( G ) The number of apoptotic SGN per section is significantly increased in E13 . 5 and E14 . 5 Elp3cKO compared to WT cochleae , suggesting SGNs are properly specified but die during their differentiation process ( n = 3 - 4 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 ; * * p < 0 . 01 ; F = 23 . 74 ; DF = 7 ; mean ± SD ) .

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(G) The number of apoptotic SGN per section is significantly increased in E13.5 and E14.5 Elp3cKO compared to WT cochleae, suggesting SGNs are properly specified but die during their differentiation process (n=3-4 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; **p<0.01; F=23.74; DF=7; mean±SD).


( A ) Proteostat staining of WT and Elp3cKO E13 . 5 cochlear sections reveals the presence of numerous aggresome - like structures in Elp3 - deficient SGNs . Scale bar = 100µm .

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(A) Proteostat staining of WT and Elp3cKO E13.5 cochlear sections reveals the presence of numerous aggresome-like structures in Elp3-deficient SGNs. Scale bar =100µm.


( B ) Transmission electron micrographs from E13 . 5 Elp3cKO SGNs confirm the presence of apoptotic neurons ( marked by a red arrowhead ) and a large electron dense juxtanuclear structures typical of aggresome ( dashed circle ) . Scale bars = 10µm and 2µm ( last panel ) .

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(B) Transmission electron micrographs from E13.5 Elp3cKO SGNs confirm the presence of apoptotic neurons (marked by a red arrowhead) and a large electron dense juxtanuclear structures typical of aggresome (dashed circle). Scale bars =10µm and 2µm (last panel).


( C ) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae ( n = 8 / 6 for WT / KO ; unpaired t - test two - tailed ; p = 0 . 0001 / 0 . 0015 / 0 . 0010 ; t = 9 . 535 / 4 . 090 / 4 . 307 ; DF = 12 / 12 / 12 for Elp3 , Chop and Chac1 respectively ; mean ± SEM presented ) indicating that the pro - apoptotic arm of the unfolded protein response ( UPR ) is activated in Elp3 - deficient SGNs .

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(C) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae (n=8/6 for WT/KO; unpaired t-test two-tailed; p=0.0001/0.0015/0.0010; t=9.535/4.090/4.307; DF=12/12/12 for Elp3, Chop and Chac1 respectively; mean±SEM presented) indicating that the pro-apoptotic arm of the unfolded protein response (UPR) is activated in Elp3-deficient SGNs.


( D - E ) Proteostat staining of whole - mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3 . Orthogonal slices ( Orthog ) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs . Scale bars = 5µm . # IHC ; * OHC1 ; * * OHC2 ; * * * OHC3 .

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(D-E) Proteostat staining of whole-mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs. Scale bars =5µm. #IHC; *OHC1; **OHC2; ***OHC3.


( F ) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3 , confirming proteostasis disruption .

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(F) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3, confirming proteostasis disruption.


( G ) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti ( n = 3 animals per genotype ; unpaired t - test two - tailed ; p = 0 . 0003 / 0 . 4296 / 0 . 056 ; t = 11 . 62 / 0 . 8778 / 2 . 662 ; DF = 4 / 4 / 4 for Elp3 , Chop and Chac1 respectively ; mean ± SEM presented ) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR - mediated apoptosis .

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(G) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti (n=3 animals per genotype; unpaired t-test two-tailed; p=0.0003/0.4296/0.056; t=11.62/0.8778/2.662; DF=4/4/4 for Elp3, Chop and Chac1 respectively; mean±SEM presented) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR-mediated apoptosis.


( A ) Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity , as misaligned HCs and dysmorphic stereociliary bundles are visible ( red arrows and white arrowheads , respectively ) . Scale bars =

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

(A) Scanning electron micrographs of cochlear tissue from P19 animals indicate no HC loss in the absence of Elp3 but illustrate some defects of polarity, as misaligned HCs and dysmorphic stereociliary bundles are visible (red arrows and white arrowheads, respectively). Scale bars=5µ


( B ) Surface view of P1 actin - stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio - lateral axis ( M - L ) or misshaped . Scale bars = 5µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Surface view of P1 actin-stained organ of Corti confirms defective establishment of polarity in Elp3cKO as numerous stereociliary bundles are misaligned along the medio-lateral axis (M-L) or misshaped. Scale bars=5µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3


( C ) Elp3cKOs exhibit a broader distribution of hair cell orientation . The angles of deviation between the stereociliary bundle and the medio - lateral axis are plotted in rose diagrams , for each OHC row . Whereas the majority of WT bundles are pointing to the most lateral region of the tissue ( within a range of 10 ° ) , many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation ( WT / Elp3cKO OHC1 : n = 99 / 99 , p > 0 . 1 , DF = 99 ; OHC2 : n = 99 / 100 , p < 0 . 001 , DF = 99 ; OHC3 : n = 99 / 98 , p < 0 . 001 , DF = 98 ; pooled data from 6 animals per genotype ; Watson U2 test ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

(C) Elp3cKOs exhibit a broader distribution of hair cell orientation. The angles of deviation between the stereociliary bundle and the medio-lateral axis are plotted in rose diagrams, for each OHC row. Whereas the majority of WT bundles are pointing to the most lateral region of the tissue (within a range of 10°), many Elp3cKO bundles of the two last rows of OHCs present increased angles of deviation (WT/Elp3cKO OHC1: n=99/99, p>0.1, DF=99; OHC2: n=99/100, p<0.001, DF=99; OHC3: n=99/98, p<0.001, DF=98; pooled data from 6 animals per genotype; Watson U2 test). Data information: #IHC; *OHC1; **OHC2; ***OHC3


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( D ) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae ( scale bar = 2µm ) illustrating the misshaped stereociliary bundles and the shorter kinocilium ( false coloured in pink ) of Elp3cKO HCs . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (D) Scanning electron micrographs of HCs from P1 WT and Elp3cKO cochleae (scale bar=2µm) illustrating the misshaped stereociliary bundles and the shorter kinocilium (false coloured in pink) of Elp3cKO HCs. Data information: #IHC; *OHC1; **OHC2; ***OHC3


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( E ) Graph presents the mean angle of HC bundle ( n = 6 animals ; one - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , F = 13 . 69 , DF = 7 ; mean ± SD ) .

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Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (E) Graph presents the mean angle of HC bundle (n=6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, F=13.69, DF=7; mean±SD).


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( F ) Elp3cKO HCs possess a significantly larger bare zone ( WT / KO IHC : n = 23 / 24 cells ; OHC1 : n = 27 / 27 ; OHC2 : n = 27 / 29 ; OHC3 : n = 30 / 29 ; cumulative data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , * p < 0 . 05 F = 16 . 72 , DF = 7 ; mean ± SD ) .

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Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (F) Elp3cKO HCs possess a significantly larger bare zone (WT/KO IHC: n= 23/24 cells; OHC1: n=27/27; OHC2: n=27/29; OHC3: n=30/29; cumulative data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, *p<0.05 F=16.72, DF=7; mean±SD).


Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted . ( G ) Elp3cKO HCs possess a significantly smaller medial zone ( WT / KO IHC : n = 23 / 24 cells ; OHC1 : n = 27 / 27 ; OHC2 : n = 27 / 29 ; OHC3 : n = 27 / 29 ; cumulative data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test ; * * * p < 0 . 001 , * p < 0 . 05 , F = 63 . 3 , DF = 7 ; mean ± SD ) .

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Elp3cKOs HCs harbour a flatter stereociliary bundle that is centrally shifted. (G) Elp3cKO HCs possess a significantly smaller medial zone (WT/KO IHC: n= 23/24 cells; OHC1: n=27/27; OHC2: n=27/29; OHC3: n=27/29; cumulative data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test; ***p<0.001, *p<0.05, F=63.3, DF=7; mean±SD).


( H ) Kinocilia are shorter in Elp3cKO HCs ( WT / KO IHC : n = 15 / 15 ; OHC1 : n = 30 / 32 ; OHC3 : n = 31 / 30 ; OHC3 : n = 27 / 30 ; data collected from 3 animals per genotype ; One - way ANOVA , Tukey ' s multiple comparisons test , * * * p < 0 . 001 , * * p < 0 . 01F = 19 . 6 , DF = 7 ; mean ± SD ) .

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(H) Kinocilia are shorter in Elp3cKO HCs (WT/KO IHC: n=15/15; OHC1: n=30/32; OHC3: n=31/30; OHC3: n=27/30; data collected from 3 animals per genotype; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, **p<0.01F=19.6, DF=7; mean±SD).


( I ) High magnification of representative HCs from P1 WT and Elp3cKO cochlea stained for F - actin and gamma - tubulin ( white ) . The basal body of kinocilia is shifted centrally at the apical surface of Elp3cKO HCs . Scale bars = 2µm . Polar plotting of kinocilium basal body position for each row of HCs of WT and Elp3cKO ( WT / KO IHC : n = 42 / 32 ; OHC1 : n = 47 / 48 ; OHC2 : n = 47 / 45 ; OHC3 : n = 44 / 44 ; cumulative data collected from 3 animals per genotype ) . Cellular orientations were tested using Watson U2 test ( p < 0 . 001 between WT / Elp3cKO , for all HC rows ) and the mean vector lengths , determined from the cell centre to the basal body , were compared using t - tests ( p < 0 . 001 between WT / Elp3cKO , for all HC rows ) .

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(I) High magnification of representative HCs from P1 WT and Elp3cKO cochlea stained for F-actin and gamma-tubulin (white). The basal body of kinocilia is shifted centrally at the apical surface of Elp3cKO HCs. Scale bars=2µm. Polar plotting of kinocilium basal body position for each row of HCs of WT and Elp3cKO (WT/KO IHC: n= 42/32; OHC1: n= 47/48; OHC2: n= 47/45; OHC3: n= 44/44; cumulative data collected from 3 animals per genotype). Cellular orientations were tested using Watson U2 test (p<0.001 between WT/Elp3cKO, for all HC rows) and the mean vector lengths, determined from the cell centre to the basal body, were compared using t-tests (p<0.001 between WT/Elp3cKO, for all HC rows).


The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with LGN ( J ) and F - actin . The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with LGN (J) and F-actin. The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with Gαi3 ( K ) and F - actin . The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with Gαi3 (K) and F-actin. The enrichment of these apical polarity regulators at the lateral edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


The localization of apical polarity proteins is affected by Elp3 loss . Surface views of mid - basal turn of P1 cochleae from WT or Elp3cKO mice stained with aPKC ( L ) and F - actin . The enrichment of these apical polarity regulators at the medial ( L ) edges of HCs is disrupted in Elp3cKO HCs . Scale bars = 5µm ( upper panels ) and 2µm ( lower panels ) . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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The localization of apical polarity proteins is affected by Elp3 loss. Surface views of mid-basal turn of P1 cochleae from WT or Elp3cKO mice stained with aPKC (L) and F-actin. The enrichment of these apical polarity regulators at the medial (L) edges of HCs is disrupted in Elp3cKO HCs. Scale bars=5µm (upper panels) and 2µm (lower panels). Data information: #IHC; *OHC1; **OHC2; ***OHC3


( A - B ) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown . ( A ) HEK293T cells transfected with siRNAs for Elp3 , Alkbh8 or control and immunolabelled with LGN , acetylated alpha - tubulin and DAPI . Scale bar = 5µm . ( B ) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified ( siRNA Control / Elp3 / Alkbh8 : n = 27 / 30 / 27 ; pooled from 3 independent experiments ; mean ± SEM ) .

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(A-B) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown. (A) HEK293T cells transfected with siRNAs for Elp3, Alkbh8 or control and immunolabelled with LGN, acetylated alpha-tubulin and DAPI. Scale bar=5µm. (B) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified (siRNA Control/Elp3/Alkbh8: n=27/30/27; pooled from 3 independent experiments; mean±SEM).


( C - D ) The length of the primary cilium is reduced in non - dividing HEK293T cells upon Elp3 or Albh8 knockdown . ( C ) Control , Elp3 or Alkbh8 siRNA - treated HEK293T cells were immunolabelled for basal body marker ALMS1 , axoneme marker Arl13b and DAPI . Scale bar = 5µm . ( D ) Primary cilium length of non - dividing HEK293T was measured and pooled data were plotted ( siRNA Control / Elp3 / Alkbh8 : n = 94 / 81 / 90 ; data collected from 3 independent experiments ; One - way ANOVA , Tukey ' s multiple comparisons test , * * * p < 0 . 001 , F = 21 . 46 , DF = 4 ; minimum , first quartile , median , third quartile and maximum presented ) .

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(C-D) The length of the primary cilium is reduced in non-dividing HEK293T cells upon Elp3 or Albh8 knockdown. (C) Control, Elp3 or Alkbh8 siRNA-treated HEK293T cells were immunolabelled for basal body marker ALMS1, axoneme marker Arl13b and DAPI. Scale bar=5µm. (D) Primary cilium length of non-dividing HEK293T was measured and pooled data were plotted (siRNA Control/Elp3/Alkbh8: n=94/81/90; data collected from 3 independent experiments; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, F=21.46, DF=4; minimum, first quartile, median, third quartile and maximum presented).


( E - F ) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells ( n = 3 ; One - way ANOVA , Tukey ' s multiple comparisons test , * * p < 0 . 01 , * p < 0 . 05 , F = 16 . 51 , DF = 2 ; mean ± SD ) .

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(E-F) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells (n=3; One-way ANOVA, Tukey's multiple comparisons test, **p<0.01, *p<0.05, F=16.51, DF=2; mean±SD).


( G ) Increased formation of aggresome - like structures in Elp3 - or Alkbh8 - depleted HEK293T . Aggresomes ( red ) ; DAPI ( blue ) . Enlarged images depict examples of non - dividing ( left ) and dividing ( right ) cells . Scale bar = 10µm .

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(G) Increased formation of aggresome-like structures in Elp3- or Alkbh8-depleted HEK293T. Aggresomes (red); DAPI (blue). Enlarged images depict examples of non-dividing (left) and dividing (right) cells. Scale bar=10µm.


Inducing aggresomes with MG - 132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells . ( H ) HEK293T cells treated with DMSO vehicle or MG - 132 and stained for aggresomes ( left panels ) or LGN , F - actin and DAPI ( green , red and blue , respectively in right panels ) . Scale bar = 10µm .

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Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (H) HEK293T cells treated with DMSO vehicle or MG-132 and stained for aggresomes (left panels) or LGN, F-actin and DAPI (green, red and blue, respectively in right panels). Scale bar=10µm.


Inducing aggresomes with MG - 132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells . ( I ) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells . Mean fluorescence values per percentage of cell length with SEM are plotted . Fluorescence intensity profiles collected from 3 independent experiments ( DMSO / MG - 132 : n = 24 / 24 ) .

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Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (I) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells. Mean fluorescence values per percentage of cell length with SEM are plotted. Fluorescence intensity profiles collected from 3 independent experiments (DMSO/MG-132: n= 24/24).


( J ) Proteostat staining of MG132 - treated whole - mounted P0 organs of Corti reveals the presence of aggresome - like structures in HCs . Orthogonal slices ( Orthog ) reveal aggregated proteins localize at the apical surface of MG132 - treated HCs . Scale bars = 5µm . # IHC ; * OHC1 ; * * OHC2 ; * * *

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(J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3


( K ) Localization of LGN at the lateral apical surface of E16 . 5 WT HCs is disrupted by MG - 132 . LGN ( white / green ) and F - actin ( red ) . Scale bars = 5µm ; magnified views = 2µm .

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(K) Localization of LGN at the lateral apical surface of E16.5 WT HCs is disrupted by MG-132. LGN (white/green) and F-actin (red). Scale bars=5µm; magnified views=2µm.


LGN and Gαi3 protein levels are not affected by Elp3 loss . ( A ) Western blots with total lysates from control and Elp3 siRNA - treated HEK293T cells and corresponding quantifications of normalized LGN , Gαi3 and Elp3 levels ( n = 6 from 2 independent cultures ; Unpaired two - tailed t - test , LGN : p = 0 . 749 , t = 0 . 328 , DF = 10 , Gαi3 : p = 0 . 275 , t = 1 . 155 , DF = 10 , Elp3 : * * * p = 0 . 0003 , t = 5 . 438 , DF = 10 , mean ± SEM ) .

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

LGN and Gαi3 protein levels are not affected by Elp3 loss. (A) Western blots with total lysates from control and Elp3 siRNA-treated HEK293T cells and corresponding quantifications of normalized LGN, Gαi3 and Elp3 levels (n=6 from 2 independent cultures; Unpaired two-tailed t-test, LGN: p=0.749, t=0.328, DF=10, Gαi3: p=0.275, t=1.155, DF=10, Elp3: ***p=0.0003, t=5.438, DF=10, mean±SEM).


LGN and Gαi3 protein levels are not affected by Elp3 loss . ( B ) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels ( n = 3 , each lysate obtained from cochleae pooled from 2 animals ; Unpaired two - tailed t - test , LGN : p = 0 . 586 , t = 0 . 591 , DF = 4 , Gαi3 : p = 0 . 963 , t = 0 . 0491 , DF = 4 , mean ± SEM ) .

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

LGN and Gαi3 protein levels are not affected by Elp3 loss. (B) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels (n=3, each lysate obtained from cochleae pooled from 2 animals; Unpaired two-tailed t-test, LGN: p=0.586, t=0.591, DF=4, Gαi3: p=0.963, t=0.0491, DF=4, mean±SEM).


( C ) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion . HEK293T transfected with Elp3 siRNA were co - stained for aggresomes , Dapi and LGN or Gαi3 . Scale bar = 5µm .

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

(C) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion. HEK293T transfected with Elp3 siRNA were co-stained for aggresomes, Dapi and LGN or Gαi3. Scale bar=5µm.


Microtubular transport speed is reduced by the presence of aggresome - like structures in HEK293T cells and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HEK293T cells labelled with LysoTracker ® . ( D ) : DMSO / MG - 132 / MG - 132 + 4PBA n = 555 / 337 / 453 , pooled from 3 experiments , * * * p < 0 . 001 , * * p < 0 . 01 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (D): DMSO/MG-132/MG-132+4PBA n=555/337/453, pooled from 3 experiments, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Microtubular transport speed is reduced by the presence of aggresome - like structures in HEK293T cells and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HEK293T cells labelled with LysoTracker ® . ( E ) : siCtl / + 4PBA n = 622 / 677 ; siElp3 / + 4PBA n = 674 / 577 ; siAlkbh8 / + 4PBA n = 460 / 484 , pooled from 4 experiments , * * * p < 0 . 001 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (E): siCtl/+4PBA n=622/677; siElp3/+4PBA n=674/577; siAlkbh8/+4PBA n=460/484, pooled from 4 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Microtubular transport speed is reduced by the presence of aggresome - like structures in cultured HCs , and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HCs , at the level of their apical surface labelled with LysoTracker ® . ( F ) : DMSO / MG - 132 / MG - 132 + 4PBA n = 210 / 225 / 266 , pooled from 3 experiments , * * * p < 0 . 001 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (F): DMSO/MG-132/MG-132+4PBA n=210/225/266, pooled from 3 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Microtubular transport speed is reduced by the presence of aggresome - like structures in cultured HCs , and is improved upon 4 - PBA treatment . The mean velocity of lysosomes was measured on time - lapse recordings of HCs , at the level of their apical surface labelled with LysoTracker ® . ( G ) : WT / WT + 4PBA / Elp3cKO / Elp3cKO + 4PBA n = 732 / 763 / 730 / 738 , pooled from 4 OCs per condition , * * * p < 0 . 001 , * * p < 0 . 01 . All data analysed with Kruskal - Wallis ANOVA , Dunn ' s multiple comparisons test . Box / whisker plots depict minimum , first quartile , median , third quartile , and maximum .

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

Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (G): WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA n=732/763/730/738, pooled from 4 OCs per condition, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.


Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( A ) LGN and F - actin immunostainings of E16 . 5 + 1DIV OCs reveal that MG - 132 perturbs LGN distribution at the apical surface of HCs , while co - treatment with 4 - PBA ameliorates its enrichment at the lateral edge . Scale bars = 5µm ; magnified views = 2µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3


Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( B ) Acetylated alpha - tubulin immunostaining reveals that the kinocilium is centrally shifted by MG - 132 and partially restored by 4 - PBA in E16 . 5 + 1DIV HCs ( yellow arrowheads = kinocilium base ; scale bar = 2µm ) .

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

Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm).


Intrinsic polarity is disrupted upon MG - 132 treatment of cultured HCs and restored by the chemical chaperone 4 - PBA . ( C ) Kinocilium position plotted as a fraction of HC diameter for each HC row ( DMSO / MG - 132 / MG - 132 + 4PBA IHC : n = 57 / 67 / 53 , * * * p < 0 . 001 , F = 34 . 52 , DF = 2 ; OHC1 : n = 61 / 66 / 53 , * * * p < 0 . 001 , F = 57 . 88 , DF = 2 ; OHC2 : n = 55 / 73 / 58 , * * * p < 0 . 001 , F = 124 . 7 , DF = 2 ; OHC3 : n = 48 / 66 / 43 , * * * p < 0 . 001 , F = 67 . 16 , DF = 2 ; pooled from 4 explants per condition ; mean ± SD ; One - way ANOVA with Tukey ' s multiple comparisons test ) .

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

Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).


In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( D ) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration . Scale bars = 5µm ; magnified views = 2µm . Data information : # IHC ; * OHC1 ; * * OHC2 ; * * *

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

In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3


In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( E ) Acetylated alpha - tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4 - PBA ( yellow arrowheads = base of kinocilium ; scale bar = 2µm ) .

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

In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm).


In vivo administration of 4 - PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs . Pregnant mice were treated daily with saline or 4 - PBA from E13 . 5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17 . 5 . ( F ) Kinocilium position plotted as a fraction of HC diameter for each HC row ( WT / WT + 4PBA / Elp3cKO / Elp3cKO + 4PBA IHC : n = 35 / 31 / 35 / 36 , p = 0 . 6812 , F = 4 . 510 , DF = 3 ; OHC1 : n = 36 / 32 / 37 / 40 , * * * p < 0 . 001 , * * p < 0 . 01 , F = 27 . 60 , DF = 3 ; OHC2 : n = 35 / 33 / 38 / 42 , * * * p < 0 . 001 , F = 76 . 62 , DF = 3 ; OHC3 : n = 36 / 32 / 38 / 39 , * * * p < 0 . 001 , * p < 0 . 05 , F = 27 . 86 , DF = 3 ; pooled from 3 animals per condition ; mean ± SD ; One - way ANOVA with Tukey ' s multiple comparisons test ) .

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

In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).


( 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 ) .

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

(A) 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 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(C) 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 .

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(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 .

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

(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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.


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 .

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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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 )

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 ) .

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(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 ) .

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(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 ) .

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(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 .

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(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.


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 .

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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.


( 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 ) ) .

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(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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.


( 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 .

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

(A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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).


( 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 ) .

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

(A) 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 ) .

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

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

(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.


Hippocampal 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 ) .

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

Hippocampal 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 .

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

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 ) .

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

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 ) .

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

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

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.


( 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 .

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

(A-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 ) .

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

(A-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 ) .

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

(A-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).


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

(G) 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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

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

(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 ) .

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

(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).