Figure 1 . ( A ) HMGB1 translocates from the nucleus to the cytosol during autophagy , but not apoptosis . Mouse embryonic fibroblasts ( MEFs ) and the human Panc2 . 03 tumor cell line were treated with 1 µM rapamycin ( Rap ) for 12 h , starvation ( HBSS ) for 3 h , or UV irradiation at 50 mJ / cm2 for 5 min before a 12 - h recovery and then were immunostained with HMGB1 - specific antibody ( green ) and Hoechst 33342 ( blue ) . The mean nuclear ( Nuc ) and cytosolic ( Cyt ) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods . * , P < 0 . 05 and * * , P < 0 . 005 versus untreated ( UT ) group ; n = 3 . Representative images are depicted ( right ) . ( B ) Inhibition of autophagy blocks HMGB1 translocation . Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5 - specific shRNA for 48 h and were stimulated with starvation ( HBSS ) for 3 h and immunostained with HMGB1 - or LC3 - specific antibody and Hoechst 33342 . The mean nuclear / cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis . A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted ( right ) . In parallel , the indicated cells were transfected with GFP - LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP - LC3 punctae . * , P < 0 . 05 , * * , P < 0 . 005 , and * * * , P < 0 . 0005 versus HBSS group ; n = 3 . Ctrl , control . ( C ) Knockdown of ATG5 inhibits LC3 - II expression . Western blot analysis of LC3 - I / II expression in Panc02 cells under the conditions described in B . Actin was used as a loading control . ( D ) Hmgb1 − / − does not influence ATG5 staining . Hmgb1 − / − and Hmgb1 + / + MEFs were immunostained with HMGB1 - specific antibody ( green ) , ATG - 5 - specific antibody ( red ) , and Hoechst 33342 ( dark blue ) . Representative images are depicted in the right panels . ( E ) mETC inhibitors promote ROS production , autophagy , and HMGB1 translocation . Panc2 . 03 cells were stimulated with rotenone ( Rot ) , thenoyltrifluoroacetone ( TTFA ) , and antimycin A ( AA ) at indicated doses for 12 h , and ROS production was assessed by measuring the fluorescent intensity of CM - H2DCFDA in a fluorescent plate reader . In parallel experiments , cells were then immunostained with HMGB1 - or LC3 - specific antibody and Hoechst 33342 . The mean nuclear / cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis . * , P < 0 . 05 , * * , P < 0 . 005 , and * * * , P < 0 . 0005 versus untreated group ; n = 3 . ( F ) mETC inhibitors increase LC3 - II expression and promote HMGB1 translocation . Western blot analysis of LC3 - I / II and nuclear / cytosolic HMGB1 expression as indicated in E . Fibrillarin is a nuclear fraction control , and tubulin is a cytoplasmic fraction control . ( G ) Antioxidant and SOD RNAi limit starvation - induced autophagy and HMGB1 translocation . Panc2 . 03 cells were pretreated with the antioxidant ( NAC ) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h . Then cells were starved ( HBSS ) for 3 h and analyzed by imaging cytometry to determine the mean nuclear / cytosolic HMGB1 intensity and LC3 punctae per cell . * , P < 0 . 05 ; * * , P < 0 . 005 ; and * * * , P < 0 . 0005 versus HBSS group ; n = 3 . A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here . ( H ) Antioxidant and SOD RNAi limit starvation - induced autophagy as measured by LC3 - II expression . Western blot analysis of LC3 - I / II expression under the conditions indicated in G . Actin was used as a loading control . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(A) HMGB1 translocates from the nucleus to the cytosol during autophagy, but not apoptosis. Mouse embryonic fibroblasts (MEFs) and the human Panc2.03 tumor cell line were treated with 1 µM rapamycin (Rap) for 12 h, starvation (HBSS) for 3 h, or UV irradiation at 50 mJ/cm2 for 5 min before a 12-h recovery and then were immunostained with HMGB1-specific antibody (green) and Hoechst 33342 (blue). The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods. *, P < 0.05 and **, P < 0.005 versus untreated (UT) group; n = 3. Representative images are depicted (right).(B) Inhibition of autophagy blocks HMGB1 translocation. Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5-specific shRNA for 48 h and were stimulated with starvation (HBSS) for 3 h and immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted (right). In parallel, the indicated cells were transfected with GFP-LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP-LC3 punctae. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus HBSS group; n = 3. Ctrl, control.(C) Knockdown of ATG5 inhibits LC3-II expression. Western blot analysis of LC3-I/II expression in Panc02 cells under the conditions described in B. Actin was used as a loading control.(D) Hmgb1−/− does not influence ATG5 staining. Hmgb1−/− and Hmgb1+/+ MEFs were immunostained with HMGB1-specific antibody (green), ATG-5-specific antibody (red), and Hoechst 33342 (dark blue). Representative images are depicted in the right panels.(E) mETC inhibitors promote ROS production, autophagy, and HMGB1 translocation. Panc2.03 cells were stimulated with rotenone (Rot), thenoyltrifluoroacetone (TTFA), and antimycin A (AA) at indicated doses for 12 h, and ROS production was assessed by measuring the fluorescent intensity of CM-H2DCFDA in a fluorescent plate reader. In parallel experiments, cells were then immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus untreated group; n = 3.(F) mETC inhibitors increase LC3-II expression and promote HMGB1 translocation. Western blot analysis of LC3-I/II and nuclear/cytosolic HMGB1 expression as indicated in E. Fibrillarin is a nuclear fraction control, and tubulin is a cytoplasmic fraction control.(G) Antioxidant and SOD RNAi limit starvation-induced autophagy and HMGB1 translocation. Panc2.03 cells were pretreated with the antioxidant (NAC) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h. Then cells were starved (HBSS) for 3 h and analyzed by imaging cytometry to determine the mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus HBSS group; n = 3. A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here.(H) Antioxidant and SOD RNAi limit starvation-induced autophagy as measured by LC3-II expression. Western blot analysis of LC3-I/II expression under the conditions indicated in G. Actin was used as a loading control. Data are means ± SEM.


Figure 2 . ( A ) HMGB1 knockout inhibits LC3 punctae formation . HMGB1 − / − and HMGB1 + / + MEFs were treated with autophagic stimuli as indicated , and LC3 punctae formation was detected by LC3 antibody or GFP - LC3 as described in Materials and methods . * , P < 0 . 05 ; * * , P < 0 . 005 ; and * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . UT , untreated . ( B ) Representative images of LC3 punctae in Hmgb1 − / − and HMGB1 + / + MEFs with the indicated treatments are depicted . The percentage of cells showing accumulation of LC3 punctae was reported in A . ( C ) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A ( pepA ) at 10 µg / ml and E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 05 versus Hmgb1 + / + group ; n = 3 . Actin was used as a loading control . AU , arbitrary units . ( D ) Up - regulation of HMGB1 protein expression restores starvation - induced autophagy . Hmgb1 − / − MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h . LC3 punctae formation was assayed by imaging cytometric analysis . * * , P < 0 . 05 versus vector group ; n = 3 . Non , nontransfected . ( E ) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg / ml and E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 005 and * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . Representative images are depicted ( left ) . ( F ) Ultrastructural features in Hmgb1 − / − and Hmgb1 + / + MEFs with or without starvation ( HBSS for 3 h ) treatment ( a - e point to autophagosomes and autolysosomes ) . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(A) HMGB1 knockout inhibits LC3 punctae formation. HMGB1−/− and HMGB1+/+ MEFs were treated with autophagic stimuli as indicated, and LC3 punctae formation was detected by LC3 antibody or GFP-LC3 as described in Materials and methods. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. UT, untreated. (B) Representative images of LC3 punctae in Hmgb1−/− and HMGB1+/+ MEFs with the indicated treatments are depicted. The percentage of cells showing accumulation of LC3 punctae was reported in A.(C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units.(D) Up-regulation of HMGB1 protein expression restores starvation-induced autophagy. Hmgb1−/− MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h. LC3 punctae formation was assayed by imaging cytometric analysis. **, P < 0.05 versus vector group; n = 3. Non, nontransfected.(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. Representative images are depicted (left).(F) Ultrastructural features in Hmgb1−/− and Hmgb1+/+ MEFs with or without starvation (HBSS for 3 h) treatment (a-e point to autophagosomes and autolysosomes). Data are means ± SEM.


Figure 3 . Inhibition of autophagy by cytoplasmic HMGB1 . Hmgb1 − / − and Hmgb1 + / + MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h , and LC3 punctae formation was detected by a confocal microscope . ( A ) Representative images of LC3 punctae ( white arrows ) and HMGB1 ( red arrows ) in cytoplasts of Hmgb1 − / − and Hmgb1 + / + MEFs are depicted . ( B ) The percentage of cells showing accumulation of LC3 punctae was reported ( * , P < 0 . 05 ; n = 3 ) . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.


Figure 4 . ( A ) MEK inhibitors block starvation - induced p - ERK and Bcl - 2 . HMGB1 − / − and HMGB1 + / + MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h . Protein expression levels were assessed as indicated by co - IP or Western blotting . ( B ) Knockout of HMGB1 limits the disassociation of the Bcl - 2 - Beclin1 complex during treatment with autophagic stimuli . HMGB1 − / − and HMGB1 + / + MEFs were starved in the presence or absence of 10 µM U0126 for 3 h . Protein expression levels were then assayed as indicated by co - IP or Western blotting . ( C ) HMGB1 interacts directly with Beclin1 during autophagy . HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co - IP or Western blotting . ( D ) HMGB1 direct interactions with Bcl - 2 are dependent on Beclin1 . Knockdown of Beclin1 and Bcl - 2 by siRNA in HMGB1 wild - type MEFs was performed , and protein expression levels were then assayed as indicated by co - IP or Western blotting . Ctrl , control . ( E and F ) Quantitative data demonstrating the interaction between HMGB1 - Beclin1 and Beclin1 - HMGB1 using densitometry software to assay the relative protein band intensity in co - IP experiments as shown in A - D . * * , P < 0 . 005 and * * * , P < 0 . 0005 ; n = 3 . UT , untreated . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting.(B) Knockout of HMGB1 limits the disassociation of the Bcl-2-Beclin1 complex during treatment with autophagic stimuli. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting.(C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co-IP or Western blotting.(D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1 and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl, control.(E and F) Quantitative data demonstrating the interaction between HMGB1-Beclin1 and Beclin1-HMGB1 using densitometry software to assay the relative protein band intensity in co-IP experiments as shown in A-D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.


Figure 5 . ( A ) The C106 mutation ( C106S ) of HMGB1 impairs its nuclear localization . Hmgb1 − / − MEFs were transfected with wild - type and cysteine mutant HMGB1 - GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved ( HBSS ) for 3 h . The mean nuclear ( Nuc ) and cytosolic ( Cyt ) HMGB1 intensity per cell was analyzed by imaging cytometric analysis . * , P < 0 . 05 versus HMGB1 + / + group ; n = 3 . Representative images of HMGB1 location are shown on the left ( green , HMGB1 ; blue , nucleus ) . The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C - terminal domain , with the cysteine mutation locations identified . ( B and C ) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated and then were starved ( HBSS ) for the indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h and then starved as indicated . LC3 punctae formation was assayed by imaging cytometric analysis ( B ) ( B and C ) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated and then were starved ( HBSS ) for the indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h and then starved as indicated . Apoptosis was assayed by FACS ( C ) ( C ) as described in Materials and methods . * , P < 0 . 05 and * * , P < 0 . 005 ; n = 3 . UT , untreated . Non , nontransfection . ( D ) C23 / C45 is required for the binding of HMGB1 to Beclin1 . Hmgb1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated and stimulated with starvation ( HBSS ) for 3 h . These cells were then assayed for protein expression levels as indicated by IP or Western blotting . Blots are representative of three independent experiments with similar results . ( E ) Reducing reagents disrupt the interaction between wild - type / C106 HMGB1 and Beclin1 . As a control , before IP , samples were incubated with 50 mM DTT ( + DTT ) and assayed for protein expression levels as indicated by IP or Western blotting . Blots are representative of two independent experiments with similar results . ( F ) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol . Cells were stimulated with HBSS , rapamycin ( Rap ) , rotenone ( Rot ) , or thenoyltrifluoroacetone ( TTFA ) for 3 h or 12 h , and LC3 punctae formation was assayed as indicated . * * , P < 0 . 005 versus Beclin1 shRNA group ; n = 3 . Ctrl , control . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.(A) The C106 mutation (C106S) of HMGB1 impairs its nuclear localization. Hmgb1−/− MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine mutation locations identified.(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B)(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/−MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. Apoptosis was assayed by FACS (C) (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfection.(D) C23/C45 is required for the binding of HMGB1 to Beclin1. Hmgb1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and stimulated with starvation (HBSS) for 3 h. These cells were then assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of three independent experiments with similar results.(E) Reducing reagents disrupt the interaction between wild-type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results.(F) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3. Ctrl, control. Data are means ± SEM.


Figure 6 . ( A and B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . Cells as indicated were stimulated with 1 µg / ml HMGB1 protein ( rHMGB1 ) for 24 h , and LC3 expression was detected by Western blotting ( A ) . ( A and B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . Cells as indicated were stimulated with 1 µg / ml HMGB1 protein ( rHMGB1 ) for 24 h , LC3 punctae formation ( arrows ) was assayed by immunofluorescence ( B ; n = 3 ; * , P < 0 . 05 ) . ( C and D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with and without pUNO1 - HMGB1 transfection . Cells as indicated were stimulated with Earle ' s balanced salt solution for 3 h , and LC3 expression was detected by Western blotting ( C ) . ( C and D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with and without pUNO1 - HMGB1 transfection . Cells as indicated were stimulated with Earle ' s balanced salt solution for 3 h , and LC3 punctae formation ( arrows ) was assayed by immunofluorescence ( D ; n = 3 ; * , P < 0 . 05 ) . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, and LC3 expression was detected by Western blotting (A).(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, LC3 punctae formation (arrows) was assayed by immunofluorescence (B; n = 3; *, P < 0.05).(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 expression was detected by Western blotting (C).(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 punctae formation (arrows) was assayed by immunofluorescence (D; n = 3; *, P < 0.05). Data are means ± SEM.


Figure 7 . Expression of HMGB1 or the C106S mutant , but not C23S and C45S mutants , restore autophagic flux in Hmgb1 − / − MEFs . ( A ) HMGB1 − / − MEFs were transfected with wild - type ( WT ) or the cysteine mutant HMGB1 - GFP plasmids as indicated , starved ( HBSS ) for 3 h in the presence or absence of 100 nM bafilomycin A1 , and then immunostained with LAMP2 - specific antibody / Alexa Fluor 594 secondary antibody ( shown in red ) , LC3 - specific antibody / Alexa Fluor 647 secondary antibody ( shown in green ) , and Hoechst 33342 ( shown in blue ) . Images were acquired digitally from a randomly selected pool of 10 - 15 fields under each condition . ( B ) Quantitative analysis of the percentage of LAMP2 / LC3 colocalization was detected by Image - Pro Plus 5 . 1 software . Data are means ± SEM .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.Expression of HMGB1 or the C106S mutant, but not C23S and C45S mutants, restore autophagic flux in Hmgb1−/− MEFs. (A) HMGB1−/− MEFs were transfected with wild-type (WT) or the cysteine mutant HMGB1-GFP plasmids as indicated, starved (HBSS) for 3 h in the presence or absence of 100 nM bafilomycin A1, and then immunostained with LAMP2-specific antibody/Alexa Fluor 594 secondary antibody (shown in red), LC3-specific antibody/Alexa Fluor 647 secondary antibody (shown in green), and Hoechst 33342 (shown in blue). Images were acquired digitally from a randomly selected pool of 10-15 fields under each condition. (B) Quantitative analysis of the percentage of LAMP2/LC3 colocalization was detected by Image-Pro Plus 5.1 software. Data are means ± SEM.


Figure 1 ( A ) Yeast two - hybrid assays to test for interaction between Munc18 - 1 and ERK1 / 2 . pGAL4 - Munc18 - 1 ( M18 ) and pGAL4 - Empty ( control ) were used as baits and ERK1 and ERK2 as prey . Both ERK1 and ERK2 bind Munc18 - 1 and allow growth on selection plates ( minus Histidine ( - HIS ) and minus Histidine and Adenosine ( - ADE ) ) . ( B ) In vitro kinase assays showing Munc18 - 1 phosphorylation by ERK . Radioactive phosphoblot ( 32P ) and immunoblot ( IB ) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT , Munc184A ( M18T78A , S158A , S241A , T574A ) , Munc183A ( M18S158A , S241A , T574A ) , and Munc18AA ( M18S241A , T574A ) from HEK293T cells incubated with recombinant ERK2 . The phosphoblot shows phosphorylated Munc18 - 1 ( pMunc18 - 1 ) and auto - phosphorylated ERK2 ( pERK2 ) . ( C ) Quantification of phospho - Munc18 - 1 intensity in wild - type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18 - 1 normalized to total Munc18 - 1 levels . Phosphorylation of Munc18WT was set to 100 % . ( Munc18WT : 100 ± 3 % ; Munc18AA : 24 ± 2 % , n = 3 , * p = 0 . 01 ) . ( D ) ERK1 in vitro kinase assay showing ERK - dependent 32P - incorporation in Munc18WT immuno - precipitated from HEK293T cells . Western Blot stained with Munc18 - 1 antibody shows total precipitated Munc18 - 1 levels were identical in all conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)).(B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01).(D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions.


Figure 2 ( A ) Experimental setup of fear conditioning paradigm . Mice placed in a fear - conditioning box received no shock ( NS ) , an immediate shock ( IS ) or delayed shock ( DS ) and were decapitated 30 min after returning to the home cage . Dorsal and ventral hippocampi were subjected to cellular fractionation ( P2 ) . DS and IS significantly increased the levels of phosphorylated ERK1 / 2 , a hallmark of ERK activation . ( B ) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group . ( D ) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241 , total Munc18 - 1 , pSynapsin , total synapsin , pERK and GAPDH as loading control . ( E ) Quantification Munc18 - 1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18 - 1 levels . No shock was set to 1 ( NS : 1 . 0 ± 0 . 19 , n = 5 ; DS : 4 . 01 ± 0 . 39 , n = 5 ; IS : 2 . 88 ± 0 . 37 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( F ) Quantification of Munc18 - 1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18 - 1 levels . ( NS : 1 . 0 ± 0 . 32 , n = 5 ; DS : 4 . 39 ± 0 . 55 , n = 5 ; IS : 3 . 62 ± 0 . 50 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( G ) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels . ( NS : 1 . 0 ± 0 . 31 , n = 5 ; DS : 1 . 88 ± 0 . 43 , n = 5 ; IS : 1 . 18 ± 0 . 34 , n = 5 ; n . s . ) . Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels . ( NS : 1 . 0 ± 0 . 11 , n = 5 ; DS : 3 . 51 ± 0 . 41 , n = 5 ; IS : 3 . 98 ± 0 . 24 , n = 5 ; * * p < 0 . 01 ) . ( H ) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels . ( NS : 1 . 0 ± 0 . 20 , n = 5 ; DS : 3 . 32 ± 0 . 46 , n = 5 ; IS : 3 . 30 ± 0 . 60 , n = 5 ; * * p < 0 . 01 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Experimental setup of fear conditioning paradigm. Mice placed in a fear- conditioning box received no shock (NS), an immediate shock (IS) or delayed shock (DS) and were decapitated 30 min after returning to the home cage. Dorsal and ventral hippocampi were subjected to cellular fractionation (P2). DS and IS significantly increased the levels of phosphorylated ERK1/2, a hallmark of ERK activation.(B) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group.(D) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241, total Munc18-1, pSynapsin, total synapsin, pERK and GAPDH as loading control.(E) Quantification Munc18-1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18-1 levels. No shock was set to 1 (NS: 1.0±0.19, n=5; DS: 4.01±0.39, n=5; IS: 2.88±0.37, n=5; **p<0.01; ***p<0.001).(F) Quantification of Munc18-1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18-1 levels. (NS: 1.0±0.32, n=5; DS: 4.39±0.55, n=5; IS: 3.62±0.50, n=5; **p<0.01; ***p<0.001).(G) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.31, n=5; DS: 1.88±0.43, n=5; IS: 1.18±0.34, n=5; n.s.). Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels. (NS: 1.0±0.11, n=5; DS: 3.51±0.41, n=5; IS: 3.98±0.24, n=5; **p<0.01).(H) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.20, n=5; DS: 3.32±0.46, n=5; IS: 3.30±0.60, n=5; **p<0.01).


Figure 3 ( A ) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 ( 10 M ) and DMSO , the GABAA receptor blocker GABAzine ( GBZ , 10 M ) or the voltage - gated sodium channel blocker TTX ( 2 M ) for 4 h . Blots were stained for pMunc18S241 , Munc18 - 1 and tubulin as loading control . ( B ) Quantification of ( A ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18 - 1 levels . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 4 % ; GBZ : 1 . 73 ± 0 . 12 ; TTX : 0 . 052 ± 0 . 002 , n = 4 , * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 ( 10 M ) and MEK inhibitor PD98059 ( 10 M ) for 4 h . Proteasome block increases the levels of phosphorylated Munc18 - 1 and ERK1 / 2 , which can be prevented by MEK inhibition . Blots were stained for pMunc18S241 , Munc18 , pERK1 / 2 and ERK1 / 2 . ( D ) Quantification of ( C ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18 - 1 levels . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 8 % ; MG132 : 375 ± 14 . 2 % ; MG132 + PD98059 : 24 ± 3 % , n = 3 , * * p < 0 . 01 ) . ( E ) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 ( 1 M ) for 5 min or BDNF ( 100ng / ml ) for 48 h in the presence of 2 M TTX and stained for pMunc18S241 , Munc18 , pERK and tubulin as loading control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01).(C) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01).(E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.


Figure 4Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( A ) Amplitude of excitatory synaptic response ( EPSC ) ( M18WT : 1 . 37 ± 0 . 21 nA , n = 35 ; M18S241A : 2 . 53 ± 0 . 31 nA , n = 45 ; M18S241D : 0 . 24 ± 0 . 05 nA , n = 17 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) . Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( B ) Percentage of cells that do not respond to electrical stimulation ( M18WT : 15 . 5 % ; M18S241A : 0 % ; M18S241D : 56 . 9 % ) . These cells are not included in ( A ) . Insert : representative recording of single EPSCs . Scale bar represents 10 ms and 0 . 5 nA . Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( C ) Typical examples of spontaneous vesicle release . Scale bar represents 0 . 1 s and 25 pA . ( D ) Spontaneous release frequency ( M18WT : 7 . 05 ± 1 . 28 Hz , n = 32 ; M18S241A : 14 . 26 ± 2 . 04 Hz , n = 37 ; M18S241D : 0 . 64 ± 0 . 29 Hz , n = 11 ; * * * p < 0 . 001 , * * p < 0 . 01 ) . ( E ) Amplitude of spontaneous release events ( M18WT : 15 . 40 ± 1 . 06 pA , n = 17 ; M18S241A : 15 . 03 ± 0 . 61 pA , n = 20 ; M18S241D : 10 . 98 ± 0 . 72 pA , n = 10 ; * * p < 0 . 01 ) . Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( F ) Size of the readily releasable pool ( RRP ) assessed with single hyperosmotic sucrose application ( 500 mM , 3 . 5 s , M18WT : 0 . 74 ± 0 . 15 nC , n = 7 ; M18S241A : 1 . 6 ± 0 . 33 nC , n = 10 ; M18S241D : could not be determined ; * p < 0 . 05 ) . Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( G ) Neurons were stimulated with two consecutive pulses with varying inter - stimulus interval to assess release probability . Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter - stimulus interval ( M18WT , n = 25 ; M18S241A , n = 19 ; M18S241D , n = 17 ) ; * p < 0 . 05 , * * p < 0 . 01 ) . All data are expressed as mean ± SEM . See also Figure EV2 and EV3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(A) Amplitude of excitatory synaptic response (EPSC) (M18WT: 1.37±0.21 nA, n=35; M18S241A: 2.53±0.31 nA, n=45; M18S241D: 0.24±0.05 nA, n=17, ***p<0.001, **p<0.01, *p<0.05).Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(B) Percentage of cells that do not respond to electrical stimulation (M18WT: 15.5%; M18S241A: 0%; M18S241D: 56.9%). These cells are not included in (A). Insert: representative recording of single EPSCs. Scale bar represents 10 ms and 0.5 nA.Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(C) Typical examples of spontaneous vesicle release. Scale bar represents 0.1 s and 25 pA.(D) Spontaneous release frequency (M18WT: 7.05 ± 1.28 Hz, n=32; M18S241A: 14.26 ± 2.04 Hz, n=37; M18S241D: 0.64 ± 0.29 Hz, n=11; ***p<0.001, **p<0.01).(E) Amplitude of spontaneous release events (M18WT: 15.40 ± 1.06 pA, n=17; M18S241A: 15.03 ± 0.61 pA, n=20; M18S241D: 10.98 ± 0.72 pA, n=10; **p<0.01).Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(F) Size of the readily releasable pool (RRP) assessed with single hyperosmotic sucrose application (500 mM, 3.5 s, M18WT: 0.74 ± 0.15 nC, n=7; M18S241A: 1.6 ± 0.33 nC, n=10; M18S241D: could not be determined; * p < 0.05).Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(G) Neurons were stimulated with two consecutive pulses with varying inter-stimulus interval to assess release probability. Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter-stimulus interval (M18WT, n=25; M18S241A, n=19; M18S241D, n=17); * p < 0.05, ** p < 0.01). All data are expressed as mean ± SEM. See also Figure EV2 and EV3.


Figure 5 ( A - F ) Munc18 - 1 protein levels were measured in glia free cultures of munc18 - 1null mutant neurons expressing M18WT , M18S241A or M18S241D . ( A ) Typical examples showing Munc18 - 1 protein localization and intensity in neurites of M18WT , M18S241A or M18S241Dneurons . MAP2 in blue , VAMP2 in red and Munc18 - 1 in green . Scale bar represents 2 m . ( B ) Mean somatic Munc18 - 1 intensity ( M18WT : 1682 ± 206 a . u . , n = 46 ; M18S241A : 1719 ± 238 a . u . , n = 36 ; M18S241D : 582 ± 98 a . u . , n = 33 ; * * * p < 0 . 001 ) . ( C ) Mean synapticMunc18 - 1 intensity ( M18WT : 1281 ± 161 a . u . ; M18S241A : 1390 ± 180 a . u . ; M18S241D : 210 ± 30 a . u . ; * * * p < 0 . 001 ) . ( D ) Mean synapticMunc18 - 1 intensity as a function of radial distance from the soma . ( A - F ) Munc18 - 1 protein levels were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( E ) Mean synaptic Munc18 - 1 intensity in cultures with 3 times the standard number of neurons to increase network activity ( M18WT : 1072 ± 123 a . u . , n = 36 ; M18S241A : 1480 ± 170 a . u . , n = 32 ; M18S241D : 251 ± 70 a . u . , n = 34 ; * p < 0 . 05 , * * * p < 0 . 001 ) . ( A - F ) Munc18 - 1 protein levels were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( F ) Mean synaptic Munc18 - 1 intensity in cultures treated with GABAA receptor antagonist Bicuculline ( 40 μM , 48 h ) . ( M18WT : 761 ± 121 a . u . , n = 26 ; M18S241A : 1482 ± 150 a . u . , n = 31 , * * p < 0 . 01 ) . ( G ) Mean synaptic syntaxin intensity in autaptic cultures ( M18WT : 875 ± 35 , n = 51 ; M18S241A : 1058 ± 42 , n = 46 ; M18S241D : 458 ± 19 , n = 40 , * * p < 0 . 01 , * * * p < 0 . 001 ) . ( H ) Western Blot of lysates from high - density neuronal cultures from munc18 - 1 null mutant mice rescued with M18WT , M18S241A or M18S241D . Prior to lysis , cells were treated for 6 h with MG132 ( 10 M ) or DMSO as control . Western Blots were stained for Munc18 - 1 and tubulin as loading control . ( I ) Quantification of ( H ) . Munc18 - 1 levels were normalized to tubulin levels . Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant ( M18WT : 1 . 62 ± 0 . 41 ; M18S241A : 1 . 26 ± 0 . 16 ; M18S241D : 7 . 53 ± 1 . 17 , n = 3 ; * * p < 0 . 01 ) . ( J ) Ubiquitination of different Munc18 - 1 mutants in HEK293T cells . Western Blot of denatured Munc18 - 1 immuno - precipitations from cells expressing Munc18 - 1 mutants and HA - tagged ubiquitin . Prior to lysis , cells were treated for 8 h with MG132 ( 10 M ) . Ubiquitin covalently attached to Munc18 - 1 can be observed as a smear above the characteristic 68 kD Munc18 - 1 band on Western blots stained for HA - tag . ( K ) Quantification of immuno - precipitation in ( J ) . Level of Munc18 - 1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18 - 1 levels . M18WT was set to 1 ( M18WT : 1 ± 0 . 06 ; M18S241A : 0 . 84 ± 0 . 57 ; M18S241D : 2 . 70 ± 0 . 07 , n = 3 ; * * p < 0 . 01 ) . All data are expressed as mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1null mutant neurons expressing M18WT, M18S241A or M18S241D.(A) Typical examples showing Munc18-1 protein localization and intensity in neurites of M18WT, M18S241A or M18S241Dneurons. MAP2 in blue, VAMP2 in red and Munc18-1 in green. Scale bar represents 2 m.(B) Mean somatic Munc18-1 intensity (M18WT: 1682 ± 206 a.u., n = 46; M18S241A: 1719 ± 238 a.u., n = 36; M18S241D: 582 ± 98 a.u., n = 33; *** p < 0.001).(C) Mean synapticMunc18-1 intensity (M18WT: 1281 ± 161 a.u.; M18S241A: 1390 ± 180 a.u.; M18S241D: 210 ± 30 a.u.; *** p < 0.001).(D) Mean synapticMunc18-1 intensity as a function of radial distance from the soma.(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(E) Mean synaptic Munc18-1 intensity in cultures with 3 times the standard number of neurons to increase network activity (M18WT: 1072 ± 123 a.u., n = 36; M18S241A: 1480 ± 170 a.u., n = 32; M18S241D: 251 ± 70 a.u., n = 34; * p < 0.05, *** p < 0.001).(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01).(G) Mean synaptic syntaxin intensity in autaptic cultures (M18WT: 875 ± 35, n = 51; M18S241A: 1058 ± 42, n = 46; M18S241D: 458 ± 19, n=40, ** p < 0.01, ***p<0.001).(H) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01).(J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.


Figure 6 ( A ) Western blot from whole cell lysate of munc18 - 1 null mutant neurons rescued with M18WT and incubated with WIN55 , 212 - 2 ( 5 M ) for 30 s . Cells were harvested after the indicated time . CB1R activation leads to a rapid and transient increase in pERK activity . ( B - D ) Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A . CB1R agonist WIN55 , 212 - 2 ( 5 M ) was applied at t = 60 s for 30 s . ( B ) Amplitude of excitatory synapse response normalized to t = 0 s . Neurons were stimulated by a single action potential every 30 s for 4 min ( M18WTn = 21 , M18S241An = 24 , * p < 0 . 05 ) . Note the broken y - axis . ( C ) Ratio of the frequency of spontaneous release events after ( t = 180 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 58 . 0 ± 2 . 3 % , n = 21 ; M18S241A : 84 . 2 ± 5 . 6 % , n = 24 , * p < 0 . 05 ) . ( D ) Ratio of amplitude of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 85 . 0 ± 2 . 7 % , n = 21 ; M18S241A : 83 . 1 ± 2 . 6 % , n = 24 ) . ( E - G ) Autaptic neurons of munc18 - 1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55 , 212 - 2 ( 1 M ) , WIN55 , 212 - 2 ( 1 M ) plus AM251 ( 1 M ) or vehicle only ( DMSO ) . ( E ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) or vehicle only ( DMSO ) were applied at t = 60 s for 30 s . ( WT + WIN n = 13 ; WT + DMSO n = 16 , * p < 0 . 05 ) . Note the broke Y - axis . ( F ) Paired pulse ratio ( 20 ms interval ) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55 , 212 - 2 ( 1 M ) at t = 60 s for 30 s . WIN55 , 212 - 2 application increases PP ratios indicative of a reduction of synaptic release probability ( t = 0 s : 0 . 94 ± 0 . 1 , n = 13 ; t = 240 s : 1 . 36 ± 0 . 2 , n = 16 , * p < 0 . 05 ) . ( G ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) was applied together with the CB1R antagonist AM251 ( 1 M ) at t = 60 s for 30 s . ( WT + WIN n = 13 , same trace as 6F ; WT + WIN + AM251 n = 15 , * p < 0 . 05 ) . Note the broke Y - axis . ( H - J ) Hippocampal autaptic neurons of munc18 - 1 null mutant mice were rescued with M18WT or M18S241A . mGluR2 / 3 agonist LY379268 ( 1 M ) was applied by bath - perfusion for 4 min . ( H ) Ratio of amplitude of evoked release events after ( t = 240 s ) and before ( t = 0 s ) LY379268 application ( M18WT : 55 . 0 ± 4 . 7 % , n = 14 ; M18S241A : 81 . 1 ± 3 . 6 % , n = 16 , * p < 0 . 05 ) . ( I ) Ratio of frequency of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) LY379268 application ( M18WT : 63 . 0 ± 5 . 7 % , n = 14 ; M18S241A : 85 . 1 ± 3 . 4 % , n = 16 , * p < 0 . 05 ) . ( J ) Ratio of the amplitude of spontaneous release events after ( t = 240 s ) and before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 81 . 0 ± 1 . 3 % , n = 14 ; M18S241A : 82 . 1 ± 3 . 3 % , n = 16 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Western blot from whole cell lysate of munc18-1 null mutant neurons rescued with M18WT and incubated with WIN55,212-2 (5 M) for 30 s. Cells were harvested after the indicated time. CB1R activation leads to a rapid and transient increase in pERK activity.(B-D) Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A. CB1R agonist WIN55,212-2 (5 M) was applied at t = 60 s for 30 s.(B) Amplitude of excitatory synapse response normalized to t=0 s. Neurons were stimulated by a single action potential every 30 s for 4 min (M18WTn = 21, M18S241An = 24, *p<0.05). Note the broken y-axis.(C) Ratio of the frequency of spontaneous release events after (t = 180 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 58.0 ± 2.3 %, n = 21; M18S241A: 84.2 ± 5.6 %, n = 24, * p < 0.05).(D) Ratio of amplitude of spontaneous release events after (t=240 s) and before (t=0 s) WIN55,212-2 application (M18WT: 85.0 ± 2.7 %, n = 21; M18S241A: 83.1 ± 2.6 %, n = 24).(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.(H-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).


Figure 7 ( A - D ) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre - treatment with PD98059 ( PD ; 10 µM ) compared to 0 . 1 % DMSO ( control ) . ( A ) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups ( DMSO : 196 . 36 + / - 16 . 51 pA , n = 10 ; PD : 196 . 42 + / - 18 . 61 pA , n = 11 , p = 0 . 9 ) . ( B ) DSE in neurons of DMSO pre - treated slices , induced by a 10s depolarization to 0 mV ( arrow ) . Inset : example traces of 2 averaged EPSCs before ( black ) and after depolarization ( grey ) . ( C ) DSE in slices pre - treated with PD . Insets : example traces of 2 averaged EPSCs before ( red ) and after depolarization ( grey ) . ( D ) Comparison of DSE between control and PD . PD pre - treatment limits amplitude and duration of DSE . * * = p < 0 . 01 ; unpaired T - test with Welch ' s correction . Numbers in bars represent the number of neurons in each group . ( E - H ) ERK - pathway inhibition by pre - treatment with PD98059 ( PD ; 10 µM ) blocks the effect of WIN55 , 212 - 2 ( WIN ; 2 µM ) on the amplitude of EPSCs recorded in CA1 pyramidal neurons . ( E ) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups ( DMSO : 295 . 69 + / - 28 . 73 pA , n = 6 ; PD : 270 . 86 + / - 32 . 14 pA , n = 6 , p = 0 . 6 ) . ( F ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of 0 . 1 % DMSO ( control ) pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( black ) and the last 5 min ( grey ) . ( G ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of PD pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( red ) and the last 5 minutes ( grey ) . ( H ) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference . * = p < 0 . 05 ; unpaired T - test . Numbers in bars represent number of slices in each group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A-D) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre-treatment with PD98059 (PD; 10 µM) compared to 0.1% DMSO (control).(A) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 196.36 +/- 16.51 pA, n = 10; PD: 196.42 +/- 18.61 pA, n = 11, p = 0.9).(B) DSE in neurons of DMSO pre-treated slices, induced by a 10s depolarization to 0 mV (arrow). Inset: example traces of 2 averaged EPSCs before (black) and after depolarization (grey).(C) DSE in slices pre-treated with PD. Insets: example traces of 2 averaged EPSCs before (red) and after depolarization (grey).(D) Comparison of DSE between control and PD. PD pre-treatment limits amplitude and duration of DSE. ** = p < 0.01; unpaired T-test with Welch's correction. Numbers in bars represent the number of neurons in each group.(E-H) ERK-pathway inhibition by pre-treatment with PD98059 (PD; 10 µM) blocks the effect of WIN55,212-2 (WIN; 2 µM) on the amplitude of EPSCs recorded in CA1 pyramidal neurons.(E) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 295.69 +/- 28.73 pA, n = 6; PD: 270.86 +/- 32.14 pA, n = 6, p= 0.6).(F) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of 0.1% DMSO (control) pre-treated slices. Inset: example traces of averaged EPSCs of baseline (black) and the last 5 min (grey).(G) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of PD pre-treated slices. Inset: example traces of averaged EPSCs of baseline (red) and the last 5 minutes (grey).(H) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference. * = p < 0.05; unpaired T-test. Numbers in bars represent number of slices in each group.


Figure 1 ( A ) Western blot analysis of the immunoproteasome subunits LMP2 , LMP7 and β - actin ( loading control ) in three PolgA + / + and three PolgAmut / mut cell lines . ( B ) RT - qPCR analysis of immunoproteasome subunits Psmb8 , Psmb9 , Psmb10 in three different PolgA + / + and three PolgAmut / mut cell lines . ( C ) Activity assay for the catalytic subunits of the proteasome using the pan activity - based probe ( ABP ) MV151 , the β1 / LMP2 specific LW124 , and the β5 / LMP7 specific MVB127 ABPs in three PolgA + / + and three PolgAmut / mut cell lines . Resolution of β5 / LMP7 in the SDS gel is hampered as both subunits are of similar molecular weight and thus do not separate in our gels . Total protein staining was used as a loading control .

O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Western blot analysis of the immunoproteasome subunits LMP2, LMP7 and β-actin (loading control) in three PolgA+/+ and three PolgAmut/mut cell lines.(B) RT-qPCR analysis of immunoproteasome subunits Psmb8, Psmb9, Psmb10 in three different PolgA+/+ and three PolgAmut/mut cell lines.(C) Activity assay for the catalytic subunits of the proteasome using the pan activity-based probe (ABP) MV151, the β1/LMP2 specific LW124, and the β5/LMP7 specific MVB127 ABPs in three PolgA+/+ and three PolgAmut/mut cell lines. Resolution of β5/LMP7 in the SDS gel is hampered as both subunits are of similar molecular weight and thus do not separate in our gels. Total protein staining was used as a loading control.


Figure 2 ( D ) representative Western blots for the analysis of the cGAS / STING signaling pathway in PolgA + / + and PolgAmut / mut cells with β - actin serving as loading control . ( E ) Secreted IFNβ in supernatants from three distinct PolgA + / + and PolgAmut / mut cell lines . The 15 . 81 pg / ml detected by ELISA in PolgAmut / mut cell supernatants corresponds to 18 . 97 IU / ml recombinant mouse ( rm ) IFN - β according to the R & D Systems cytokine conversion table ( F ) RT - qPCR analysis of Stat1 in three different PolgA + / + and three PolgAmut / mut cell lines .

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Figure 2(D) representative Western blots for the analysis of the cGAS/STING signaling pathway in PolgA+/+ and PolgAmut/mut cells with β-actin serving as loading control.(E) Secreted IFNβ in supernatants from three distinct PolgA+/+ and PolgAmut/mut cell lines. The 15.81 pg/ml detected by ELISA in PolgAmut/mut cell supernatants corresponds to 18.97 IU/ml recombinant mouse (rm) IFN-β according to the R&D Systems cytokine conversion table(F) RT-qPCR analysis of Stat1 in three different PolgA+/+ and three PolgAmut/mut cell lines.


Figure 3 ( A ) Western blot analysis of LMP2 , LMP7 , STAT1 and β - actin ( loading control ) in PolgA + / + cells after incubation with either PolgA + / + or PolgAmut / mut cell - conditioned medium for 48 h . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control set to 1 in PolgA + / + incubated with either PolgA + / + or PolgAmut / mut cell - conditioned medium from three different cell lines . ( B ) Western blot analysis of LMP2 , LMP7 , STAT1 , and β - actin ( loading control ) upon siRNA - mediated silencing of cGAS or STING in PolgAmut / mut cells compared to untransfected ( Ctrl ) and non - sense scrambled transfection control ( NC ) in three independent experiments . ( E ) Western blot analysis of LMP2 , LMP7 , STAT1 , and β - actin ( loading control ) in PolgA + / + cells transfected with mouse wildtype mtDNA , mtDNA pretreated with DNase I or only DNase I for 48 h . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control in PolgA + / + cells . Values are given as fold over PolgA + / + control with the mean of normalized PolgA + / + signal normalized to 1 . ( F ) Western blot analysis of LMP2 , LMP7 , STAT1 and β - actin ( loading control ) in PolgA + / + cells transfected with cGAMP for 24 h . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control ( Ctrl ) set to 1 . ( G ) Protein expression of LMP2 , LMP7 and β - actin ( loading control ) in wildtype ( WT ) , cGAS KO or STINGgt / gt losss of function MEFs that had been transfected with 200 ng / ml mtDNA for 48 h .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Western blot analysis of LMP2, LMP7, STAT1 and β-actin (loading control) in PolgA+/+ cells after incubation with either PolgA+/+ or PolgAmut/mut cell-conditioned medium for 48 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control set to 1 in PolgA+/+ incubated with either PolgA+/+ or PolgAmut/mut cell-conditioned medium from three different cell lines.(B) Western blot analysis of LMP2, LMP7, STAT1, and β-actin (loading control) upon siRNA-mediated silencing of cGAS or STING in PolgAmut/mut cells compared to untransfected (Ctrl) and non-sense scrambled transfection control (NC) in three independent experiments.(E) Western blot analysis of LMP2, LMP7, STAT1, and β-actin (loading control) in PolgA+/+ cells transfected with mouse wildtype mtDNA, mtDNA pretreated with DNase I or only DNase I for 48 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control in PolgA+/+ cells. Values are given as fold over PolgA+/+ control with the mean of normalized PolgA+/+ signal normalized to 1. (F) Western blot analysis of LMP2, LMP7, STAT1 and β-actin (loading control) in PolgA+/+ cells transfected with cGAMP for 24 h. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control (Ctrl) set to 1.(G) Protein expression of LMP2, LMP7 and β-actin (loading control) in wildtype (WT), cGAS KO or STINGgt/gt losss of function MEFs that had been transfected with 200 ng/ml mtDNA for 48 h.


Figure 5 ( B ) UTY - assay of CD8 + reporter T cells upon co - culture with different ratios of PolgA + / + or PolgAmut / mut cells . Data are combined results of three independent experiments and normalized to the signal of maximum induction of PolgA + / + cells in co - culture with CD8 + reporter T cells . ( E ) UTY - assay of CD8 + reporter T cells upon 1 : 2 co - culture ( splenocytes : T cells ) with splenocytes isolated from either WT or LMP7 KO mice and transfected with mtDNA . Splenocytes were isolated from 4 mice each in two different preparations .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) UTY-assay of CD8+ reporter T cells upon co-culture with different ratios of PolgA+/+ or PolgAmut/mut cells. Data are combined results of three independent experiments and normalized to the signal of maximum induction of PolgA+/+ cells in co-culture with CD8+ reporter T cells.(E) UTY-assay of CD8+ reporter T cells upon 1:2 co-culture (splenocytes:T cells) with splenocytes isolated from either WT or LMP7 KO mice and transfected with mtDNA. Splenocytes were isolated from 4 mice each in two different preparations.


Figure 6 ( A ) Western blot analysis of immunoproteasome subunits LMP2 , LMP7 , and of STAT1 in pmAT2 cells transfected with mtDNA or mtDNA pretreated with DNase I for 48 h or treated with IFNβ for 24 h . β - actin was used as a loading control . Densitometric analysis of LMP2 , LMP7 and STAT1 expression normalized to β - actin loading control in pmAT2 from three independent pmAT2 preparations . ( C ) Representative Western blot analysis of phLF for LMP2 , LMP7 , STAT1 , and β - actin ( loading control ) upon transfection of mtDNA or mtDNA pretreated with DNase I for 24 h , 48 h or 72 h . ( G ) Western blot analysis of STING , pTBK1 , TBK1 , pIRF3 , IRF3 , pIκBα , IκBα , pP65 , P65 and β - actin ( loading control ) in phLFs transfected with mtDNA for 2 h , 6 h , or 24 h , respectively .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Western blot analysis of immunoproteasome subunits LMP2, LMP7, and of STAT1 in pmAT2 cells transfected with mtDNA or mtDNA pretreated with DNase I for 48 h or treated with IFNβ for 24 h. β-actin was used as a loading control. Densitometric analysis of LMP2, LMP7 and STAT1 expression normalized to β-actin loading control in pmAT2 from three independent pmAT2 preparations.(C) Representative Western blot analysis of phLF for LMP2, LMP7, STAT1, and β-actin (loading control) upon transfection of mtDNA or mtDNA pretreated with DNase I for 24 h, 48 h or 72 h.(G) Western blot analysis of STING, pTBK1, TBK1, pIRF3, IRF3, pIκBα, IκBα, pP65, P65 and β-actin (loading control) in phLFs transfected with mtDNA for 2 h, 6 h, or 24 h, respectively.


Figure 7 ( B ) Flow cytometric detection of Influenza - M1 / HLA - A2 complex in phLF upon transfection with mtDNA , mtDNA pretreated with DNase I , or treatment with IFNβ ( n = 3 independent experiments ) . ( D ) CD8 + T cell activation assay of Influenza - M1 / HLA - A2 restricted CD8 + T cells upon co - culture with mtDNA - transfected phLFs loaded with M1 peptide or upon treatment with IFNβ or IFNγ displaying secretion of IL - 2 from three different biological experiments .

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Figure 7(B) Flow cytometric detection of Influenza-M1/HLA-A2 complex in phLF upon transfection with mtDNA, mtDNA pretreated with DNase I, or treatment with IFNβ (n=3 independent experiments).(D) CD8+ T cell activation assay of Influenza-M1/HLA-A2 restricted CD8+ T cells upon co-culture with mtDNA-transfected phLFs loaded with M1 peptide or upon treatment with IFNβ or IFNγ displaying secretion of IL-2 from three different biological experiments.


Figure 8 ( A ) Western blot analysis of the immunoproteasome subunits LMP2 and LMP7 in wild type ( WT ) , cGAS KO and STINGgt / gt loss of function MEFs transfected with 1 μg / ml HT - DNA or plasmid - DNA for 48 h . β - actin was used as a loading control . Densitometric analysis of LMP2 and LMP7 expression normalized to β - actin loading control in wild type ( WT ) controls ( ctrl ) from three independent experiments . ( B ) UTY - assay of CD8 + reporter T cells upon 1 : 2 co - culture ( splenocytes : T cells ) with splenocytes isolated from either wildtype ( WT ) or LMP7 KO mice and transfected with htDNA . Splenocytes were isolated from 4 mice each in two different preparations . ( C ) Western blot analysis of immunoproteasome subunits LMP2 and LMP7 in wild type ( WT ) , heterozygous STAT1 mutated ( STAT1 * + / - ) and homozygous STAT1 mutated ( STAT1 * - / - ) skin fibroblasts transfected with 1 μg / ml HT - DNA or pretreated HT - DNA with DNase I for 48 h .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Western blot analysis of the immunoproteasome subunits LMP2 and LMP7 in wild type (WT), cGAS KO and STINGgt/gt loss of function MEFs transfected with 1 μg/ml HT-DNA or plasmid-DNA for 48 h. β-actin was used as a loading control. Densitometric analysis of LMP2 and LMP7 expression normalized to β-actin loading control in wild type (WT) controls (ctrl) from three independent experiments.(B) UTY-assay of CD8+ reporter T cells upon 1:2 co-culture (splenocytes:T cells) with splenocytes isolated from either wildtype (WT) or LMP7 KO mice and transfected with htDNA. Splenocytes were isolated from 4 mice each in two different preparations.(C) Western blot analysis of immunoproteasome subunits LMP2 and LMP7 in wild type (WT), heterozygous STAT1 mutated (STAT1*+/-) and homozygous STAT1 mutated (STAT1*-/-) skin fibroblasts transfected with 1 μg/ml HT-DNA or pretreated HT-DNA with DNase I for 48 h.


Figure 9 ( D - E ) Representative immunofluorescence detection of LMP2 ( red ) , CD8 + ( green ) and PD - 1 ( red ) in control and IPF lungs , arrows indicate double positive CD8 + PD - 1 + cells , magnifications 10X , 20X and 40X correspond to a scale bar of 100 µm . Arrows indicate epithelial cells ( E ) or macrophages ( M ) and cells stained positive for CD8 . ( F ) Flow cytometric analysis of CD3 , CD8 , and CCR7 expression on peripheral blood mononuclear cells ( PBMCs ) of control ( n = 21 ) and IPF ( n = 66 ) patients . Data are presented as gated % of parent . Data were analyzed with two - tailed unpaired Student ' s t - test . Asterisks indicate significance as * P < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(D-E) Representative immunofluorescence detection of LMP2 (red), CD8+ (green) and PD-1(red) in control and IPF lungs, arrows indicate double positive CD8+ PD-1+ cells, magnifications 10X, 20X and 40X correspond to a scale bar of 100 µm. Arrows indicate epithelial cells (E) or macrophages (M) and cells stained positive for CD8.(F) Flow cytometric analysis of CD3, CD8, and CCR7 expression on peripheral blood mononuclear cells (PBMCs) of control (n=21) and IPF (n=66) patients. Data are presented as gated % of parent. Data were analyzed with two-tailed unpaired Student's t-test. Asterisks indicate significance as *P<0.05.


Figure 1 ( C , D ) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs . Data are presented as mean ± SD . n = 3 ; * , p < 0 . 05 ; * * * , p < 0 . 001 unpaired Student ' s t - test . ( G ) Immunoblot of SCOT , BDH1 , CD31 and VCP in cardiac endothelial cells ( EC ) and non - endothelial cell fraction ( non - EC ) isolated from adult C57Bl / 6J mice .

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Figure 1(C, D) Quantification of SCOT and BDH1 expression levels relative to VCP in various murine organs. Data are presented as mean ± SD. n=3; *, p<0.05; ***, p<0.001 unpaired Student's t-test.(G) Immunoblot of SCOT, BDH1, CD31 and VCP in cardiac endothelial cells (EC) and non-endothelial cell fraction (non-EC) isolated from adult C57Bl/6J mice.


Figure 2 ( A , B ) Murine cardiac endothelial cells ( MCEC ) were treated with 2 mM D - β - hydroxybutyrate ( β - OHB ) or 2 mM acetoacetate ( AcAc ) for 24 hours . Quantification of concentrations of TCA cycle intermediates compared to control treatment . Data are presented as mean ± SD . n ≥ 4 ; ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test . ( D ) Quantifications of [ 13C ] - labeled fractions of amino acids ( asparagine , L - aspartate , L - glutamate , L - proline ) , putative lipid species ( nervonic acid , 1 - palmitoylglycerol , ceramide ( d18 : 1 / 16 : 0 ) ) and uridine diphosphate ( UDP ) species ( uridine monophosphate , uridine 5 ' - diphosphate , UDP - N - acetylglucosamine ) in MCEC treated with 1 mM 13C4 - β - hydroxybutyrate or 1 mM 13C4 - acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ( [ 12C ] isotope ) . Data are presented as mean ± SD . n ≥ 4 ; ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

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Figure 2(A, B) Murine cardiac endothelial cells (MCEC) were treated with 2 mM D-β-hydroxybutyrate (β-OHB) or 2 mM acetoacetate (AcAc) for 24 hours. Quantification of concentrations of TCA cycle intermediates compared to control treatment. Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.(D) Quantifications of [13C]-labeled fractions of amino acids (asparagine, L-aspartate, L-glutamate, L-proline), putative lipid species (nervonic acid, 1-palmitoylglycerol, ceramide (d18:1/16:0)) and uridine diphosphate (UDP) species (uridine monophosphate, uridine 5'-diphosphate, UDP-N-acetylglucosamine) in MCEC treated with 1 mM 13C4-β-hydroxybutyrate or 1 mM 13C4-acetoacetate for 24 hours or the same concentration of unlabeled ketone bodies ([12C] isotope). Data are presented as mean ± SD. n≥4; ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


Figure 3 ( A , B ) Murine cardiac endothelial cells ( MCEC ) were treated with different concentrations of D - β - hydroxybutyrate ( βOHB ) or acetoacetate ( AcAc ) for 24 hours . Relative BrdU absorbance was quantified compared to control treatment ( H2O for βOHB and EtOH for AcAc ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test . ( I - M ) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid - based sprouting assay . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) , 10 mM acetate , 1 mM octanoate , 1 mM butyrate , 30 mM L - β - hydroxybutyrate ( L - βOHB ) , 1 mM niacin or the respective controls ( final concentration of reagents is diluted to approximately 10 % ) and analyzed after 48 hours . The ( I , L ) average number of sprouts per spheroid , the ( J , M ) accumulated total sprout length and ( K ) average sprout length were quantified . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Murine cardiac endothelial cells (MCEC) were treated with different concentrations of D-β-hydroxybutyrate (βOHB) or acetoacetate (AcAc) for 24 hours. Relative BrdU absorbance was quantified compared to control treatment (H2O for βOHB and EtOH for AcAc). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.(I-M) Angiogenic capacity of MCEC in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc), 10 mM acetate, 1 mM octanoate, 1 mM butyrate, 30 mM L-β-hydroxybutyrate (L- βOHB), 1 mM niacin or the respective controls (final concentration of reagents is diluted to approximately 10 %) and analyzed after 48 hours. The (I, L) average number of sprouts per spheroid, the (J, M) accumulated total sprout length and (K) average sprout length were quantified. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


Figure 4 ( B , Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid - based sprouting assay . Spheroids were treated with media containing 30 mM D - β - hydroxybutyrate ( βOHB ) , 30 mM acetoacetate ( AcAc ) or the respective controls ( final concentration of reagents is diluted to approximately 10 % ) for 48 hours . The average number of sprouts per spheroid was quantified . Data are presented as mean ± SD . n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; unpaired Student ' s t - test .

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Figure 4(B, Angiogenic capacity of Oxct1 knockout cells in response to ketone body treatment was analyzed using a spheroid-based sprouting assay. Spheroids were treated with media containing 30 mM D-β-hydroxybutyrate (βOHB), 30 mM acetoacetate (AcAc) or the respective controls (final concentration of reagents is diluted to approximately 10 %) for 48 hours. The average number of sprouts per spheroid was quantified. Data are presented as mean ± SD. n=4; *, p<0.05; **, p<0.01; unpaired Student's t-test.


Figure 5 ( C , D ) Heatmaps showing expression levels of genes involved in KEGG pathways ( C ) fatty acid degradation and ( D ) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl / 6J mice that were fed a ketogenic diet or control diet for two weeks .

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Figure 5(C, D) Heatmaps showing expression levels of genes involved in KEGG pathways (C) fatty acid degradation and (D) ketone body metabolism in cardiac endothelial cells isolated from male C57Bl/6J mice that were fed a ketogenic diet or control diet for two weeks.


Figure 6 ( A , B ) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU , double positive cells are indicated by arrowheads , and quantification of double positive cells in heart sections per high power field ( HPF ) . Scale bar : 50 μm . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test . ( F ) Quantification of Ki67 + endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks ( gastrocnemius and soleus muscle ) or two weeks ( brain , lungs , subcutaneous adipose tissue , liver ) . Data are presented as mean ± SD . n ≥ 3 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 unpaired Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B) Representative images of heart sections of animals kept on a ketogenic diet or control diet for two weeks stained against ERG and Ki67 or EdU, double positive cells are indicated by arrowheads, and quantification of double positive cells in heart sections per high power field (HPF). Scale bar: 50 μm. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.(F) Quantification of Ki67+ endothelial cells in several organs of mice kept on a ketogenic diet or control diet for four weeks (gastrocnemius and soleus muscle) or two weeks (brain, lungs, subcutaneous adipose tissue, liver). Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.


Figure 7 ( B ) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG . Scale bar : 50 µm .

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Figure 7(B) Representative images of heart sections of mice kept on a control or ketogenic diet eight weeks after TAC surgery stained against Ki67 and ERG. Scale bar: 50 µm.


Figure 1B . Normalized number of filaments nucleated over time , from SPIN90 - Arp2 / 3 exposed to 2 μΜ G - actin ( 15 % labeled with Alexa488 ) and 1 μΜ profilin , with 0 or 0 . 5 µM of GST - VCA from different NPFs . Solid lines are exponential fits , yielding nucleation rates knuc = ( 1 . 06 ± 0 . 03 ) х10 - 3 s - 1 without VCA , and knuc = ( 3 . 23 ± 0 . 08 ) х10 - 3 , ( 2 . 58 ± 0 . 05 ) х10 - 3 , and ( 2 . 28 ± 0 . 03 ) х10 - 3 s - 1 with VCA from N - WASP , WASP and WASH , respectively .

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Figure 1B. Normalized number of filaments nucleated over time, from SPIN90-Arp2/3 exposed to 2 μΜ G-actin (15% labeled with Alexa488) and 1 μΜ profilin, with 0 or 0.5 µM of GST-VCA from different NPFs. Solid lines are exponential fits, yielding nucleation rates knuc=(1.06±0.03)х10-3 s-1 without VCA, and knuc=(3.23±0.08)х10-3, (2.58±0.05)х10-3, and (2.28±0.03)х10-3 s-1 with VCA from N-WASP, WASP and WASH, respectively.