( 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 .

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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 ) 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 )

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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 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 ) .

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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 − / − cells .

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


( 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 .

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(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 ) 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 .

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(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 ) 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-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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 ) and tNHTT - 150Q - EGFP ( b ) constructs into Neuro2a cells ( 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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 the inhibitory effect of RHQ on the 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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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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(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 , 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 .

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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 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 .

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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 . Protein lysates were collected at indicated times after reoxygenation and blotted for MCL ‐ 1 expression and 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 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 .

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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 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 .

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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 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 .

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(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 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 .

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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 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 .

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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 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 .

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

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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

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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 ) and MCL ‐ 1Δ / Δ ( KO ) MEFs were analysed by western blot for the 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 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 .

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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 and analysed for the 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 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 .

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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 and LM before analysing the presence of MCL ‐ 1 and Beclin ‐ 1 .

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


( B ) Expression levels of various BCL ‐ 2 homologues in the cortices of animals of the 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 morphology of MCL ‐ 1Δ / Δ . Brain sections were stained with cresyl violet and 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 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 .

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

(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 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 .

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

(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 ) 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 .

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

(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 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 .

O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(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.


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 .

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

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 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 .

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

(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 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 .

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

(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 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 .

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

(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 ) 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 ) .

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

(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 ) 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 .

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

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

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

(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 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 .

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

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

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

(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 ) 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 .

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

(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 ) and their MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − littermates ( n = 13 ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

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


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

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

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


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 .

B-EXP_ASSAY O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O

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 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 .

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

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 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 .

O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

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 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 .

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

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 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 .

B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O

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 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 .

B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O

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 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 .

O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O

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.


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 .

O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O

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 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 .

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

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 .

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

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 ) .

O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-ORGANISM O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O

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 .

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

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 .

O O O O B-ORGANISM O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

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 .

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

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 .

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

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 ) .

O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O

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 ) .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 ) .

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

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 ) .

O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE B-SMALL_MOLECULE O O 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-ORGANISM O O O O O

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 .

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

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 .

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

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 .

O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O

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 .

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

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.