F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA , RAD51 and MMS22L - TONSL or MMS22L . Averages shown , n = 2 ; error bars , SEM .

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

F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.


H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA ( 1 nM ) .

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

H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).


I A representative immunoblot showing effects of MMS22L - TONSL and MMS22L titration into reactions containing RAD51 , ssDNA ( 1 nM ) and dsDNA ( 10 nM ) , as indicated .

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

I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.


( A ) After 4 h of starvation in 1 % K ‐ acetate medium in the presence of PMSF , autophagic vesicles can be visualized in vacuoles of wild ‐ type and pep4prb1 cells using Normaski optics . In aut2Δ and aut7Δ cells no autophagic vesicles can be seen . Bar represents 20 μm .

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

(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm.


( B ) Total protein breakdown of cells starved on nitrogen ‐ free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild ‐ type cells . pep4Δ ( pra1Δ ) cells are almost completely impaired in starvation ‐ induced vacuolar protein breakdown . Cells were pulse ‐ labeled with [ 35S ] methionine and chased on non ‐ radioactive starvation medium . Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al . ( 1997 ) .

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

(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).


( C ) Survival during starvation . Like pep4Δ ( pra1Δ ) cells , which are almost completely impaired in vacuolar proteolysis , aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen ‐ free medium as compared with wild ‐ type cells . Aliquots of cells incubated in 1 % K ‐ acetate were plated and growing colonies were determined according to Straub et al . ( 1997 ) .

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

(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).


Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) and aut7Δ ( C ) cells . Further details are outlined in the text . Crude extracts were prepared from cells starved 4 h on 1 % K ‐ acetate .

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

Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.


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


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


( A ) Detection of protein - protein interactions using the two ‐ hybrid system . Intense staining indicates an interaction between Aut2p and Aut7p , Aut2p and Tub1p , and Aut2p and Tub2p . AUT7 , TUB1 and TUB2 are cloned in ‐ frame with the GAL4 ‐ activator domain . AUT2 and AUT7 were fused to the GAL4 ‐ binding domain ( see Materials and methods ) .

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

(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods).


( B ) Affinity chromatography confirms protein - protein interaction between Aut2p and Aut7p . A fusion protein of Aut2p with the 26 kDa glutathione ‐ S ‐ transferase ( GST ) domain from Schistosoma japonicum ( see Materials and methods ) was bound to glutathione - Sepharose 4B . This GST - Aut2p column was incubated with a crude extract from aut7Δ cen Met25 : : AUT7 - GFP cells . The non ‐ bound supernatant ( S ) fraction ( lane 1 ) was taken away . Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non ‐ specifically bound proteins . After the final 500 mM NaCl wash ( W , lane 2 ) , bound proteins were eluted with Laemmli buffer ( E , lane 3 ) . Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP . Using Aut2p - GST , a clear band corresponding to Aut7p - GFP is seen in the eluate fraction ( lane 3 , arrowhead ) . As a control , GST alone was attached to the column and no band was detectable ( lane 6 ) .

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

(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6).


( C ) As described in ( B ) , bovine tubulin binds to GST - Aut2p glutathione - Sepharose 4B ( lane 12 ) , but not to GST glutathione - Sepharose 4B ( lane 9 ) . Protein staining with Coomassie is shown in lanes 1 - 6 ; in lanes 7 - 12 tubulin was detected using monoclonal antibodies ( Boehringer Mannheim ) . Further details are given in Materials and methods .

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 O O O O O O O O O O O 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

(C) As described in (B), bovine tubulin binds to GST-Aut2p glutathione-Sepharose 4B (lane 12), but not to GST glutathione-Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1-6; in lanes 7-12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods.


( D ) Similar to ( C ) , bovine tubulin does not bind to GST - Aut7p glutathione - Sepharose 4B ( lane 12 ) and to GST glutathione - Sepharose 4B ( lane 9 ) . Lanes 1 - 6 , protein staining with Coomassie ; lanes 7 - 12 probed with antibodies directed against tubulin .

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

(D) Similar to (C), bovine tubulin does not bind to GST-Aut7p glutathione-Sepharose 4B (lane 12) and to GST glutathione-Sepharose 4B (lane 9). Lanes 1-6, protein staining with Coomassie; lanes 7-12 probed with antibodies directed against tubulin.


Benomyl sensitivity of wild ‐ type cells overexpressing AUT2 and AUT7 , and of aut2Δ and aut7Δ cells is not altered . Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg / ml benomyl . Plates were incubated at 30 ° C .

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

Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.


( A ) Autophagosome ‐ like vesicles ( arrowhead ) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1 % K ‐ acetate in the presence of PMSF . Bar represents 100 nm .

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

(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm.


( B ) Electron micrographs unravels the accumulation of double membrane ‐ layered autophagosome ‐ like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen ‐ free medium in the presence of PMSF . Arrowheads indicate the clearly distinguishable two membrane layers . Bar represents 100 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm.


( C and D ) Cytosol containing autophagosome ‐ like vesicles accumulating in the cytosol of aut2Δ cells ( D ) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction . In wild ‐ type cells ( C ) , no P100 is detectable . Fatty acid synthase ( Fas ) was used as a cytosolic protein . Further details are given in the text .

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

(C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text.


( D , E ) HTM - mediated quantification of MPM - 2 ( D ) and H3S10P ( E ) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 ( USP7i ) for 4 hrs . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm . The quantification is shown to the right of representative images for each analysis . ( * * * p < 0 . 05 ; t - test ) . Grey lines indicate mean values .

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

(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.


( A ) Western Blot showing the levels of USP7 , MPM - 2 , CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2 - 4 hrs ( USP7i ) , an asterisk indicates a non - specific band .

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

(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.


( B ) Immunofluorescence of CCNB1 ( red ) and CCNB1S126P ( green ) in RPE cells in control conditions ( Control ) and after treatment with 25 μM P22077 for 2 hrs ( USP7i ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

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

(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


( B ) Western blot showing the levels of MPM - 2 , pAKT , AKT , pPRC1 , pKAP , KAP , pMAPKAPK - 2 , pSTAT3 , STAT3 and β - Actin in whole cell extracts of RPE cells treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Ponceau staining is shown as loading control .

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

(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.


( D ) Immunofluorescence of PP2A - A ( green ) in U2OS cells treated with DMSO ( CONTROL ) , 50 μM P22077 ( USP7i ) for 4 hrs or 0 . 5 μM Okadaic acid ( OA ) for 1 hr 30 min . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm .

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

(D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.


( E ) Western blot showing the levels of MPM - 2 , pAKT , AKT , pPRC1 , pMAPKAPK - 2 , p4e - BP1 , 4e - BP1 and β - Actin , in whole cell extracts of RPE cells treated with DMSO ( Control ) , 25 μM P22077 ( USP7i ) and 10 μM of the PP2A activator DT - 061 for 2 - 4 hrs . Ponceau staining is shown as loading control .

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

(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.


( F ) Western blot showing the levels of MPM - 2 , phosphor - CDK substrate motif [ ( K / H ) pSP ] and β - Actin in whole cell extracts of RPE cells treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid ( OA ) for 2 hrs , 10 nM Calyculin A for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor . Ponceau staining is shown as loading control .

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

(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.


( E ) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT - 116 cells treated with DMSO ( Control ) , 50 μM P22077 for 2 - 4 hrs ( USP7i ) , 10 μM RO3306 for 4 hrs ( CDK1i ) or 50 μM P22077 + 10 μM RO3306 for 2 - 4 hrs ( USP7i + CDK1i ) . Ponceau staining is shown as loading control .

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

(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.


( B ) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control , 25 μM P22077 ( USP7i ) , 10 μM RO3306 ( CDK1i ) or 10 μM DT - 061 ( PP2Aa ) , and their combinations for the indicated times . DNA content was followed with Hoechst , and the mitochondrial membrane potential was measured by TMRE . The percentage of apoptotic cells ( TMRE negative ) is indicated for each condition . This experiment was repeated 3 times . ( * p < 0 . 05 , * * p < 0 . 01 , t - test ) . Bars represent mean values ± 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.


( C ) GLO viability assay in cells treated with 3 independent USP7 inhibitors ( P22077 , BAY 11 - 7082 or HBX 19818 ) alone or in combination with a 24hr treatment of 10 μM RO3306 ( CDK1i ) . This experiment was repeated 3 times . Data represent mean values ± SEM . ( * * * p < 0 . 05 , ANOVA ) .

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

(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).


( a ) 30 and 150 days post fertilization ( dpf ) zebrafish ventricles were isolated and relative protein levels determined ( n = 3 ) .

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

(a) 30 and 150 days post fertilization (dpf) zebrafish ventricles were isolated and relative protein levels determined (n = 3).


( c ) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue ( n = 2 - 3 technical replicates using pooled cDNA from 10 ventricles for each condition ( n = 2 for pkmb and pdk4 , and n = 3 for the other genes ) ) .

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

(c) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue (n = 2-3 technical replicates using pooled cDNA from 10 ventricles for each condition (n = 2 for pkmb and pdk4, and n = 3 for the other genes)).


d , Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS , 2 - DG or DCA ; magnified view of area in white boxes shown below ; white dashed lines mark the wound border ; arrowheads point to PCNA + CMs ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 5 ventricles ) .

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

d, Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to PCNA+ CMs; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles).


e ) Immunostaining of heart sections for MEF2 N2 . 261 and MEF2 in 5 dpci animals treated with PBS , 2 - DG or DCA ; magnified view of area in white boxes shown below ; white dashed lines mark the wound border ; arrowheads point to N2 . 261 + CMs ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 5 ventricles ) .

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

e) Immunostaining of heart sections for MEF2 N2.261 and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to N2.261+ CMs; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles).


( a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

(a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a + / + and ppargc1a - / - animals ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


d ) Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci ppargc1a + / + and ppargc1a - / - animals ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

d) Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


( e AFOG staining of heart sections from 60 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; black dashed lines outline the scar area ; scar area measured on the right ( n = 3 - 5 ventricles of each genotype ) .

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

(e AFOG staining of heart sections from 60 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


f ) AFOG staining of heart sections from 45 dpci ppargc1a + / + and ppargc1a - / - animals ; black dashed lines outline the scar area ; scar area measured on the right ( n = 3 - 5 ventricles of each genotype ) .

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

f) AFOG staining of heart sections from 45 dpci ppargc1a+/+ and ppargc1a-/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) and Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


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


Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci animals ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) and Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


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


( f AFOG staining of heart sections from 60 dpci animals ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; black dashed lines outline the scar area ; scar area measured on the right ( n = 4 - 5 ventricles of each genotype ) .

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

(f AFOG staining of heart sections from 60 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


g ) AFOG staining of heart sections from 60 dpci animals and Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; black dashed lines outline the scar area ; scar area measured on the right ( n = 4 - 5 ventricles of each genotype ) .

g) AFOG staining of heart sections from 60 dpci animals and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


( a , b ) Staining for Ki67 , CTNI and DNA ( DAPI ) in control , PDHA1STA OE or PDK3 OE RNCMs cultured in growth ( a ) or non - growth ( b ) medium ; arrowheads point to Ki67 + RNCMs ; percentage of Ki67 + RNCMs shown on the right ( n = 4 biological replicates for each condition ) .

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

(a, b) Staining for Ki67, CTNI and DNA (DAPI) in control, PDHA1STA OE or PDK3 OE RNCMs cultured in growth (a) or non-growth (b) medium; arrowheads point to Ki67+ RNCMs; percentage of Ki67+ RNCMs shown on the right (n = 4 biological replicates for each condition).


( c ) Staining for CTNI and DNA ( DAPI ) in scratch assay using control , PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch ; magnified view of area in white boxes shown below ; arrowheads point to membrane protrusions of RNCMs ; quantification of CM membrane protrusions shown on the right ( n = 4 biological replicates for each condition ) .

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

(c) Staining for CTNI and DNA (DAPI) in scratch assay using control, PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch; magnified view of area in white boxes shown below; arrowheads point to membrane protrusions of RNCMs; quantification of CM membrane protrusions shown on the right (n = 4 biological replicates for each condition).


( d ) Reactome over - representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control .

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

(d) Reactome over-representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control.


( e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs .

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

(e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs.


f ) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs .

f) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs.


( a ) H3R expression in cultured ratcortical neurons at DIV7 after OGD / R injury ( 2 h OGD and 24 h reperfusion ) shown by western blot ( n = 6 per condition ; * * P0 . 01 versus control with Student ' s t - test ) . Full - size blots are shown in Supplementary Fig . 9 .

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

(a) H3R expression in cultured ratcortical neurons at DIV7 after OGD/R injury (2 h OGD and 24 h reperfusion) shown by western blot (n=6 per condition; **P0.01 versus control with Student's t-test). Full-size blots are shown in Supplementary Fig. 9.


( b ) When the H3R antagonist thioperamide ( THIO , 10 − 6 mol l − 1 at reperfusion ) and the agonist immepip ( IMME , 10 − 6 mol l − 1 at reperfusion ) were administered , viability was tested in cultured neurons by 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyltetrazolium bromide ( MTT ) assay after OGD / R ( n = 7 per condition ; * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) 2 , 3 , 5 - triphenyltetrazolium chloride - stained brain sections showing the infarct area in WT and H3R − / − mice receiving saline , the H3R antagonists A331440 , clobenpropit ( CLOB ) and thioperamide , ( THIO ; 5 mg kg − 1 , i . p . , immediately at reperfusion and 6 h later ) , and the H3R agonist immepip ( IMME , 1 μg , i . c . v . , at reperfusion ) after I / R ( 1 h MCAO followed by 24 h reperfusion ; scale bar , 5 mm ) .

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

(c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm).


The bar graphs show infarct volume ( d ) and neurological score ( e ) ( n = 6 - 8 mice per condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 versus WT saline group with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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

The bar graphs show infarct volume (d) and neurological score (e) (n=6-8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) TUNEL staining of apoptotic cells in penumbral brain slices in vivo ( scale bar , 100 μm ) when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after tMCAO . TUNEL - positive cells are green , and all cells are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

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

(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , and cleaved caspase - 3 protein expression is shown by western blot in WT and H3R − / − mice after tMCAO ( b ) ( n = 6 - 7 per condition ; * * P0 . 01 , * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc test )

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test)


( c ) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after OGD / R . TUNEL - positive cells are green , and all cells are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

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

(c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , and cleaved caspase - 3 protein expression is shown by western blot in WT and H3R − / − mice in cultured neurons subjected to OGD / R ( d ) ( n = 8 per condition ; * P0 . 05 , * * * P0 . 001 with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 10 .

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.


( a ) 2 , 3 , 5 - Triphenyltetrazolium chloride ( TTC ) - stained brain sections from WT and HDC − / − mice showing the infarct area in those receiving saline , the H3R antagonist thioperamide ( THIO ) , the H1R antagonist pyrilamine ( PYRI ; 10 mg kg − 1 , i . p . , at reperfusion ) , the H2R antagonist cimetidine ( CIME ; 10 mg kg − 1 , i . p . , at reperfusion ) and α - FMH ( 50 mg kg − 1 , i . p . , 2 h before ischemia ) after 24 h reperfusion ( n = 6 per condition ; scale bar , 5 mm ) .

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

(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).


( b ) Infarct volumes and ( c ) neurological deficit scores ( * P0 . 05 , * * P0 . 01 , * * * P0 . 001 with analysis of variances followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Akt / GSK - 3β / mTOR / P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD / R . LiCl ( 5 mmol l − 1 immediately at reperfusion ) was used to rephosphorylate GSK - 3β / mTOR / P70S6K . The digits below represent the semiquantified optidensity of the bands , the control bands were defined as 1 . 00 .

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

(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( b ) Representative western blots showing the phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signaling in H3R − / − mice after OGD / R injury . The digits below represent the semiquantified optidensity of the bands , the control bands were defined as 1 . 00 .

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

(b) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( c ) Representative images showing LC3 - positive autophagic vacuoles ( red ) with 4 ' , 6 - diamidino - 2 - phenylindole staining ( blue ) after administration of thioperamide and LiCl with OGD / R ( scale bar , 10 μm ) .

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

(c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm).


( d ) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD / R ( n = 7 , * * P0 . 01 , * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

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

(d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( e ) Representative western blots and bar graph showing the expression of LC3 - I and LC3 - II in steady - state autophagy and autophagic flux under OGD / R injury when thioperamide ( THIO ) and immepip ( IMME ) were administered ( n = 6 ; * * P0 . 01 , * * * P0 . 001 with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 11 .

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

(e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.


( a ) Representative immunohistochemical staining of LC3 ( green ) along with NeuN staining ( red ) in WT and H3R − / − mice ( scale bar , 25 μm ) .

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

(a) Representative immunohistochemical staining of LC3 (green) along with NeuN staining (red) in WT and H3R−/− mice (scale bar, 25 μm).


( b , c ) When thioperamide was administered in vivo , LC3 expression was assessed by western blot after I / R in WT mice ( b ) ( * P0 . 05 , * * * P0 . 001 ; n = 6 mice per condition , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) , and in H3R − / − mice ( c ) ( * * * P0 . 001 versus H3R − / − sham group ; n = 5 - 6 mice per condition , with ANOVAs followed by the Bonferroni / Dunn post hoc test . ) Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 12 .

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

(b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5-6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.


( a ) Western blots showing the inhibitory effect of 3 - MA on autophagy in I / R . Full - size blots are shown in Supplementary Fig . 13 .

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

(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13.


( b ) TTC - stained sections showing the infarct area after administration of 3 - MA ( 100 nmol , i . c . v . , at reperfusion ) in WT and H3R − / − mice ( scale bar , 5 mm ) , and ( c ) bar graph showing infarct volume ( n = 6 - 8 mice per condition ; * P0 . 05 , * * * P0 . 001 , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

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

(b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( d ) TTC - stained sections ( scale bar , 5 mm ) and bar graph showing the infarct area after administration of thioperamide in Atg5 + / − mice ( n = 4 - 6 mice per condition ) .

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

(d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition).


( e ) In cultured neurons , viability was tested by MTT assay after 3 - MA ( 2 . 5 mmol l − 1 at reperfusion ) and thioperamide was administered in OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) .

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

(e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( f ) In cultured neurons , viability was tested by MTT after siRNA for Atg7 and OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) .

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

(f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( g ) Viability was tested by MTT assay on Atg5 + / + and Atg5 − / − MEFs after thioperamide administration after OGD / R ( n = 7 per condition ; * * P0 . 01 , * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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

(g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Representative co - immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD / R . Full - size blots are shown in Supplementary Fig . 14 .

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

(a) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( b ) Cell viability was tested by MTT assay after administration of Tat - H3RCT414 - 436 ( 10 − 6 mol l − 1 at reperfusion ) under OGD / R ( data are presented as mean ± s . e . m . ; n = 7 per condition ; * P0 . 05 , * * P0 . 01 , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) .

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

(b) Cell viability was tested by MTT assay after administration of Tat-H3RCT414-436 (10−6 mol l−1 at reperfusion) under OGD/R (data are presented as mean±s.e.m.; n=7 per condition; *P0.05, **P0.01, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) Representative western blots showing the effect of Tat - H3RCT414 - 436 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling and LC3 expression in OGD / R . Full - size blots are shown in Supplementary Fig . 14 .

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

(c) Representative western blots showing the effect of Tat-H3RCT414-436 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( d ) Representative images of the effect of Tat - H3RCT414 - 436 on LC3 - positive autophagic puncta ( red ) in 4 ' , 6 - diamidino - 2 - phenylindole - stained ( blue ) neurons after OGD / R ( scale bar , 10 μm ) .

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

(d) Representative images of the effect of Tat-H3RCT414-436 on LC3-positive autophagic puncta (red) in 4',6-diamidino-2-phenylindole-stained (blue) neurons after OGD/R (scale bar,10 μm).


( e ) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling and LC3 expression in OGD / R . Full - size blots are shown in Supplementary Fig . 14 .

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

(e) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( f ) Representative western blots and bar graph showing the expression of LC3 - I and LC3 - II on steady - state autophagy and autophagic flux under OGD / R when CLIC4 was knocked down ( data are presented as mean ± s . e . m . ; n = 6 ; * P0 . 05 , * * P0 . 01 ; , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Full - size blots are shown in Supplementary Fig . 14 .

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

(f) Representative western blots and bar graph showing the expression of LC3-I and LC3-II on steady-state autophagy and autophagic flux under OGD/R when CLIC4 was knocked down (data are presented as mean±s.e.m.; n=6; *P0.05, **P0.01; , with ANOVAs followed by the Bonferroni/Dunn post hoc test). Full-size blots are shown in Supplementary Fig. 14.


( g ) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD / R ( data are presented as mean ± s . e . m . ; n = 8 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) .

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

(g) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD/R (data are presented as mean±s.e.m.; n=8 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP , and FLAG - TCHP at N - and C - terminal . total lysates were immunoprecipitated with anti - FLAG antibodies , and blots were probed sequentially with anti - PCM1 , anti - FLAG , and anti - ACTIN antibodies . IgG light chains are indicated with Red Ponceau staining . The input totals were analyzed by parallel immunoblotting as a control for the level of expression .

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

Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.


Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP - FLAG C - terminal or constructs with deletions of the coiled - coil domain 1 ( TCHP ∆ 1 ) and 2 ( TCHP ∆ 2 ) , as indicated in the scheme . total lysates were immunoprecipitated with anti - FLAG antibodies , and blots were probed sequentially with anti - PCM1 , anti - FLAG , and anti - ACTIN antibodies . IgG light chains are indicated with Red Ponceau staining . The input totals were analyzed by parallel immunoblotting as a control for the level of expression .

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

Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.


Anti - PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot .

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

Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot.


Co - localization of TCHP - V5 and centriolar satellite proteins . HUVECs were transduced with TCHP - V5 lentivirus and stained for anti - PCM1 , anti - CEP290 , anti - CEP72 and anti - V5 antibodies . Scale bars , 25 µm and 5 µm in the inset . Right panel : the two - channel intensity correlation of pixels corresponding to regions identified with TCHP - V5 and satellite markers ( n = 90 cells ; Pearson colocalization coefficient ) .

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

Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 µm and 5 µm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n=90 cells; Pearson colocalization coefficient).


TCHP knock - down or control cells were stained for anti - PCM1 antibody . Panel below : quantification ( n = 50 cells ; unpaired t - test ; * * p = 0 . 004 vs control ) . Scale bars , 50 µm and 5 µm in the inset .

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

TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.


Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) and MG132 treatments for the indicated number of hours before immunoblotting . PCM1 levels were analysed in the insoluble fraction . Laminin has been used as a loading control . Below panel : Quantification of PCM1 degradation . ( one - way ANOVA ; * p = 0 . 0180 vs control time 0 ) .

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

Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0).


Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) and MG132 treatments for the indicated number of hours before immunoblotting . Condition as in ( A ) , Western blot for anti - GABARAP and anti - ACTIN antibodies . Below panel : Quantification of GABARAP degradation . ( one - way ANOVA ; * p = 0 . 0215 vs control time 0 ) .

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

Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).


Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting . Western blot was probed for anti - PCM1 and anti - V5 antibodies . Laminin has been used as a loading control .

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

Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control.


Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting . Western blot was probed for anti - V5 , anti - GABARAP and anti - ACTIN antibodies . Below panels : Quantification of C ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0003 vs control CHX ) and D ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0056 vs control CHX ) .

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

Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).


Immunofluorescent staining for LC3 and p62 in TCHP knock - down and control cells . Scale bars , 25μm . Lower panel : quantification . ( n = 90 cells , unpaired t - test ; LC3 : * * p < 0 . 0001 vs control ; p62 : * * p < 0 . 0001 vs control ) .

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

Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).


Western blot of p62 and LC3 during under normal culture condition or starved condition ( HBSS ) or the presence of BafA1 ( 2 hours ) in TCHP knock - down or control cells . Lower panels : p62 quantification . ( one - way ANOVA ; * * p = 0 . 0003 vs control , # # p = 0 . 0093 vs shTCHP ) ; LC3 quantification . ( one - way ANOVA ; * * p = 0 . 0008 vs control , # # p = 0 . 0011 vs shTCHP ) .

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

Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).


HUVECs were transduced with the tandem mCherry - EGFP - LC3 and with shRNA TCHP or control vectors . Left panels : Representative picture of mCherrry - EGFP - LC3 reporters . Scale bars , 25μm . Right panel : Quantification of the number of mCherry - only ( red bars , autolysosomes ) or double - positive ( mCherry + / EGFP + ; yellow bars , autophagosomes ) . ( n = 80 cells , one - way ANOVA ; * p = 0 . 0447 vs control ; # # p = 0 . 0007 vs shTCHP ) .

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

HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP).


Ratiometric flow cytometric analysis of mCherrry - EGFP - LC3 reporters as in C . ( one - way ANOVA ; * p = 0 . 0247 vs control ; # # p = 0 . 0088 vs shTCHP ) .

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

Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP).


Quantification of long - lived protein degradation assay in TCHP knock - down and control cells by flow cytometry ( one - way ANOVA ; * * p = 0 . 0018 vs control , # # p < 0 . 0001 vs shTCHP ) .

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

Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP).


HUVECs were transduced with TCHP - V5 and control vectors . Western blot for anti - V5 and anti - p62 antibody during under normal culture condition or HBSS or BafA1 or Torin - 1 treatment . Lower panel : quantification . ( one - way ANOVA ; * * p = 0 . 0044 ; # p = 0 . 0117 vs TCHP - V5 ) .

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

HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).


The postnuclear fraction ( PNS ) from TCHP knock - down and control HUVECs in the presence or absence of BafA1 was separated into ( low - speed pellet ) LSP and ( high - speed pellet ) HSP fractions and then analysed by immunoblots using anti p62 , LC3 and GABARAP antibodies . The sub - fractions were treated with proteinase K ( Prot . K ) with or without Triton X - 100 ( TX - 100 ) . Quantification of the Western blot : the p62 , LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non - treated samples .

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

The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.


Representative TEM images of autophagosomes in control and TCHP knock - down HUVECs in the complete medium after 3h incubation with BafA1 ( Scale bars , 600nm ) : i , quantification of mean autophagic vesicles ( AVs ) and autophagosome ( APs ) area and ii , distribution of the cross - section areas of the analysed vesicles expressed in percentage . ( n = 7 cells ; > 100 vesicles per sample ; AVs : unpaired t - test ; * * p < 0 . 0001 vs control ; APs : unpaired t - test * * p < 0 . 0001 vs control ) .

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

Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).


Left panels : Representative single - channel and merged images of HUVECs expressing GFP - LC3 and immunostained for STX17 . Scale bars , 25 µm and 2 µm in the inset . Right panels : Quantification of LC3 / STX17 positive cells in the presence or absence of BafA1 ( one - way ANOVA ; * * p = 0 . 0060 vs control ; # p = 0 . 0136 vs control BafA1 ) and quantification LC3 / STX17 positive puncta per cells ( n = 50 cells ; one - way ANOVA ; * * p < 0 . 0001 vs shTCHP BafA1 ) .

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

Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).


Expression of TCHP , p62 , IL - 1β , IL - 8 , and IL - 6 in ECs from vessels wall from patients with CAD ( n = 8 patients per group , unpaired t - test ; TCHP * * p < 0 . 0001 ; p62 * * p = 0 . 0007 ; IL - 1β * * p < 0 . 0001 ; IL - 8 * * p < 0 . 0001 ; and IL - 6 * * p < 0 . 0001 vs . Healthy subject ) .

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

Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject).


Staining , and quantification of p62 in ECs from healthy subject and patients either treated with vehicle ( DMSO ) or BAY . Scale bars , 50 µm ; ( n = 3 patients per group ; n = 80 cells ; one - way ANOVA ; * * p < 0 . 0001 vs healthy DMSO ; # # p = 0 . 003 vs patients DMSO ) .

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

Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).


Expression of IL - 6 , IL - 8 and IL - 1β ( n = 3 patients per group ; one - way ANOVA ; IL - 6 : * p = 0 . 0135 vs healthy DMSO ; # p = 0 . 0180 vs patients DMSO ; IL - 8 : * p = 0 . 0139 vs healthy DMSO ; # # p = 0 . 0024 vs patients DMSO ; IL - 1β : * p = 0 . 0104 vs healthy DMSO ; # # p = 0 . 0069 vs patients DMSO ) .

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

Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO).


migration speed , in ECs from healthy subject and patients either treated with vehicle ( DMSO ) or BAY . ( n = 3 patients per group ; one - way ANOVA ; * p = 0 . 0325 vs healthy DMSO ; # p = 0 . 0336 vs patients DMSO ) .

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

migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).


Left panels : representative images of the heart of wild - type and Tchp knock - out mice stained for CD31 ( green ) and p62 ( magenta ) . Scale bars , 100µm , and 50µm for the inset . Right panels : Quantification of the percentage of cardiac area ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 083 vs wild - type ) or vessels positive for p62 ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 058 vs wild - type ) and vessel density ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 0097 vs wild - type ) .

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

Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).


Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM . Representative of three samples examined . Scale bar 100

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

Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nm


HT29 cell survival 18h after treatment with TNF + BV - 6 + z - VAD - fmk to induce necroptosis . Cells expressed lentivirus constructs encoding 1 - 90 or 1 - 277 fragments of M45 and were cultured + / - the addition of coumermycin A1 to induce dimerisation . N = 2 for M45 - 90 gyrase + / - coumermycin A1 and 4 for M45 - 277 gyrase and for empty vector , + / - coumermycin

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

HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1


Thioflavin T fluorescence as a function of time , following dilution of WT or AAAA His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , and His - Ub - RIPK3387 ‑ 518 from 8 M to 200 mM urea - containing buffer . Buffer sample contains ThT but no protein . Samples were tested in triplicate and solid line is the average of the triplicates and the error bars ( in grey ) represent + / - s .

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

Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.d


Absorbance spectra from solutions containing Congo red with WT ( black lines ) or AAAA mutant ( dark grey lines ) His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , and His - Ub - RIPK3387 ‑ 518 that have been dialysed against Assembly Buffer . Congo red sample alone with no protein ( light grey line ) served as a control for the experiment . Experiment conducted

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

Absorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted once


Electron micrograph of material eluted in peak 1 from the three MBP fusion proteins , as labelled . These are representative micrographs of the material observed . Scale bar 100 nm in all

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

Electron micrograph of material eluted in peak 1 from the three MBP fusion proteins, as labelled. These are representative micrographs of the material observed. Scale bar 100 nm in all panels


Representative micrographs of insoluble material generated after treatment with thrombin removed the MBP . Experiment repeated three times . Scale bar 100 nm in all

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

Representative micrographs of insoluble material generated after treatment with thrombin removed the MBP. Experiment repeated three times. Scale bar 100 nm in all panel


Fluorescence polarisation profiles from incorporation of FITC - M451 ‑ 90 into excess unlabeled M451 ‑ 90 ( green ) , RIPK1497 ‑ 583 ( red ) , RIPK3387 ‑ 518 ( blue ) or ZBP1170 - 355 ( purple ) . Average polarisation profile from smoothed triplicate samples plotted , with s . d . indicated . Experiment performed twice and representative results shown

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

Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown here


Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of ( C ) mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK1497 ‑ 583 and ( D ) mCHERRY - RIPK3387 ‑ 518 and YPet - RIPK3387 ‑ 518 . E , F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of ( E ) mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑ 518 ( inset is expansion of region indicated by * ) and ( F ) mCHERRY - RIPK1497 ‑ 583 - AAAA and YPet - RIPK3387 ‑ 518 . All mixtures examined in at least three different experiments on three separate

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

Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518. E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions


Representative fluorescence intensity traces collected over 50 s for mixtures of ( A ) mCHERRY - M451 - 90 and YPet ‑ only ; ( B ) mCHERRY - M451 - 90 and YPet ‑ RIPK1497 ‑ 583 ; ( C ) mCHERRY - M451 - 90 and YPet - RIPK3387 ‑ 518 ; ( D ) mCHERRY - M451 - 90 and YPet - ZBP1170 - 355 ; ( E ) mCHERRY - M451 - 90AAAA and YPet ‑ only ; ( F ) mCHERRY - M451 - 90AAAA and YPet - RIPK1497 - 583 ; ( G ) mCHERRY - M451 - 90AAAA and YPet - RIPK3387 ‑ 518 and ( H ) mCHERRY - M451 - 90 and YPet - ZBP1170 - 355 . Independent replicates performed at least

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

Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice


Representative electron micrographs of homofibrils formed by M451 - 90 , RIPK1497 ‑ 583 , His - Ub - RIPK3387 ‑ 518 or ZBP1170 - 355 individually , and heterofibrils formed by indicated mixtures . Samples prepared twice from independent protein

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

Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations


Heterofibrils containing mCHERRY - M451 - 90 and YPet ‑ RIPK1497 ‑ 583 C Heterofibrils containing mCHERRY - M451 - 90 and YPet - RIPK3387 ‑ 518 D Heterofibrils containing mCHERRY - M451 - 90 and YPet ‑ ZBP1170 ‑ 355 E Heterofibrils containing mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑

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

Heterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583 C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518 D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355 E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑51


Heterofibrils containing mCHERRY - RIPK3387 ‑ 518 and YPet ‑ ZBP1170 ‑ 355 Pearson ' s correlation coefficients measured from two confocal images of the indicated protein mixtures .

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

Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355 Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures.


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

Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518


STED images of heteromeric fibrils formed by co - assembly of mCHERRY - M451 - 90 and YPet - RIPK3387 ‑

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

STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518


SDS AGE analysis of monomeric or assembled forms of YPet - RIPK3387 ‑ 518 and mCHERRY - M451 - 90WT or mCHERRY - M451 - 90AAAA . Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis , as indicated . Assembled samples incubated with 0 or 2 % SDS at RT for 10 min before electrophoresis . Protein components as indicated above each lane .

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

SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.


SDS AGE analysis of assemblies of mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑ 518 with or without four times molar equivalents of His - Ub - M45 .

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

SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.


SDS AGE analysis of assemblies of YPet - RIPK3387 ‑ 518 and mCHERRY - ZBP1170 - 355 with or without four times molar equivalents of His - Ub - M45 .

SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.


Confocal microscopy image of a single fibril composed of YPet - M451 -

Confocal microscopy image of a single fibril composed of YPet-M451-90


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( A ) Supernatants were harvested after 18 h , and concentrations of IL - 6 and IL - 12 were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Mean ± SD of triplicate determinations from > 3 independent experiments are shown .

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

A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( B ) mRNA was harvested after 2 h , and expression of IL - 6 and IL - 12 was measured by real - time PCR and normalized to G6pdx . Unpaired two - tailed t - test . Data ( mean ± SD ) are representative of three independent experiments .

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

A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.


C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC . Periods of oxidation were 24 h for CuSO4 ( 10 μM ) , 48 h for FeSO4 ( 10 μM ) , and 72 h for air . BMDCs were treated for 60 min with lipids prior stimulation with R837 ( 5 μg / ml ) for 18 h . Cytokine concentrations in supernatants were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Data represent mean ± SEM of triplicate determinations .

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

C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.


D PAPC ( 40 μg / ml ) oxidized with CuSO4 ( 10 μM ) for the indicated times was used for treatment of BMDCs prior to R837 ( 5 μg / ml ) stimulation and measurement of IL - 12 secretion . Mean ± SEM of triplicate determinations from three different oxidation series are shown .

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

D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.


E , F Treatment of BMDCs with the indicated concentrations of CuSO4 - oxidized PAPC suppressed IL - 6 ( E ) and IL - 12 ( F ) secretion triggered by a variety of different TLR agonists . After OxPAPC treatment for 60 min , BMDCs were stimulated for 18 h with LTA ( 500 ng / ml ) , Poly I : C ( 50 μg / ml ) , LPS ( 10 ng / ml ) , R837 ( 5 μg / ml ) , and CpG ( 100 nM ) . Cytokine concentrations in the supernatant were quantified by ELISA . Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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

E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.