradation during the chase period. 5 μM MG132, 10 mM 3-methyladenine and 15 mM ammonium chloride (NH4Cl) were used to inhibit proteasome, macroautophagy and lysosomal degradation, respectively. NH4Cl had no effect on proteasome activity and did not affect cell viability (Supplementary Fig. 4a,b). Bars in b represent relative mean values ± s.e.m. from three independent experiments. Protein levels at time 0 represent the control condition (arbitrarily set as 1).


( a , b ) Constructs identical to those described in Figure 2a , but lacking mRFP ( representations of S , HS , Q , HQ are shown in Supplementary Fig . 3a ) were co - transfected with tNHTT - 16Q - EGFP ( a ) tNHTT - 150Q - EGFP ( b ) constructs into Neuro2a ( bar , 4 μm ) .

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(a,b) Constructs identical to those described in Figure 2a, but lacking mRFP (representations of S, HS, Q, HQ are shown in Supplementary Fig. 3a) were co-transfected with tNHTT-16Q-EGFP (a) and tNHTT-150Q-EGFP (b) constructs into Neuro2a cells (bar, 4 μm).


( c ) Dot blot of lumenal fraction of purified lysosomes from Neuro2a co - transfected with tNHTT - 60Q - EGFP R , RS , RHS , RQ or RHQ . PNS , post - supernatant .

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(c) Dot blot analysis of the lumenal fraction of purified lysosomes from Neuro2a cells co-transfected with tNHTT-60Q-EGFP and R, RS, RHS, RQ or RHQ. PNS, post-nuclear supernatant.


( d ) of HTT translocation into lysosomes . of HTT detected by EM48 ( anti - huntingtin antibody ) were normalized to of β - tubulin ( in PNS ) of cathepsin D ( in lysosomes ) .

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(d) Quantification of HTT translocation into lysosomes. Levels of HTT detected by EM48 (anti-huntingtin antibody) were normalized to levels of β-tubulin (in PNS) and levels of cathepsin D (in lysosomes).


( e ) Compound generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a transfected with R , RS , RHS , RQ RHQ shRNA specific for HSC70 / or Lamp2a . ( blue , Hoechst 33258 ; green , tNHTT - 150Q - EGFP ; red , tested molecule ) .

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(e) Compound images generated by ArrayScan illustrating inclusion formation in 150Q Neuro2a cells transfected with R, RS, RHS, RQ and RHQ and shRNA specific for HSC70 and/or Lamp2a. (blue, Hoechst 33258; green, tNHTT-150Q-EGFP; red, tested molecule).


( f ) RNAi of HSC70 , Lamp2a or both alleviated inhibitory effect of RHQ on formation of inclusions containing tNHTT - 150Q - EGFP inclusions .

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(f) RNAi of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on the formation of inclusions containing tNHTT-150Q-EGFP inclusions.


( g ) Western blot of soluble polyQ in 150Q Neuro2a transfected with tested constructs shRNA for HSC70 / or Lamp2a . Full - western blots are presented in Supplementary Figure 13 .

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(g) Western blot analysis of soluble polyQ protein in 150Q Neuro2a cells transfected with tested constructs and shRNA for HSC70 and/or Lamp2a. Full-length western blots are presented in Supplementary Figure 13.


( h ) Silencing of HSC70 , Lamp2a or both alleviated inhibitory effect of RHQ on of soluble tNHTT - 150Q - EGFP . Bars in d f represent mean values ± s . e . m . from three independent experiments . Bars in h represent relative mean values ± s . e . m . from three independent experiments . For h , values for R construct were arbitrarily set to 1 .

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(h) Silencing of HSC70, Lamp2a or both alleviated the inhibitory effect of RHQ on levels of soluble tNHTT-150Q-EGFP. Bars in d and f represent mean values ± s.e.m. from three independent experiments. Bars in h represent relative mean values ± s.e.m. from three independent experiments. For h, values for the R construct were arbitrarily set to 1.


( a ) Filter trap of HTT - polyQaggregation in nine R6 / 2 mouse brains injected with three different combinations of rAAV . ( b ) of SDS - insoluble material detected by filter trap ( * , P = 0 . 037 ; * * , P = 0 . 0083 ; * * * , P = 0 . 0015 ) .

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(a) Filter trap assay analysis of HTT-polyQaggregation in nine R6/2 mouse brains injected with three different combinations of rAAV. (b) Quantification of the SDS-insoluble material detected by filter trap assay (*, P = 0.037; **, P = 0.0083; ***, P = 0.0015).


( c ) Western blot of HTT aggregation ( gel top , EM48 antibody ) , soluble HTT ( 1C2 antibody ) , higher molecular polyQ complexes by agarose gel electrophoresis for resolving ( AGERA ) . Full - western blots are presented in Supplementary Figure 13 . ( d ) of gel top - detected aggregation ( * , P = 0 . 093 ; * * , P = 0 . 0027 ; * * * , P = 0 . 0045 ) . ( e ) of soluble HTT - polyQ ( * , P = 0 . 034 ; * * , P = 0 . 00003 ; * * * , P = 0 . 0001 ) .

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(c) Western blot analysis of HTT aggregation (gel top, EM48 antibody), soluble HTT (1C2 antibody), and higher molecular polyQ complexes by agarose gel electrophoresis for resolving aggregates (AGERA). Full-length western blots are presented in Supplementary Figure 13. (d) Quantification of the gel top-detected aggregation (*, P = 0.093; **, P = 0.0027; ***, P = 0.0045). (e) Quantification of soluble HTT-polyQ (*, P = 0.034; **, P = 0.00003; ***, P = 0.0001).


( f ) Brain sections stained with anti - RFP , EM48 anti - ubiquitin antibodies ( bar , 40 μm ) . ( g ) Numbers of ubiquitin - positive inclusions in transduced areas ( * , P = 0 . 0026 ; * * , P = 5 . 535 × 10 − 8 ; * * * , P = 2 . 009 × 10 − 8 ) . Bars in b , d , e g represent relative mean values ± s . e . m . from three brains injected with different rAAV in each striatum . R in R / Q R / HQ , Q in Q / HQ brains represent control value of 1 .

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(f) Brain sections stained with anti-RFP, EM48 and anti-ubiquitin antibodies (bar, 40 μm). (g) Numbers of ubiquitin-positive inclusions in the transduced areas (*, P = 0.0026; **, P = 5.535 × 10−8; ***, P = 2.009 × 10−8). Bars in b, d, e and g represent the relative mean values ± s.e.m. from three brains injected with different rAAV in each striatum. R in R/Q and R/HQ, and Q in Q/HQ brains represent the control value of 1.


( a ) Relative changes in mice after bilateral injection with rAAV - R , rAAV - Q rAAV - HQ at 6 - 14 of ( n = 11 for three groups until 12 ; at 14 , n = 7 for R / R , n = 10 for Q / Q , n = 11 for HQ / HQ ) ( * , P = 0 . 0401 ; * * , P = 0 . 0081 ; * * * , P = 0 . 0182 ; * * * * , P = 0 . 0225 ) .

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(a) Relative body weight changes in mice after bilateral injection with rAAV-R, rAAV-Q and rAAV-HQ at 6-14 weeks of age (n = 11 for all three groups until 12 weeks; at 14 weeks, n = 7 for R/R, n = 10 for Q/Q, n = 11 for HQ/HQ) (*, P = 0.0401; **, P = 0.0081; ***, P = 0.0182; ****, P = 0.0225).


( b ) Clasping scores . n = 11 for three groups ( scale : 0 , no clasping ; 1 , clasping of forelimbs only ; 2 , clasping of both fore hind limbs once or twice ; 3 , clasping of both fore hind limbs more than 3 times or more than 5 s ) .

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(b) Clasping scores. n = 11 for all three groups (scale: 0, no clasping; 1, clasping of the forelimbs only; 2, clasping of both fore and hind limbs once or twice; 3, clasping of both fore and hind limbs more than 3 times or more than 5 s).


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(c) Rotarod performance; n = 11 for all three groups (*, P = 0.0055; **, P = 0.002; ***, P = 0.0455; ****, P = 0.0242).


( d ) Survival curves for mice after bilateral injection with rAAV - R , rAAV - Q , rAAV - HQ ; n = 11 for groups . Median survival ( days ) : R / R - 108 ; Q / Q - 123 ; HQ / HQ - 140 . Mean survival ( days ) : R / R - 105 . 5 ± 3 . 9 ; Q / Q - 122 . 3 ± 4 . 8 ; HQ / HQ - 136 . 2 ± 4 . 4 . Log - rank , P 0 . 0001 ; Wilcoxon , P 0 . 0001 .

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(d) Survival curves for mice after bilateral injection with rAAV-R, rAAV-Q, and rAAV-HQ; n = 11 for all groups. Median survival (days): R/R-108; Q/Q-123; HQ/HQ-140. Mean survival (days): R/R-105.5 ± 3.9; Q/Q-122.3 ± 4.8; HQ/HQ-136.2 ± 4.4.Log-rank test, P 0.0001; Wilcoxon test, P 0.0001.


( A ) HeLa were incubated for 4 h in either EBSS or media without glucose / pyruvate . Lysates were then analysed by western blot for presence of MCL ‐ 1 , as well as actin as a loading control .

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(A) HeLa cells were incubated for 4 h in either EBSS or media without glucose/pyruvate. Lysates were then analysed by western blot for the presence of MCL‐1, as well as actin as a loading control.


( B ) CGNs were subjected to 17 h hypoxia after 7 daysin vitro . lysates were collected at indicated times after reoxygenation blotted for MCL ‐ 1 expression for actin as a loading control .

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(B) CGNs were subjected to 17 h hypoxia after 7 daysin vitro. Protein lysates were collected at indicated times after reoxygenation and blotted for MCL‐1 expression and for actin as a loading control.


( C ) H1299 stably expressing GFP - LC3 were incubated for 2 h in EBSS , expression of various BCL ‐ 2 homologues was analysed by western blot . Alternatively , cortical neurons were incubated for 4 h in media without glucose / pyruvate .

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(C) H1299 cells stably expressing GFP-LC3 were incubated for 2 h in EBSS, and expression of various BCL‐2 homologues was analysed by western blot. Alternatively, primary cortical neurons were incubated for 4 h in media without glucose/pyruvate.


( D ) H1299 stably expressing GFP - LC3 were incubated for 3 h in media without glucose / pyruvate , followed by re ‐ addition of glucose pyruvate for indicated times .

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(D) H1299 cells stably expressing GFP-LC3 were incubated for 3 h in media without glucose/pyruvate, followed by re‐addition of glucose and pyruvate for the indicated times.


( E ) HeLa were infected with 5 p . f . u . per of either Ad HA ‐ tBID or Ad LacZ for 7 h , media being changed to EBSS for last 3 h where indicated . Alternatively , were treated with 50 μM camptothecin for 3 h .

(E) HeLa cells were infected with 5 p.f.u. per cell of either Ad HA‐tBID or Ad LacZ for 7 h, the media being changed to EBSS for the last 3 h where indicated. Alternatively, cells were treated with 50 μM camptothecin for 3 h.


( F , G ) HeLa were treated as in E in presence of 50 μM zVAD ‐ FMK analysed by immunofluorescence microscopy for active BAX ( 6A7 epitope ; F ) or cyt c ( G ) . Data are expressed as average of three experiments ± s . d . * P0 . 01 .

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(F, G) HeLa cells were treated as in E in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence microscopy for active BAX (6A7 epitope; F) or cyt c (G). Data are expressed as the average of three experiments±s.d. *P0.01.


( A ) H1299 stably expressing GFP - LC3 were incubated for indicated times in EBSS in presence of 50 μM zVAD ‐ FMK analysed by immunofluorescence for cyt c or active BAX ( 6A7 epitope ) . As a positive control , were infected with 5 p . f . u . per Ad HA ‐ tBID for 7 h . Data are expressed as average of three experiments ± s . d .

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(A) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA‐tBID for 7 h. Data are expressed as the average of three experiments±s.d.


( B ) H1299 stably expressing GFP - LC3 were incubated for 4 h in either EBSS or media without glucose / pyruvate . were then fixed , stained with an antibody against cyt c analysed by immunofluorescence . Scale bars = 50 μm .

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(B) H1299 cells stably expressing GFP-LC3 were incubated for 4 h in either EBSS or media without glucose/pyruvate. Cells were then fixed, stained with an antibody against cyt c and analysed by immunofluorescence. Scale bars=50 μm.


( Alternatively ( C ) , were analysed by western blot for presence of MCL ‐ 1 autophagic markers GFP - LC3 ( anti ‐ GFP antibody ) p62 .

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( Alternatively (C), the cells were analysed by western blot for the presence of MCL‐1 and the autophagic markers GFP-LC3 (anti‐GFP antibody) and p62.


( D ) H1299 stably expressing GFP - LC3 were incubated for indicated times in either EBSS or media without glucose / pyruvate analysed by western blot for presence of MCL ‐ 1 autophagic markers .

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(D) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in either EBSS or media without glucose/pyruvate and analysed by western blot for the presence of MCL‐1 and autophagic markers.


( E , F ) H1299 stably expressing GFP - LC3 were pre ‐ incubated for 1 h with 5 mM 3MA ( E ) or 200 nM bafilomycin ( F ) , then incubated for 2 h in complete media or EBSS in presence of 3MA or bafilomycin . Lysates were then analysed for presence of MCL ‐ 1 , autophagy marker p62 actin as a loading control .

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(E, F) H1299 cells stably expressing GFP-LC3 were pre‐incubated for 1 h with 5 mM 3MA (E) or 200 nM bafilomycin (F), and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin. Lysates were then analysed for the presence of MCL‐1, the autophagy marker p62 and actin as a loading control.


( A ) H1299 stably expressing GFP - LC3 were infected with 10 p . f . u . per of either Ad LacZ or Ad Myc ‐ MCL ‐ 1 for 24 h then incubated in EBSS for 4 h . Alternatively , were transfected with control or MCL ‐ 1 siRNA 24 h before EBSS treatment . were stained with an antibody against cyt c analysed by immunofluorescence . Representative are shown . Scale bars = 50 μm .

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(A) H1299 cells stably expressing GFP-LC3 were infected with 10 p.f.u. per cell of either Ad LacZ or Ad Myc‐MCL‐1 for 24 h then incubated in EBSS for 4 h. Alternatively, cells were transfected with control or MCL‐1 siRNA 24 h before EBSS treatment. Cells were stained with an antibody against cyt c and analysed by immunofluorescence. Representative images are shown. Scale bars=50 μm.


( B ) of surface covered by GFP - LC3 vesicles was quantified using Image J software . n > 70 in three experiment , * P0 . 01 .

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(B) The percent of the cell surface area covered by GFP-LC3 vesicles was quantified using the Image J software. n>70 cells in three experiment, *P0.01.


( C , D ) H1299 stably expressing GFP - LC3 were transfected as in A incubated with 200 nM bafilomycin for 1 h where indicated . of endogenous LC3 ‐ II relative to actin were quantified by western blot in C . Data are expressed as average of three experiments ± s . d , * P0 . 05 .

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(C, D) H1299 cells stably expressing GFP-LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated. The levels of endogenous LC3‐II relative to actin were quantified by western blot in C. Data are expressed as the average of three experiments±s.d, *P0.05.


( E , F ) H1299 stably expressing GFP - LC3 were treated as in A analysed by western blot for presence of p62 GFP - LC3 .

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(E, F) H1299 cells stably expressing GFP-LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP-LC3.


( G ) cortical neurons of indicated genotypes were infected with a lentivirus expressing Cre at time of plating analysed for presence of MCL ‐ 1 p62 by western blot 3 days later .

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(G) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL‐1 and p62 by western blot 3 days later.


( H ) Transformed wild ‐ type ( WT ) MCL ‐ 1Δ / Δ ( KO ) MEFs were analysed by western blot for expression of endogenous LC3 .

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(H) Transformed wild‐type (WT) and MCL‐1Δ/Δ (KO) MEFs were analysed by western blot for the expression of endogenous LC3.


( I , J ) Colocalization of GFP - LC3 vesicles with lysosomal marker LAMP1 was analysed by immunofluorescence in treated as in A . number of LC3 ‐ positive vesicles that colocalised with LAMP1 was quantified in I . Data are expressed as of LC3 vesicles per that are LAMP1 ‐ positive ± s . e . m . , with at least 14 per condition . Representative are shown in J . Arrows indicate LC3 ‐ positive vesicles colocalizing with LAMP1 . Scale bars = 10 μm .

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(I, J) Colocalization of GFP-LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A. The number of LC3‐positive vesicles that colocalised with LAMP1 was quantified in I. Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1‐positive±s.e.m., with at least 14 cells per condition. Representative images are shown in J. Arrows indicate LC3‐positive vesicles colocalizing with LAMP1. Scale bars=10 μm.


( A ) MCL ‐ 1 interacts with Beclin ‐ 1 . Lysates from H1299 stably expressing GFP - LC3 were immunoprecipitated using control or MCL ‐ 1 ‐ specific antibodies analysed for presence of Beclin ‐ 1 by western blot .

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(A) MCL‐1 interacts with Beclin‐1. Lysates from H1299 stably expressing GFP-LC3 were immunoprecipitated using control or MCL‐1‐specific antibodies and analysed for the presence of Beclin‐1 by western blot.


( B ) H1299 stably expressing GFP - LC3 were fractionated into HM ( containing mitochondria ) , LM ( containing ER ) . Fractions were analysed for presence of MCL ‐ 1 , BCL ‐ 2 , Beclin ‐ 1 , mitochondrial marker mtHSP70 ER marker calreticulin .

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(B) H1299 cells stably expressing GFP-LC3 were fractionated into HM (containing mitochondria), and LM (containing ER). Fractions were analysed for the presence of MCL‐1, BCL‐2, Beclin‐1, the mitochondrial marker mtHSP70 and the ER marker calreticulin.


( C ) MCL ‐ 1 was immunoprecipitated from HM LM before analysing presence of MCL ‐ 1 Beclin ‐ 1 .

(C) MCL‐1 was immunoprecipitated from HM and LM before analysing the presence of MCL‐1 and Beclin‐1.


( B ) Expression of various BCL ‐ 2 homologues in cortices of of indicated genotypes .

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(B) Expression levels of various BCL‐2 homologues in the cortices of animals of the indicated genotypes.


( C ) Brain of MCL ‐ 1Δ / Δ . Brain sections were stained with cresyl violet imaged using a × 1 objective .

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(C) Brain morphology of MCL‐1Δ/Δ. Brain sections were stained with cresyl violet and imaged using a × 1 objective.


( D - G ) Cortical neurons around lesion in MCL ‐ 1Δ / Δ are positive for autophagic marker LC3 . Brain sections from with indicated genotypes ( P14 for E , G ; P7 for H ) were stained for LC3 ( Green ) neuronal marker NeuN ( in H , red ) , along with stain Hoechst ( Blue ) . Confocal were taken using × 10 ( E ) or × 63 ( G , H ) objectives . Scale bars = 200 μm ( E ) ; 50 μm ( G , H ) . of LC3 is shown in D . total number of was determined by counting Hoechst ‐ positive nuclei . Data are expressed as of LC3 ‐ positive in at least three per genotype ± s . d . * P0 . 01 . ( F ) of total surface occupied by autophagosomes . Data are expressed as of total cortical covered by autophagosomes in at least 16 EM per genotype ± s . d . P0 . 005 ( I ) Representative EM from MCL ‐ 1 + / Δ MCL ‐ 1Δ / ΔP14mice . N , nucleus ; * , autophagosomal structures ; arrowheads , double ; arrow , mitochondrion inside a vesicle .

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(D-G) Cortical neurons around the lesion in MCL‐1Δ/Δ animals are positive for the autophagic marker LC3. Brain sections from animals with the indicated genotypes (P14 for E, G; P7 for H) were stained for LC3 (Green) and the neuronal marker NeuN (in H, red), along with the nuclear stain Hoechst (Blue). Confocal images were taken using × 10 (E) or × 63 (G, H) objectives. Scale bars=200 μm (E); 50 μm (G, H). Quantification of the LC3 staining is shown in D. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of LC3‐positive cells in at least three animals per genotype±s.d. *P0.01. (F) Quantification of the total surface area occupied by autophagosomes. Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype±s.d. P0.005 (I) Representative EM images from MCL‐1+/Δ and MCL‐1Δ/ΔP14mice. N, nucleus; *, autophagosomal structures; arrowheads, double membrane; arrow, mitochondrion inside a vesicle.


( A ) Cortices from indicated genotypes ages were analysed by western blot for presence of active caspase ‐ 3 , p62 MCL ‐ 1 . cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase ‐ 3 .

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(A) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase‐3, p62 and MCL‐1. Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase‐3.


( B ) Active BAX ( 6A7 ) active caspase ‐ 3 were also quantified by cortex of 1 ‐ to 14 ‐ day ‐ old MCL ‐ 1Δ / Δ mice . Representative from at least three sections from each were counted . total number of was determined by counting Hoechst ‐ positive nuclei . Data are expressed as of positive for at least three per genotype ± s . d . * P0 . 05 .

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(B) Active BAX (6A7) and active caspase‐3 were also quantified by staining the cortex of 1‐ to 14‐day‐old MCL‐1Δ/Δ mice. Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for at least three animals per genotype±s.d. *P0.05.


( C ) Active BAX is present in a subset of LC3 ‐ positive . Sections from P7 P14 MCL ‐ 1Δ / Δ brains were stained for LC3 ( Green ) active BAX ( 6A7 ; Red ) . Confocal were taken using × 63 objective . Arrows point to BAX ‐ positive , LC3 ‐ negative . Representative are shown . Scale bars = 50 μm .

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(C) Active BAX is present in a subset of LC3‐positive cells. Sections from P7 and P14 MCL‐1Δ/Δ brains were stained for LC3 (Green) and active BAX (6A7; Red). Confocal images were taken using × 63 objective. Arrows point to BAX‐positive, LC3‐negative cells. Representative images are shown. Scale bars=50 μm.


cortical neurons of indicated genotypes were infected with a lentivirus expressing Cre at time of plating analysed for presence of caspase ‐ fragment of caspase ‐ 3 substrate CAS5 days later . Alternatively ( E ) , neurons were analysed for cytochrome c release by immunofluorescence . Data are expressed as of positive for at least three per genotype ± s . d .

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Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate CAS5 days later. Alternatively (E), the neurons were analysed for cytochrome c release by immunofluorescence. Data are expressed as percent of positive cells for at least three embryos per genotype±s.d.


( A ) Brains from E12 . 5 Foxg1 Cre MCL ‐ 1 were analysed by western blot for presence of caspase ‐ fragment of caspase ‐ 3 substrate p130CAS autophagic markers LC3 p62 .

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(A) Brains from E12.5 Foxg1 Cre MCL‐1 animals were analysed by western blot for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS and the autophagic markers LC3 and p62.


( B ) Autophagy in Foxg1 Cre MCL ‐ 1Δ / Δ mice . Brain sections from E15 . 5 with indicated genotypes were stained for LC3 ( green ) . Confocal were taken using × 20 ( bottom panel ) or × 63 ( top middle panels ) objectives . Scale bars = 50 μm .

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(B) Autophagy in Foxg1 Cre MCL‐1Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top and middle panels) objectives. Scale bars=50 μm.


( C ) Colocalization of LC3 vesicles with lysosomal marker LAMP1 was analysed by immunohistochemistry in E15 . 5 wild ‐ type . Data are expressed as of LC3 vesicles that are LAMP1 ‐ positive ± s . d . About 100 LC3 vesicles were counted per section , with four sections per . VZ , ventricular zone .

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(C) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15.5 wild‐type embryos. Data are expressed as the percentage of LC3 vesicles that are LAMP1‐positive±s.d. About 100 LC3 vesicles were counted per section, with four sections per embryo. VZ, ventricular zone.


( D ) cortical neurons neuronal progenitor were analysed for presence of indicated apoptosis ( BIM , Puma ) autophagy ( ATG7 , ATG5 ‐ ATG12 ) related as well as Tuj ‐ 1 ( neuronal marker ) , Nestin ( neural progenitor marker ) HSC70 ( loading control ) .

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(D) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis (BIM, Puma) and autophagy (ATG7, ATG5‐ATG12) related proteins as well as Tuj‐1 (neuronal marker), Nestin (neural progenitor marker) and HSC70 (loading control).


( E ) neurospheres were treated for 8 h with EBSS analysed by western blot for presence of MCL ‐ 1 actin as a loading control .

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(E) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL‐1 and actin as a loading control.


( F ) neurons neurospheres were treated as in E analysed by western blot for p130CAS , LC3 actin as a loading control .

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(F) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130CAS, LC3 and actin as a loading control.


Alternatively , they were analysed by immunofluorescence for c release ( G ) in presence of 50 μM zVAD ‐ FMK to prevent apoptotic from detaching from coverslip . Data are expressed as average of 3 - 7 ± s . d . * P0 . 001 .

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Alternatively, they were analysed by immunofluorescence for c release (G) in the presence of 50 μM zVAD‐FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3-7 embryos±s.d. *P0.001.


( A ) Brain of P14 MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − mice . Brain sections were stained with cresyl violet imaged using a × 1 objective .

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(A) Brain morphology of P14 MCL‐1Δ/Δ Beclin‐1+/− mice. Brain sections were stained with cresyl violet and imaged using a × 1 objective.


( B - D ) Active BAX ( 6A7 ; B ) active caspase ‐ 3 ( C ) were quantified by cortex of 14 ‐ day ‐ old MCL ‐ 1Δ / Δ MCL ‐ 1Δ / Δ Beclin - 1 + / − mice . number of BAX ‐ positive LC3 ‐ negative was also quantified . ( D ) Representative from at least three sections from each were counted . total number of was determined by counting Hoechst ‐ positive nuclei . Data are expressed as of positive for least three per genotype ± s . d . * P0 . 05 .

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(B-D) Active BAX (6A7; B) and active caspase‐3 (C) were quantified by staining the cortex of 14‐day‐old MCL‐1Δ/Δ and MCL‐1Δ/Δ Beclin-1+/− mice. The number of BAX‐positive LC3‐negative cells was also quantified. (D)Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for least three animals per genotype±s.d. *P0.05.


( E ) Survival data from MCL ‐ 1Δ / Δ ( n = 14 ) their MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − ( n = 13 ) .

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(E) Survival data from MCL‐1Δ/Δ (n=14) and their MCL‐1Δ/Δ Beclin‐1+/− littermates (n=13).


Confocal demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C . elegans hypodermal mitochondria . Scale bar 10 µm .

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Confocal images demonstrate overlap of GFP tagged mtON with MitoTrackerTM Red CMXRos stained C. elegans hypodermal mitochondria. Scale bar 10 µm.


Immunoblot comparing supernatant mitochondria - enriched pellet of isolation fractions . GFP - tagged mtON migrates at predicted molecular of 82 kDa accounting for mitochondria - target , proton pump , GFP . mtON is observed only in mitochondrial fraction compared to marker HSP60 ( mitochondria ) actin ( cytosol ) . blots are from same lanes on one .

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Immunoblot comparing the cytosolic supernatant and the mitochondria-enriched pellet of isolation fractions. GFP-tagged mtON migrates at the predicted molecular weight of 82 kDa accounting for the mitochondria-target sequence, the proton pump, and GFP. mtON is observed only in the mitochondrial fraction compared to marker proteins HSP60 (mitochondria) and actin (cytosol). All blots are from the same lanes on one membrane.


Representative TMRE traces in arbitrary units ( a . u . ) before after Δψm dissipation with FCCP . Dashed lines indicate where FCCP was added . + / - ATR traces were performed in absence of succinate . mtON activation was continuous throughout traces . green trace is from mitochondria with ATR , gray traces are without . of change in ( ΔF ) normalized to maximum change in given by succinate respiration ( ΔFmax ) for each mitochondrial preparation . Data are from maximum dose in Fig . EV 3A . Two - way ANOVA with Sidak ' s for multiple comparisons , * p = 0 . 0469 , + ATR succinate vs . + ATR + p = 0 . 9978 , n = 6 mitochondrial isolations . Data show mean ± .

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Representative TMRE fluorescence traces in arbitrary units (a.u.) before and after Δψm dissipation with FCCP. Dashed lines indicate where FCCP was added. +/- ATR traces were performed in the absence of succinate. mtON activation was continuous throughout the traces. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in fluorescence (ΔF) normalized to the maximum change in fluorescence given by succinate respiration (ΔFmax) for each mitochondrial preparation. Data are from the maximum light dose in Fig. EV 3A. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0469, +ATR succinate vs. +ATR +light p = 0.9978, n = 6 mitochondrial isolations. Data show mean ± SEM.


Representative BCECF - AM 490 / 440 nm trace . of 545 nm at either 440 or 490 nm excitation . Dashed lines are where or FCCP treatment occurred . green trace is from mitochondria with ATR , gray traces are without . of change in BCECF - AM normalized to maximum change given by succinate matrix alkalization . Two - way ANOVA with Sidak ' s for multiple comparisons , - ATR succinate vs . - ATR , + * p = 0 . 0212 , + ATR succinate vs . + ATR p = 0 . 999 , + ATR succinate vs . + ATR , - p = 0 . 0237 , + ATR , + vs , + ATR , - p = 0 . 0474 , n = 3 mitochondrial isolations . Some points overlap . Data show mean ± .

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Representative BCECF-AM 490/440 nm ratio trace. Ratio of 545 nm fluorescence intensity at either 440 or 490 nm excitation. Dashed lines are where light or FCCP treatment occurred. Light green trace is from mitochondria with ATR, gray traces are without. Quantification of change in BCECF-AM fluorescence ratio normalized to maximum change given by succinate matrix alkalization. Two-way ANOVA with Sidak's test for multiple comparisons, -ATR succinate vs. -ATR, +light *p = 0.0212, +ATR succinate vs. +ATR light p = 0.999, +ATR succinate vs. +ATR,-light p = 0.0237, +ATR, +light vs, +ATR, -light p = 0.0474, n = 3 mitochondrial isolations. Some individual points overlap. Data show mean ± SEM.


ATP normalized to total ATP synthesis given by succinate respiration . Data from Fig . EV 3B . Succinate data shown for comparison after normalization . Two - way ANOVA with Sidak ' s for multiple comparisons , * p = 0 . 0011 , + ATR succinate vs + ATR p = 0 . 5680 , n = 3 , 7 , 3 , 7 , biological replicates for each bar from left to right . Some points overlap . Data show mean + .

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ATP levels normalized to total ATP synthesis given by succinate respiration. Data from Fig. EV 3B. Succinate data shown for comparison after normalization. Two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0011, +ATR succinate vs +ATR light p = 0.5680, n = 3,7,3,7, biological replicates for each bar from left to right. Some individual points overlap. Data show mean + SEM.


O2 required to consume 50 nmoles of ADP after mtON activation . Data are maximum illumination from Fig . EV 4D using time - matched conditions , One - way ANOVA * p = 0 . 013 ( - ATR , - vs . + ATR , + p = 0 . 010 . - ATR , + vs . + ATR , + p = 0 . 013 . ) - ATR , - n = 5 , n = 6 mitochondrial preparations . Data show mean + .

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O2 required to consume 50 nmoles of ADP after mtON activation. Data are the maximum illumination from Fig. EV 4D using time-matched dark conditions, One-way ANOVA *p = 0.013 (-ATR, -light vs. +ATR, +light p = 0.010. -ATR, +light vs. +ATR, +light p = 0.013.) -ATR, -light n = 5, rest n = 6 mitochondrial preparations. Data show mean + SEM.


Day 1 adult expressing mtON were exposed to 50 µM rotenone ( ETC I inhibitor ) for 5 hours survival was scored . Illumination was continuous throughout toxin exposure ( see methods ) . ATR alone was protective ( - ATR , - vs . + ATR , - p = 0 . 016 . ) effect of mtON activation was greater than ATR alone effect , one - way ANOVA with Tukey ' s post hoc * p = 0 . 01 . ( - ATR , - vs + ATR , + p = 0 . 0002 . - ATR , + vs . + ATR , + p = 0 . 0009 . n = 3 plates each condition with at least 15 per plate ) . Data show mean ± . mtON - expressing were exposed to 50 µM antimycin A ( III inhibitor ) survival was scored 18 hours later . One - way ANOVA with Tukey ' s post hoc * p = 0 . 02 ( - ATR , - vs + ATR , + p = 0 . 03 , - ATR , + vs . + ATR , + p = 0 . 01 . n = 5 plates each condition with at least 15 per plate ) . Data show mean ± . mtON - expressing were exposed to 0 . 25 M azide ( IV inhibitor ) for 1 hour scored for survival 1 hour after recovering , one - way ANOVA with Tukey ' s post hoc * p = 0 . 0002 . ( - ATR , - vs + ATR , + p < 0 . 0001 , - ATR , + vs . + ATR , + p < 0 . 0001 . n = 3 plates each condition with at least 15 per plate ) . Data show mean ± . mtON - expressing were exposed to 31 µM oligomycin A ( ATP synthase inhibitor ) survival was scored 18 hours later . No significant differences were found by one - way ANOVA ( n = 3 plates each condition with at least 15 per plate ) . Data show mean ± .

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Day 1 adult animals expressing mtON were exposed to 50 µM rotenone (ETC complex I inhibitor) for 5 hours and survival was scored. Illumination was continuous throughout toxin exposure (see methods). ATR alone was protective (-ATR, -light vs. +ATR, -light p = 0.016.) The effect of mtON activation was greater than the ATR alone effect, one-way ANOVA with Tukey's post hoc test *p = 0.01. (-ATR, -light vs +ATR, +light p = 0.0002. -ATR, +light vs. +ATR, +light p = 0.0009. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 50 µM antimycin A (complex III inhibitor) and survival was scored 18 hours later. One-way ANOVA with Tukey's post hoc test *p = 0.02 (-ATR, -light vs +ATR, +light p = 0.03, -ATR, +light vs. +ATR, +light p = 0.01. n = 5 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 0.25 M azide (complex IV inhibitor) for 1 hour and scored for survival 1 hour after recovering, one-way ANOVA with Tukey's post hoc test *p = 0.0002. (-ATR, -light vs +ATR, +light p < 0.0001, -ATR, +light vs. +ATR, +light p <0.0001. n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM. mtON-expressing animals were exposed to 31 µM oligomycin A (ATP synthase inhibitor) and survival was scored 18 hours later. No significant differences were found by one-way ANOVA (n = 3 plates each condition with at least 15 animals per plate). Data show mean ± SEM.


mtON - expressing in a I mutant background ( - 1 ) were scored for locomotion speed in presence of food by counting bends per minute . mtON activation rescued decreased locomotion of - 1 mutant background , one - way ANOVA with Tukey ' s post hoc , - ATR , + vs . + ATR , + * p = 0 . 0145 , + ATR , + vs . wildtype ( WT ) * p = 0 . 0008 ( - ATR , - vs + ATR , + p = 0 . 0272 , + ATR , - vs . + ATR , + p = 0 . 0332 , conditions vs WT , p < 0 . 001 , n = 30 , 30 , 30 , 41 , 41 each bar from left to right ) . Data show mean ± standard deviation .

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mtON-expressing animals in a complex I mutant background (gas-1) were scored for locomotion speed in the presence of food by counting body bends per minute. mtON activation rescued the decreased locomotion of the gas-1 mutant background, one-way ANOVA with Tukey's post hoc test, -ATR, +light vs. +ATR, +light *p = 0.0145, +ATR, +light vs. wildtype (WT) *p = 0.0008 (-ATR, -light vs +ATR, +light p = 0.0272, +ATR, -light vs. +ATR, +light p = 0.0332, all conditions vs WT, p < 0.001, n = 30, 30,30,41,41 animals each bar from left to right). Data show mean ± standard deviation.


Top : Immunoblot assessing effect of mtON activation on AMPK phosphorylation status . Top bands ( ~ 62 kDa ) are phosphorylated AMPK signal , bottom bands ( ~ 43 kDa ) are actin signal . Image is from same cut to separately probe for phosphorylated AMPK ( pAMPK ) actin . Bottom : Densitometry showing decreased pAMPK to actin , as there is no known antibody directed against total AMPK in C . elegans . Phosphorylation increases in absence of food , but low phosphorylation is preserved when mtON is activated , two - way ANOVA with Sidak ' s for multiple comparisons , * p = 0 . 0203 , n = 3 , 3 , 3 , 4 , 4 biological replicates each bar from left to right . Data show mean + standard deviation .

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Top: Immunoblot assessing the effect of mtON activation on AMPK phosphorylation status. Top bands (~62 kDa) are phosphorylated AMPK signal, and bottom bands (~43 kDa) are actin signal. Image is from the same membrane cut to separately probe for phosphorylated AMPK (pAMPK) and actin. Bottom: Densitometry analysis showing decreased pAMPK to actin ratio, as there is no known antibody directed against total AMPK in C. elegans. Phosphorylation increases in the absence of food, but low phosphorylation is preserved when mtON is activated, two-way ANOVA with Sidak's test for multiple comparisons, *p = 0.0203, n = 3,3,3,4,4 biological replicates each bar from left to right. Data show mean + standard deviation.


Locomotion was assessed by counting bends per minute . Wild type were compared to aak - 2 ( ok524 ) mutant . 2 - sample , 2 - unpaired t - * p < 0 . 0001 , wild type n = 35 , aak - 2 n = 39 across at least 3 days . Data show mean ± standard deviation .

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Locomotion was assessed by counting body bends per minute. Wild type animals were compared to aak-2(ok524) mutant animals. 2-sample, 2-tailed unpaired t-test *p < 0.0001, wild type n = 35, aak-2 n = 39 animals across at least 3 days. Data show mean ± standard deviation.


Locomotion in response to mtON activation . Illumination was continuous throughout bends measurement ( see methods ) . For AAK - 2 activation , were exposed to 1 mM AICAR for 4 hours before bends measurement . One - way ANOVA with Tukey ' s post hoc , - ATR , + vs + ATR , + * p < 0 . 0001 , + ATR , + vs . AICAR * p < 0 . 0001 , + ATR , + vs AICAR + * p < 0 . 0001 . n = 36 , 39 , 37 , 46 , 36 , 36 each bar from left to right . Data show mean ± standard deviation .

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Locomotion in response to mtON activation. Illumination was continuous throughout body bends measurement (see methods). For AAK-2 activation, animals were exposed to 1 mM AICAR for 4 hours before body bends measurement. One-way ANOVA with Tukey's post hoc test, -ATR, +light vs +ATR, +light *p < 0.0001, +ATR, +light vs. AICAR *p < 0.0001, +ATR,+light vs AICAR +light *p < 0.0001. n = 36, 39, 37, 46, 36, 36 animals each bar from left to right. Data show mean ± standard deviation.


Survival after HR in day 1 adult is shown with hypoxic preconditioning ( PC , represented by diagonal stripes ) 2 - sample 2 - unpaired t - , ctrl vs . PC * p = 0 . 0035 , n = 4 , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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Survival after HR in day 1 adult animals is shown with hypoxic preconditioning (PC, represented by diagonal stripes) 2-sample 2-tailed unpaired t-test, ctrl vs. PC *p = 0.0035, n = 4, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Protection ( % ) is survival minus survival of control condition . PC data calculated from panel B . FCCP final concentrations 0 . 001 , 0 . 01 , 0 . 1 , 1 nM . One - way ANOVA with Tukey ' s post hoc , PC vs . 0 . 001 nM FCCP , * p = 0 . 0012 , 0 . 001 nM FCCP vs 0 . 01 FCCP , * p = 0 . 0142 , n = 4 , 3 , 3 , 3 , 3 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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Protection (%) is percent survival minus percent survival of control condition. PC data calculated from panel B. FCCP final concentrations 0.001, 0.01, 0.1, 1 nM. One-way ANOVA with Tukey's post hoc test, PC vs. 0.001 nM FCCP, *p = 0.0012, 0.001 nM FCCP vs 0.01 FCCP, *p = 0.0142, n = 4,3,3,3,3 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


Illumination was continuous throughout PC alone ; control illumination was for same duration under normoxic conditions ( see methods & panel A ) . Two - way ANOVA comparing + ATR vs - ATR in each group . * p = 0 . 016 , n = 11 , 11 , 4 , 4 , 6 , 6 independent experiments each bar from left to right , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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Illumination was continuous throughout PC alone; control illumination was for the same duration under normoxic conditions (see methods & panel A). Two-way ANOVA comparing +ATR vs -ATR in each group. *p = 0.016, n = 11,11,4,4,6,6 independent experiments each bar from left to right, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


during hypoxia . 2 sample 2 - unpaired t - , * p = 0 . 0276 , n = 7 independent experiments , each averaged from 3 technical replicates with at least 15 per replicate . Data show mean ± .

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light during hypoxia. 2 sample 2-tailed unpaired t-test, *p = 0.0276, n = 7 independent experiments, each averaged from 3 technical replicates with at least 15 animals per replicate. Data show mean ± SEM.


A , Rb1 ( RB ) was expressed in uterine luminal epithelium stroma during early pregnancy in mice . Scale bar = 100μm ; arrowhead , . B , Phosphorylated RB ( pRB ) , inactive form of RB , was expressed in luminal epithelium on days 2 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 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 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 , 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 ± , Student ' s t ) .

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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 development of preimplantation were normal in Rb1d / d mice ( A & C , mean ± , Student ' s t ; n = 5 mice for each group ; B , Fisher ' s exact ; 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 on day 4 in uterine epithelium of Rb1d / d mice was higher than that of Rb1f / f mice ( mean ± , Student ' s t ; 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 , attachment occurred normally in Rb1d / d mice at 0900h on day 5 ( mean ± , Student ' s t ; 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 resorption on day 8 ( * P < 0 . 05 ; Fisher ' s exact ; n = 45 implantation sites for Rb1f / f mice n = 46 implantation sites for Rb1d / d mice ) . Arrow , implantation site ; scale bar = 1 cm .

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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 showed resorption in Rb1d / d mice on day 8 of pregnancy . Scale bar = 200 μm ; arrowhead , ; arrow , broken with blood 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 , of implantation site was comparable between Rb1f / f Rb1d / d mice on day 6 , but reduced on day 8 in Rb1d / d mice compared to Rb1f / f mice ( mean ± , Student ' s t ; 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 Bmp2 , markers of decidualization , was comparable in between Rb1d / d Rb1f / f mice at 0900h on day 6 in implantation sites ( mean ± , Student ' s t ; 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 lineage , showed that epithelial alignment is collapsed trophoblast invades stroma at implantation sites in Rb1f / f mice , while epithelial alignment is persistent trophoblast invasion into stroma is compromised in Rb1d / d mice . Scale bar = 200μm ; arrowhead , an .

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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 ( ) showed that uterine epithelium surrounding disappears trophoblast contacts directly with stroma at 2000h on day 5 in Rb1f / f mice , while uterine epithelium is persistent does not attach to stroma in Rb1d / d mice . Scale bar = 10µm ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , 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 , showed that fragmented luminal epithelial with lipid droplets are engulfed by trophoblast in control mice ( Rb1f / f mice ) . Scale bar = 2µm . Arrowhead , fragments engulfed by trophoblast ; yellow dotted circle , lipid droplets in luminal epithelium ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , 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.


D , showed that microvilli , markers of uterine receptivity , were observed at surface of intact luminal epithelium on day 4 of pregnancy epithelial with lipid droplets were observed at 2000h on day 5 of pregnancy in control mice ( Rb1f / f mice ) . Scale bar = 2µm ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , 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 , showed that MFG - E8 is not expressed at implantation sites of Rb1f / f mice at 0900h on day 5 Rb1d / d mice at 0900h 2000h on day 5 , but is expressed at 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 , .

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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 ) on days 4 6 of pregnancy were comparable between Rb1f / f Rb1d / d mice ( mean ± , Student ' s t ; 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 on days 4 6 of pregnancy were comparable between Rb1f / f Rb1d / d mice . In Rb1d / d mice , pre - implantation P4 treatment on days 2 3 increased serum P4 on day 4 but did not affect those on day 6 ( mean ± , Student ' s t ; 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 epithelium of Rb1d / d mice on day 4 was recovered by pre - implantation P4 treatment on days 2 3 . Scale bar = 100μm ; le , luminal epithelium ; s , stroma . E , of Ki67 - positive epithelial in Rb1d / d mice was completely suppressed by pre - implantation P4 supplementation on days 2 3 . same data of Rb1f / f Rb1d / d mice without P4 treatment in Fig 2D were used ( mean ± , Student ' s t ; n = 5 different mice for each group ) . Three different high - powered fields per mouse were analyzed each of 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 ) 2 ( n = 30 different implantation sites ) , but not in Group 3 ( n = 40 different implantation sites ) . same data of Rb1f / f Rb1d / d mice without P4 treatment in Fig 2I were used . H & E was performed using sections that shows implantation sites , resorption rate was evaluated . * P < 0 . 05 vs Rb1f / f mice ; * * P < 0 . 05 vs Rb1d / d mice ; Fisher ' s exact .

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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 3 ( Group 2 ) ( mean ± , Student ' s t ) . same data of Rb1f / f Rb1d / d dams without P4 treatment in Fig 1E were used . numbers of pups in 20 Rb1f / f 37 Rb1d / d dams without P4 treatment 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 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 , ; green dotted , luminal epithelium ; red dotted , stroma .

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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 expression of pMLKL , a critical mediator of necroptosis , in mouse uterine epithelial . 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 control mice ( Rb1f / f mice ) on days 5 6 compared to on day 4 ( mean ± , Student ' s t ; n = 3 mice for each group ) . As for uterine samples on days 5 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 , implanting uterus produced TNFα at implantation site of both Rb1f / f Rb1d / d mice . Immunostaining of TNFα at implantation sites of Rb1f / f Rb1d / d mice were performed . Scale bar = 100μm ; arrowhead , .

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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 mouse uterine epithelial demonstrated that TNFα induces expression of annexin V , a marker of PS presentation at outer of Rb1f / f epithelial but does not in Rb1d / d ones , P4 restores TNFα - primed annexin V expression in Rb1d / d uterine epithelial ( mean ± , Student ' s t ) . Using three lines of mouse epithelial obtained from both Rb1f / fand Rb1d / d mice , in vivo experiments were performed three times . Each of 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 using mouse uterine epithelial showed that TNFα administration increases expression of annexin V in growth - arrested mouse epithelial with thymidine treatment , but does not in control ( mean ± , Student ' s t ) . Using three lines of mouse epithelial obtained from both Rb1f / fand Rb1d / d mice , in vivo experiments were performed three times . Each of 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 , expression of TNF receptor type 2 ( TNFR2 ) was upregulated in growth - arrested epithelial with thymidine P4 supplementation ( mean ± , Student ' s t ) . Using three lines of mouse epithelial 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 , fragmented uterine epithelial with lipid droplets were engulfed by trophoblast . Scale bar = 2µm . Arrowhead , fragments engulfed by trophoblast ; dotted circle , lipid droplets in cytoplasm ; tr , trophoblast ; le , luminal epithelium ; s , stroma ; red dotted , stroma ; green dotted , luminal epithelium ; blue dotted , 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 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 , . C , Pre - implantation P4 treatment recovered 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 , .

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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 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 .

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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 eControl dams ( mean ± , Student ' s t ; 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 , expression patterns of Ki67 on day 4 in uterine epithelium were comparable between Rb1 eKO Rb1 eControl mice . Scale bar , 100μm ; le , luminal epithelium ; s , stroma .

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

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

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.