( A ) CARD14 signaling induces CYLD and A20 cleavage . HEK293T cells were transfected with different concentrations of FLAG - CARD14 , with or without MALT1 as indicated . CARD11 ( L232LI ) transfection was used as a positive control . Endogenous CYLD and A20 processing was analyzed by immunoblotting . CYLD and A20 cleavage fragments are indicated by an arrow .

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(A) CARD14 signaling induces CYLD and A20 cleavage. HEK293T cells were transfected with different concentrations of FLAG-CARD14, with or without MALT1 as indicated. CARD11(L232LI) transfection was used as a positive control. Endogenous CYLD and A20 processing was analyzed by immunoblotting. CYLD and A20 cleavage fragments are indicated by an arrow.


( B ) CARD14 signaling induces CYLD cleavage after R324 . HEK293T cells were transfected with different concentrations of FLAG - CARD14sh and either wild - type ( CYLDWT ) or non - cleavable CYLD ( CYLDR324A ) . CYLD processing was analyzed by immunoblotting . The CYLD cleavage fragment is indicated by an arrow .

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(B) CARD14 signaling induces CYLD cleavage after R324. HEK293T cells were transfected with different concentrations of FLAG-CARD14sh and either wild-type (CYLDWT) or non-cleavable CYLD (CYLDR324A). CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow.


( C ) Expression of catalytically active MALT1 is necessary for CARD14 - induced CYLD cleavage . MALT1 - deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild - type ( MALT1WT ) or catalytically inactive MALT1 ( MALT1C / A ) , were co - transfected with FLAG - CARD14sh and CYLD as indicated . CYLD processing was analyzed by immunoblotting . The CYLD cleavage fragment is indicated by an arrow .

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(C) Expression of catalytically active MALT1 is necessary for CARD14-induced CYLD cleavage. MALT1-deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A), were co-transfected with FLAG-CARD14sh and CYLD as indicated. CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow.


( D ) Psoriasis - associated CARD14 mutation enhances activation of MALT1 proteolytic activity . HEK293T cells were transfected with wild - type ( WT ) FLAG - CARD14 or different psoriasis - associated CARD14 mutants ( 20ng ) , with or without MALT1 ( 20 ng ) as indicated . Endogenous CYLD and A20 processing , as well as IκBα phosphorylation , were analyzed by immunoblotting . The CYLD and A20 cleavage fragments are indicated by an arrow .

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(D) Psoriasis-associated CARD14 mutation enhances activation of MALT1 proteolytic activity. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14 or different psoriasis-associated CARD14 mutants (20ng), with or without MALT1 (20 ng) as indicated. Endogenous CYLD and A20 processing, as well as IκBα phosphorylation, were analyzed by immunoblotting. The CYLD and A20 cleavage fragments are indicated by an arrow.


( E ) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14 - induced gene expression in HaCaT keratinocytes . CARD14 expression was induced by doxycycline ( dox ) treatment in the absence or presence of different concentrations of mepazine as indicated . The next morning , cell culture medium was refreshed and IL - 8 and MCP - 1 concentration in the cell supernatant was measured 8 h later by ELISA . TNF stimulation ( MALT1 - independent ) was used as a negative control . Values are the mean of triplicates ± s . e . Data are representative of two independent experiments . Significance levels , * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 by student t test .

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(E) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14-induced gene expression in HaCaT keratinocytes. CARD14 expression was induced by doxycycline (dox) treatment in the absence or presence of different concentrations of mepazine as indicated. The next morning, cell culture medium was refreshed and IL-8 and MCP-1 concentration in the cell supernatant was measured 8 h later by ELISA. TNF stimulation (MALT1-independent) was used as a negative control. Values are the mean of triplicates ± s.e. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.


( A - B ) CYLD and A20 inhibit CARD14sh - induced NF - kB activation . HEK293T cells were transfected with NF - kB reporter plasmid and FLAG - tagged CARD14sh ( 30ng ) , with or without the indicated concentrations of CYLD ( A ) or A20 ( B ) . Luciferase activity in cell lysates was measured 24 h later . Closed arrowheads indicate full length A20 and CYLD ; arrows indicate cleaved A20 and CYLD . Data are representative of three independent experiments .

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(A-B) CYLD and A20 inhibit CARD14sh-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid and FLAG-tagged CARD14sh (30ng), with or without the indicated concentrations of CYLD (A) or A20 (B). Luciferase activity in cell lysates was measured 24 h later. Closed arrowheads indicate full length A20 and CYLD; arrows indicate cleaved A20 and CYLD. Data are representative of three independent experiments.


( C ) A20 and ABIN - 1 inhibit CARD14 - induced NF - kB activation . HEK293T cells were transfected with NF - kB reporter plasmid , A20 ( 3 ng ) or ABIN1 ( 15ng ) and the indicated concentrations of FLAG - tagged CARD14sh . Luciferase activity in cell lysates was measured 24 h later . Values are the mean of triplicates ± s . e . Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels .

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(C) A20 and ABIN-1 inhibit CARD14-induced NF-kB activation. HEK293T cells were transfected with NF-kB reporter plasmid, A20 (3 ng) or ABIN1 (15ng) and the indicated concentrations of FLAG-tagged CARD14sh. Luciferase activity in cell lysates was measured 24 h later. Values are the mean of triplicates ± s.e. Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels.


( D ) A20 inhibits CARD14 - induced NF - kB activation through its ZnF domains . HEK293T cells were transfected with NF - kB reporter plasmid , FLAG - CARD14sh ( 20ng ) and wild - type ( WT ) or mutant A20 expression plasmids ( 3ng ) as indicated . Luciferase activity in cell lysates was measured after 24 h . CARD14 and A20 expression was measured by immunoblotting ( bottom panel ) . Data are representative of two independent experiments .

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(D) A20 inhibits CARD14-induced NF-kB activation through its ZnF domains. HEK293T cells were transfected with NF-kB reporter plasmid, FLAG-CARD14sh (20ng) and wild-type (WT) or mutant A20 expression plasmids (3ng) as indicated. Luciferase activity in cell lysates was measured after 24 h. CARD14 and A20 expression was measured by immunoblotting (bottom panel). Data are representative of two independent experiments.


( A ) CARD14 ( E138A ) induces cytokine and chemokine expression in human primary keratinocytes . Cells were transfected with empty vector ( EV ) or CARD14 ( E138A ) as indicated . 24 h later , the secretion of 36 different human cytokines , chemokines , and acute phase proteins was analyzed via a multiplex antibody array . Quantification was done using ImageJ software and is expressed as relative CARD14 / EV values for a selected number of proteins ( bottom panel ) .

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(A) CARD14(E138A) induces cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with empty vector (EV) or CARD14(E138A) as indicated. 24 h later, the secretion of 36 different human cytokines, chemokines, and acute phase proteins was analyzed via a multiplex antibody array. Quantification was done using ImageJ software and is expressed as relative CARD14/EV values for a selected number of proteins (bottom panel).


( B ) MALT1 deficiency inhibits CARD14 ( E138A ) - induced cytokine and chemokine expression in human primary keratinocytes . Cells were transfected with scrambled ( scr ) or MALT1 - targeting siRNA as indicated , 48 h later followed by transfection with CARD14 ( E138A ) . After 24 h , cytokine and chemokine expression was analyzed and is expressed as described in ( A ) .

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(B) MALT1 deficiency inhibits CARD14(E138A)-induced cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14(E138A). After 24 h, cytokine and chemokine expression was analyzed and is expressed as described in (A).


( C ) Pathogenic CARD14 mutation enhances MALT1 - dependent CARD14 - induced IL - 8 expression in human primary keratinocytes . Cells were transfected with scrambled ( scr ) or MALT1 - targeting siRNA as indicated , 48 h later followed by transfection with CARD14 wild - type ( WT ) , CARD14 ( G117S ) or CARD14 ( E138A ) as indicated . TNF treatment was used as a control . After 24 h , IL - 8 concentration in the cell supernatant was analyzed via ELISA . Values are the mean of triplicates ± s . e . Expression levels of MALT1 and CARD14 variants is verified by immunoblotting ( bottom panel ) .

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(C) Pathogenic CARD14 mutation enhances MALT1-dependent CARD14-induced IL-8 expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14 wild-type (WT), CARD14(G117S) or CARD14(E138A) as indicated. TNF treatment was used as a control. After 24 h, IL-8 concentration in the cell supernatant was analyzed via ELISA. Values are the mean of triplicates ± s.e. Expression levels of MALT1 and CARD14 variants is verified by immunoblotting (bottom panel).


( D ) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14 - induced IL - 8 production and A20 processing in human primary keratinocytes . Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated . TNF treatment was used as a control . 24 h later , IL - 8 levels in the cell supernatant were measured by ELISA . Values are the mean of triplicates ± s . e . Expression of different CARD14 variants and CARD14 - induced processing of endogenous A20 were analyzed by immunoblotting . Data are representative of two independent experiments . Significance levels , * P < 0 . 05 , * * P < 0 . 001 , * * * P < 0 . 001 by student t test .

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(D) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14-induced IL-8 production and A20 processing in human primary keratinocytes. Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated. TNF treatment was used as a control. 24 h later, IL-8 levels in the cell supernatant were measured by ELISA. Values are the mean of triplicates ± s.e. Expression of different CARD14 variants and CARD14-induced processing of endogenous A20 were analyzed by immunoblotting. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.


A ) Treadmill analysis ( n = 8 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by unpaired , two - tailed Student ' s t test * * * p = 0 . 005 ; * * * p = 0 . 0007 ( T3 ) and * * * * p < 0 . 0001 ( T4 ) . Only p values for rapamycin - treated vs . untreated Cox15sm / sm mice are shown

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A) Treadmill analysis (n=8/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two-tailed Student's t test ***p=0.005; ***p=0.0007 (T3) and ****p<0.0001 (T4). Only p values for rapamycin-treated vs. untreated Cox15sm/sm mice are shown


B ) Histological and histochemical characterization of skeletal muscle in rapamycin - treated and untreated Cox15sm / sm and WT mice . H & E : hematoxylin and eosin ; PAS : periodic acid Schiff reaction ; COX : cytochrome c oxidase ; SDH : succinate dehydrogenase . White bars correspond to 50µm

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B) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; PAS: periodic acid Schiff reaction; COX: cytochrome c oxidase; SDH: succinate dehydrogenase. White bars correspond to 50µm


C ) Analysis of the cross - sectional area of muscle fibers in the different genotypes ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test : * * * p = 0 . 001 ; * * * p = 0 . 006 , * * * * p < 0 . 0001

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C) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: ***p=0.001; ***p=0.006, ****p<0.0001


D ) Analysis of the number of centralized nuclei in muscle fibers ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0018 , * * p = 0 . 0052

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D) Analysis of the number of centralized nuclei in muscle fibers (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0018, **p=0.0052


E ) Analysis of PAS - reaction intensity in muscle fibers ( n = 4 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * p = 0 . 0293 ( WT vs . Cox15sm / sm ) and * p = 0 . 0150 ( Cox15sm / sm vs . Cox15sm / sm Rapa )

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E) Analysis of PAS-reaction intensity in muscle fibers (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0293 (WT vs. Cox15sm/sm) and *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm Rapa)


F ) Analysis of COX - reaction intensity in muscle fibers ( n = 3 ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * * p < 0 . 0001 ; * * p = 0 . 0031

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F) Analysis of COX-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001; **p=0.0031


G ) Analysis of SDH - reaction intensity in muscle fibers ( n = 3 ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * p = 0 . 0199 , * * p = 0 . 0091

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G) Analysis of SDH-reaction intensity in muscle fibers (n=3). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199, **p=0.0091


A ) Spectrophotometric activities of the respiratory chain ( n = 4 - 5 / group ) . CS : citrate synthase ; CIV : complex IV . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by two way ANOVA with Tukey ' s correction * * * * p < 0 . 0001 ( CS : WT vs . Cox15sm / sm ) , * * * p = 0 . 0010 ( CS : Cox15sm / sm vs . Cox15sm / sm rapamycin ) , * * * * p < 0 . 0001 ( CIV : WT vs . Cox15sm / sm ) , * * p = 0 . 0060 ( CIV / CS : WT vs . Cox15sm / sm )

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A) Spectrophotometric activities of the respiratory chain (n=4-5/group). CS: citrate synthase; CIV: complex IV. Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by two way ANOVA with Tukey's correction ****p<0.0001 (CS: WT vs. Cox15sm/sm), ***p=0.0010 (CS: Cox15sm/sm vs. Cox15sm/sm rapamycin), ****p<0.0001 (CIV: WT vs. Cox15sm/sm), **p=0.0060 (CIV/CS: WT vs. Cox15sm/sm)


B ) Western blot immunovisualization of subunits of the respiratory complexes . Note the increased protein levels in Cox15sm / sm vs . WT samples , which are reduced to normal levels upon rapamycin treatment . Additional samples were run on a separate gel ( not shown )

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B) Western blot immunovisualization of subunits of the respiratory complexes. Note the increased protein levels in Cox15sm/sm vs. WT samples, which are reduced to normal levels upon rapamycin treatment. Additional samples were run on a separate gel (not shown)


C ) BNGE in gel activity for cIV . Sc : supercomplexes . Note that the COX reaction is slightly increased in rapamycin - treated vs . untreated Cox15sm / sm samples

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C) BNGE in gel activity for cIV. Sc: supercomplexes. Note that the COX reaction is slightly increased in rapamycin-treated vs. untreated Cox15sm/sm samples


D ) Immunoblot of 1D - BNGE using an anti - cIV antibody ( COX1 ) . Note that COX amount is slightly increased in rapamycin treated vs . untreated Cox15sm / sm muscles . SDHB was used as a loading control

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D) Immunoblot of 1D-BNGE using an anti-cIV antibody (COX1). Note that COX amount is slightly increased in rapamycin treated vs. untreated Cox15sm/sm muscles. SDHB was used as a loading control


E ) State III ( succinate - driven ) oxygen consumption ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by unpaired , two tail Student ' s t test * * p = 0 . 0016 ( WT vs . Cox15sm / sm ) ; * p = 0 . 047 ( Cox15sm / sm vs . Cox15sm / sm rapamycin )

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E) State III (succinate-driven) oxygen consumption (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by unpaired, two tail Student's t test **p=0.0016 (WT vs. Cox15sm/sm); *p=0.047 (Cox15sm/sm vs. Cox15sm/sm rapamycin)


F ) Analysis of mtDNA amount ( n = 9 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey ' s correction : * * p = 0 . 0020 ; * p = 0 . 0296

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F) Analysis of mtDNA amount (n=9/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey's correction: **p=0.0020; *p=0.0296


Note the accumulation of profoundly altered mitochondria in Cox15sm / sm muscles ( white arrows ) , possibly as a result of partially digested organelles within endolysosomes . Black arrows indicate examples of normal mitochondria . In rapamycin - treated Cox15sm / sm animals only a few of these structures are present , which can be detected also in rapamycin - treated WT samples . The bar corresponds to 1136 nm ( 4400X )

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Note the accumulation of profoundly altered mitochondria in Cox15sm/sm muscles (white arrows), possibly as a result of partially digested organelles within endolysosomes. Black arrows indicate examples of normal mitochondria. In rapamycin-treated Cox15sm/sm animals only a few of these structures are present, which can be detected also in rapamycin-treated WT samples. The bar corresponds to 1136 nm (4400X)


A ) Representative western blot of autophagy markers at steady state in skeletal muscle . GAPDH was used as a loading control . B ) Densitometric analysis of immunoblots similar to that shown in A ( n = 4 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0068 ( LC3 - II WT vs . Cox15sm / sm ) , * * p = 0 . 0063 ( P62 WT vs . Cox15sm / sm ) * p = 0 . 025 ( P62 Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * * p = 0 . 0098 ( S6 WT vs . Cox15sm / sm ) , * p = 0 . 034 ( P - S6 WT vs WT + rapamycin ) , * * * p = 0 . 0002 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * p = 0 . 014 ( P - S6 WT vs . WT + rapamycin ) * * * p = 0 . 0007 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin )

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A) Representative western blot of autophagy markers at steady state in skeletal muscle. GAPDH was used as a loading control. B) Densitometric analysis of immunoblots similar to that shown in A (n=4/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0068 (LC3-II WT vs. Cox15sm/sm), **p=0.0063 (P62 WT vs. Cox15sm/sm) *p=0.025 (P62 Cox15sm/sm vs. Cox15sm/sm+rapamycin), **p=0.0098 (S6 WT vs. Cox15sm/sm), *p=0.034 (P-S6 WT vs WT+rapamycin), ***p=0.0002 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), *p=0.014 (P-S6 WT vs. WT+rapamycin) ***p=0.0007 (Cox15sm/sm vs. Cox15sm/sm+rapamycin)


C ) Analysis of the autophagic flux ( n = 3 / group ) . Upper panel : representative western blot of Cox15sm / sm and WT muscles treated with colchicine , rapamycin , or rapamycin plus colchicine . Note that LC3 - II was increased in Cox15sm / sm vs . WT samples . Colchicine did not increase LC3 - II levels suggesting a block in the autophagic flux ; rapamycin plus colchicine treated Cox15sm / sm mice showed higher levels of LC3 - II , suggesting that rapamycin increased the autophagic flux in Cox15sm / sm muscles . Black : WT ; dark grey : colchicine - treated WT ; middle grey : rapamycin - treated WT ; light grey : colchicine plus rapamycin treated mice ; red : untreated Cox15sm / sm ; orange : colchicine - treated ; blue : rapamycin - treated Cox15sm / sm ; purple : colchicine plus rapamycin treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0049 ( WT vs . WT + colchicine ) , * * p = 0 . 0046 ( WT + rapamycin vs . WT + rapamycin + colchicine ) , * p = 0 . 0225 ( WT vs . Cox15sm / sm ) , * * p = 0 . 0057 ( Cox15sm / sm colchicine vs . Cox15sm / sm + rapamycin + colchicine ) , * * * p = 0 . 0003 ( Cox15sm / sm rapamycin vs . Cox15sm / sm + rapamycin + colchicine )

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C) Analysis of the autophagic flux (n=3/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rapamycin, or rapamycin plus colchicine. Note that LC3-II was increased in Cox15sm/sm vs. WT samples. Colchicine did not increase LC3-II levels suggesting a block in the autophagic flux; rapamycin plus colchicine treated Cox15sm/sm mice showed higher levels of LC3-II, suggesting that rapamycin increased the autophagic flux in Cox15sm/sm muscles. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0049 (WT vs. WT+colchicine), **p=0.0046 (WT+rapamycin vs. WT+rapamycin+colchicine), *p=0.0225 (WT vs. Cox15sm/sm), **p=0.0057 (Cox15sm/sm colchicine vs. Cox15sm/sm+rapamycin+colchicine), ***p=0.0003 (Cox15sm/sm rapamycin vs. Cox15sm/sm+rapamycin+colchicine)


A ) Analysis of the autophagic flux ( n = 4 / group ) . Upper panel : representative western blot of Cox15sm / sm and WT muscles treated with colchicine , rilmenidine , or rilmenidine plus colchicine . Colchicine alone increased LC3 - II in WT but not Cox15sm / sm mice ; rilmenidine had no effect on WT LC3 - II levels , but reduced them in Cox15sm / sm mice . Rilmenidine plus colchicine increased LC3 - II levels in both WT and Cox15sm / sm mice . Black : WT ; dark grey : colchicine - treated WT ; middle grey : rapamycin - treated WT ; light grey : colchicine plus rapamycin treated mice ; red : untreated Cox15sm / sm ; orange : colchicine - treated ; blue : rapamycin - treated Cox15sm / sm ; purple : colchicine plus rapamycin treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test : * * p = 0 . 0093 ( WT vs . WT + colchicine ) , * * * * p < 0 . 0001 ( WT + rilmenidine vs . WT + rilmenidine + colchicine ) , * * p = 0 . 0001 ( WT vs . Cox15sm / sm ) , * * p = 0 . 0083 ( Cox15sm / sm vs . Cox15sm / sm + rilmenidine ) , * p = 0 . 0112 ( Cox15sm / sm vs . Cox15sm / sm + rilmenidine + colchicine ) , * * * * p < 0 .

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A) Analysis of the autophagic flux (n=4/group). Upper panel: representative western blot of Cox15sm/sm and WT muscles treated with colchicine, rilmenidine, or rilmenidine plus colchicine. Colchicine alone increased LC3-II in WT but not Cox15sm/sm mice; rilmenidine had no effect on WT LC3-II levels, but reduced them in Cox15sm/sm mice. Rilmenidine plus colchicine increased LC3-II levels in both WT and Cox15sm/sm mice. Black: WT; dark grey: colchicine-treated WT; middle grey: rapamycin-treated WT; light grey: colchicine plus rapamycin treated mice; red: untreated Cox15sm/sm; orange: colchicine-treated; blue: rapamycin-treated Cox15sm/sm; purple: colchicine plus rapamycin treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test: **p=0.0093 (WT vs. WT+colchicine), ****p<0.0001 (WT+rilmenidine vs. WT+rilmenidine+colchicine), **p=0.0001 (WT vs. Cox15sm/sm), **p=0.0083 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine), *p=0.0112 (Cox15sm/sm vs. Cox15sm/sm+rilmenidine+colchicine), ****p<0.000


B ) Treadmill analysis ( n = 4 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent SEM

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B) Treadmill analysis (n=4/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM


C ) Histological and histochemical characterization of skeletal muscle in rapamycin - treated and untreated Cox15sm / sm and WT mice . H & E : hematoxylin and eosin ; GT : Gomori trichrome ; PAS : periodic acid Schiff reaction

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C) Histological and histochemical characterization of skeletal muscle in rapamycin-treated and untreated Cox15sm/sm and WT mice. H&E: hematoxylin and eosin; GT: Gomori trichrome; PAS: periodic acid Schiff reaction


D ) Analysis of the cross - sectional area of muscle fibers in the different genotypes ( n = 3 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * p = 0 . 0002 , * * * * p < 0 . 0001

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D) Analysis of the cross-sectional area of muscle fibers in the different genotypes (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ***p=0.0002, ****p<0.0001


E ) Analysis of the number of centralized nuclei in muscle fibres ( n = 3 / group ) . Black : untreated WT ; grey : rilmenidine - treated WT ; red : untreated Cox15sm / sm ; blue : rilmenidine - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * p = 0 . 0091

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E) Analysis of the number of centralized nuclei in muscle fibres (n=3/group). Black: untreated WT; grey: rilmenidine-treated WT; red: untreated Cox15sm/sm; blue: rilmenidine-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test **p=0.0091


A ) anti - TFEB immunofluorescence on rapamycin - and rilmenidine - treated WT and Cox15sm / sm muscles vs . untreated ( UT ) samples . CD11C ( blue signal ) indicates inflammatory cells . Right panel : quantification of n = 3 animals / group . The scale bars correspond to 30µm . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test B ) Quantification of the TFEB - positive nuclei ( n = 3 / group ) . Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * p = 0 . 0199 ( WT vs . WT + rapamycin ) , * p = 0 . 0150 ( Cox15sm / sm vs . Cox15sm / sm + rapamycin ) , * * * p = 0 . 0001

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A) anti-TFEB immunofluorescence on rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. CD11C (blue signal) indicates inflammatory cells. Right panel: quantification of n=3 animals/group. The scale bars correspond to 30µm. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test B) Quantification of the TFEB-positive nuclei (n=3/group). Quantification was performed using Imaris spots surface excluding the CD11C positive nuclei. Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test *p=0.0199 (WT vs. WT+rapamycin), *p=0.0150 (Cox15sm/sm vs. Cox15sm/sm+rapamycin), ***p=0.0001


A ) Real - time PCR analysis of Lamp1 transcript ( n = 3 / group ) . Black : untreated WT ; grey : rapamycin - treated WT ; red : untreated Cox15sm / sm ; blue : rapamycin - treated Cox15sm / sm . Error bars represent SEM . The asterisks represent the significance levels calculated by Student ' s t test * * p = 0 . 008 , * p = 0 . 038

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A) Real-time PCR analysis of Lamp1 transcript (n=3/group). Black: untreated WT; grey: rapamycin-treated WT; red: untreated Cox15sm/sm; blue: rapamycin-treated Cox15sm/sm. Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test **p=0.008, *p=0.038


B ) Anti - LAMP1 staining in rapamycin - and rilmenidine - treated WT and Cox15sm / sm muscles vs . untreated ( UT ) samples . Rapamycin , but not rilmendine , increases the number of LAMP1 - positive vesicles in Cox15sm / sm samples ( inset ) . The scale bars correspond to 30µm

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B) Anti-LAMP1 staining in rapamycin- and rilmenidine-treated WT and Cox15sm/sm muscles vs. untreated (UT) samples. Rapamycin, but not rilmendine, increases the number of LAMP1-positive vesicles in Cox15sm/sm samples (inset). The scale bars correspond to 30µm


C ) Quantification of LAMP1 - positive vesicles ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by one - way ANOVA with Tukey post - hoc multiple comparison test * * * * p < 0 . 0001

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C) Quantification of LAMP1-positive vesicles (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by one-way ANOVA with Tukey post-hoc multiple comparison test ****p<0.0001


D ) Cathepsin L specific activity ( n = 3 / group ) . Error bars represent SEM . The asterisks represent the significance levels calculated by Student ' s t test * p = 0 . 0133

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D) Cathepsin L specific activity (n=3/group). Error bars represent SEM. The asterisks represent the significance levels calculated by Student's t test *p=0.0133


( C ) Cleavage of supercoiled ( SC ) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants . Efficient full length linearization ( FLL ) of the substrate resulting from a complete double - strand break was only observed when using sgRNAs bearing the 5 ' most end of the tracrRNA ( variants 1 and 3 ) . Moreover , the terminator - like Hairpin 3 is not required for target cleavage ( variant 1 ) . OC - open circular ; FLL - full length linear ; SC - supercoiled .

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(C) Cleavage of supercoiled (SC) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.


( B ) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l . wt - wildtype .

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(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.


( C ) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition . The molar ratio of RNP to substrate was kept low ( 5 : 1 ) to increase reaction stringency . Asp2Cas12l predominantly recognizes a 5 ' - CCY - 3 ' PAM and Asp3Cas12l a 5 ' - CCB - 3 ' PAM . N = 3 . Data information : In ( C ) , individual data points are plotted , where n = 4 replicates from independent experiments ( Asp2Cas12l ) and n = 3 replicates from independent experiments ( Asp3Cas12l ) . The data points were fitted to a single exponential association curve ( solid lines ) .

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(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3. Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).


( A ) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single - stranded ( ss ) DNA or dsDNA ( including PAM ) activators with target sequences complementary to the gRNA spacer . NS activator - non specific activator ( ssDNA oligonucleotide or dsDNA duplex ) with no sequence complementarity to the spacer of the gRNA .

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(A) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single-stranded (ss) DNA or dsDNA (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.


( B ) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes . Background - subtracted traces from fluorescent reporter assays with favored ssDNA ( 5 ' - CCCCCCCC - 3 ' ) or ssRNA ( 5 ' - CCCCCCCC - 3 ' ) probes . RFU - relative fluorescence units . ( C ) Background - subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes . ( D ) Rates of trans - degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets . Collateral ssDNAse activity is about 3 - fold higher than the rate of ssRNA degradation . Fluorescence intensities in B - C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters . Data information : In ( B - D ) , individual data points from n = 3 replicates from independent experiments are plotted . In ( D ) , bars represent the mean .

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(B) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA (5'-CCCCCCCC-3') or ssRNA (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units. (C) Background-subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes. (D) Rates of trans-degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation. Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters. Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.


Distribution of 25 - 35 mer reads mapping to the mouse genome . Proportion of 30 - 35 nt genomic reads matching mouse tRNA sequences . The sequencing was done in duplicates . Error bars represent SEM , significance was calculated using unpaired t - test .

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Distribution of 25-35 mer reads mapping to the mouse genome. Proportion of 30-35 nt genomic reads matching mouse tRNA sequences. The sequencing was done in duplicates. Error bars represent SEM, significance was calculated using unpaired t-test.


Perbase sequence coverage of 30 - 35 nt reads across parental tRNA sequence . Anticodon positions are demarcated by discontinuous lines ( Blue = LIF - treated , Grey = Wnt3 + LIF - treated , Red = RA - treated mESCs ) . Perbase coverage of 30 - 35 nt reads across the 468 individual parental tRNAs plotted as heatmaps .

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Perbase sequence coverage of 30-35 nt reads across parental tRNA sequence. Anticodon positions are demarcated by discontinuous lines (Blue = LIF-treated, Grey = Wnt3+LIF- treated, Red = RA- treated mESCs). Perbase coverage of 30-35 nt reads across the 468 individual parental tRNAs plotted as heatmaps.


Northern blot of candidate tRNAs and tsRNAs tsGlnCTG , tsGlyGCC , tsGluTTC , tsValCAC and tsLysTTT . The black arrows represent the tRNAs and the red arrows mark the tsRNAs .

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Northern blot of candidate tRNAs and tsRNAs tsGlnCTG, tsGlyGCC, tsGluTTC, tsValCAC and tsLysTTT. The black arrows represent the tRNAs and the red arrows mark the tsRNAs.


Northern Blot of 5 ' - tsRNAs ; tsGlyGCC , tsGlnCTG , tsGluTTC , tsLysTTT , tsValCAC at various timepoints of RA - induced mESCs differentiation showing dynamic expression of these 5 ' - tsRNAs . Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates . The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs .

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Northern Blot of 5'-tsRNAs; tsGlyGCC, tsGlnCTG, tsGluTTC, tsLysTTT, tsValCAC at various timepoints of RA-induced mESCs differentiation showing dynamic expression of these 5'-tsRNAs. Relative quantification of the tsRNA bands from northern hybridization tested across two biological replicates. The solid lines represent the expression of tsRNAs and the dashed lines represent tRNAs.


Effect of ASO - mediated inhibition of 5 ' - tsRNAs on alkaline phosphatase activity in RA - treated mESCs . ( n = 3 ) , Error bars represent SEM , significance was calculated using unpaired t - test .

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Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test.


Relative expression of stemness markers in RA - induced differentiation mESCs blocked for 5 ' - tsRNA function with ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test.


Volcano plot of the transcriptome depicting the overexpressed transcripts ( in green ) and downregulated transcripts ( in pink ) in ASO treated - differentiating cells .

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Volcano plot of the transcriptome depicting the overexpressed transcripts (in green) and downregulated transcripts (in pink) in ASO treated-differentiating cells.


QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5 ' - tsRNA knockdowns using ASOs . ( n = 2 ) Error bars represent SEM , significance was calculated using unpaired t - test .

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QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5'-tsRNA knockdowns using ASOs. (n =2) Error bars represent SEM, significance was calculated using unpaired t-test.


Scatter plot showing Log2 fold - change enrichment of protein interactome between LIF - and RA - treated mESCs . All peptides identified by LC - MS / MS had < 1 % FDR ( Fig EV4E ) . LC - MS / MS was conducted twice ( R2 = 0 . 85 between LIF duplicates and R2 = 0 . 82 between RA - treated mESCs duplicates ) .

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Scatter plot showing Log2 fold-change enrichment of protein interactome between LIF- and RA-treated mESCs. All peptides identified by LC-MS/MS had < 1% FDR (Fig EV4E). LC-MS/MS was conducted twice (R2 = 0.85 between LIF duplicates and R2 = 0.82 between RA-treated mESCs duplicates).


Scatter plot showing Log2 fold - change enrichment of mRNAs associated with tsGlnCTG over Scramble ( Scr1 ) in LIF - versus RA - treated mESCs .

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Scatter plot showing Log2 fold-change enrichment of mRNAs associated with tsGlnCTG over Scramble (Scr1) in LIF- versus RA-treated mESCs.


Bar plots and representative gene lists depicting association of tsGlnCTG with ' pluripotency - associated ' ( D ) and ' differentiation - responsive ' ( D ' ) genes in LIF - or RA - treated mESCs .

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Bar plots and representative gene lists depicting association of tsGlnCTG with 'pluripotency-associated' (D) and 'differentiation-responsive' (D') genes in LIF- or RA-treated mESCs.


In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo ( ASO ) against tsGlnCTG . ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1 . ( n = 2 ) , Error bars represent S . E . M .

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In vitro binding analysis of tsGlnCTG to Igf2bp1 in the presence and absence of antisense oligo (ASO) against tsGlnCTG. ASOs effectively disrupt the binding of tsGlnCTG to Igf2bp1. (n = 2), Error bars represent S.E.M.


Quantification of the Igf2bp1 bound c - Myc mRNA between LIF - versus RA - treated mESCs ( n = 2 ; see Fig . EV5F for duplicate data ) .

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Quantification of the Igf2bp1 bound c-Myc mRNA between LIF- versus RA-treated mESCs (n = 2; see Fig. EV5F for duplicate data).


Association of c - Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition . ( n = 2 ) , Error bars represent SD , significance was calculated by one - tailed unpaired t - test .

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Association of c-Myc transcript in different translating pools in RA treated mESCs as compared to LIF condition. (n = 2), Error bars represent SD, significance was calculated by one-tailed unpaired t-test.


Relative enrichment of c - Myc mRNA in translating ( 80S and Polysome ) and non - translating ( mRNPs ) pools ( fractionated from polysome profiling ) between ASO - treated and Mock - treated RA - induced differentiating mESCs . ( n = 2 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

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Relative enrichment of c-Myc mRNA in translating (80S and Polysome) and non-translating (mRNPs) pools (fractionated from polysome profiling) between ASO-treated and Mock-treated RA-induced differentiating mESCs. (n =2) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Relative levels of c - Myc mRNA in ASO - treated and Mock - treated RA - induced differentiating mESCs compared to LIF treated mESCs . ( n = 3 ) Error bars represent SD , significance was calculated using one - tailed unpaired t - test .

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Relative levels of c-Myc mRNA in ASO-treated and Mock-treated RA-induced differentiating mESCs compared to LIF treated mESCs. (n = 3) Error bars represent SD, significance was calculated using one-tailed unpaired t-test.


Epistatic analysis of 5 ' - tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity . ( n = 3 ) , Error bars represent SD , significance was calculated using unpaired t - test .

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Epistatic analysis of 5'-tsRNAs and IGF2BP1 in regulating Myc transcriptional reporter activity. (n = 3), Error bars represent SD, significance was calculated using unpaired t-test.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Time course of antibody response . Serum samples were diluted 40 - fold prior to ELISA analysis . Animals in the high - dose and low - dose group were immunized with 1 × 109 and 1 × 108 pfu virus each time , respectively . Arrows indicate the time point for immunization .

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Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Time course of antibody response. Serum samples were diluted 40-fold prior to ELISA analysis. Animals in the high-dose and low-dose group were immunized with 1×109 and 1×108 pfu virus each time, respectively. Arrows indicate the time point for immunization.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Determination of virus neutralization antibody titer using 96 - well microneutralization assay . An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples . Statistical significance between different data sets was calculated using t - test .

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Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Determination of virus neutralization antibody titer using 96-well microneutralization assay. An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples. Statistical significance between different data sets was calculated using t-test.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . ELISPOT analysis of IFN - γ response in PBMC samples . Pool A and B contain peptides corresponding to S1 - orf8 fusion and N , respectively . Data represent two animals from each group .

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Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. ELISPOT analysis of IFN-γ response in PBMC samples. Pool A and B contain peptides corresponding to S1-orf8 fusion and N, respectively. Data represent two animals from each group.


Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine . Average food consumption after live virus challenge .

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. Average food consumption after live virus challenge.


Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS - CoV . All animals were euthanized 7 days after viral challenge , and anti - SARS - CoV monoclonal antibody was used to identify SARS antigens in the tissue . Representative data from lung sections were shown . Histology samples include : a . Control group . b . High - dose group . c . Placebo group . The arrowheads point to alveoli space filled with proteinaceous fluid . IHC samples include : d . Control group . e . High - dose group . f . Placebo group . Arrows point to cells that were positive for SARS antigen expression .

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Histology and IHC study on postmortem lung sections from rhesus macaques infected with live SARS-CoV. All animals were euthanized 7 days after viral challenge, and anti-SARS-CoV monoclonal antibody was used to identify SARS antigens in the tissue. Representative data from lung sections were shown. Histology samples include: a. Control group. b. High-dose group. c. Placebo group. The arrowheads point to alveoli space filled with proteinaceous fluid. IHC samples include: d. Control group. e. High-dose group. f. Placebo group. Arrows point to cells that were positive for SARS antigen expression.


( B ) Bar graph representing the normalized average value of the neuronal ATP / ATP + ADP ratio on the basal condition , on glutamate stimulation for 5 minutes , on glutamate stimulation with anisomycin ( 25μM ) and on glutamate stimulation with cycloheximide ( 350μM ) . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , n = 5 independent platings . One - way ANOVA followed by Bonferroni ' s multiple comparison test .

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(B) Bar graph representing the normalized average value of the neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with anisomycin (25μM) and on glutamate stimulation with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. **p<0.01, ***p<0.001, ****p<0.0001, n=5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.


( C ) Bar graph representing the normalized average value of neuronal ATP / ATP + ADP ratio on the basal condition , on glutamate stimulation for 5 minutes , on glutamate stimulation with MPEP , on glutamate stimulation with CNQX + D - AP5 and on glutamate stimulation with CNQX + D - AP5 + anisomycin . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . . * p < 0 . 05 , * * * p < 0 . 001 , n = 4 - 5 independent platings . One - way ANOVA followed by Bonferroni ' s multiple comparison test .

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(C) Bar graph representing the normalized average value of neuronal ATP/ATP+ADP ratio on the basal condition, on glutamate stimulation for 5 minutes, on glutamate stimulation with MPEP, on glutamate stimulation with CNQX + D-AP5 and on glutamate stimulation with CNQX + D-AP5 + anisomycin. Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment.. *p<0.05, ***p<0.001, n=4-5 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.


( D ) Representative current - clamp and voltage - clamp traces showing a single neuron firing spontaneous bursts of action potentials . The traces on the right shows a single representative burst at a higher temporal resolution .

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(D) Representative current-clamp and voltage-clamp traces showing a single neuron firing spontaneous bursts of action potentials. The traces on the right shows a single representative burst at a higher temporal resolution.


( E ) Bar graph representing the burst frequency and burst duration both in the voltage and current - clamp mode . n = 16 neurons from ≥ 3 independent platings . Data presented as mean ± SEM with scattered data points .

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(E) Bar graph representing the burst frequency and burst duration both in the voltage and current-clamp mode. n=16 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.


( F ) Representative voltage - clamp traces of miniature EPSCs recorded ( Vhold = - 70mV ) from a neuron in presence of TTX .

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(F) Representative voltage-clamp traces of miniature EPSCs recorded (Vhold= -70mV) from a neuron in presence of TTX.


( G ) Bar graph representing the baseline mEPSC frequency and amplitude in the current - clamp mode . N = 11 neurons from ≥ 3 independent platings . Data presented as mean ± SEM with scattered data points .

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(G) Bar graph representing the baseline mEPSC frequency and amplitude in the current-clamp mode. N=11 neurons from ≥3 independent platings. Data presented as mean ± SEM with scattered data points.


( I ) Immunoblots depicting levels of PSD 95 , GFAP , Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations . Quantification representing the average fold enrichments of PSD 95 , Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate . Data presented as mean ± SEM with scattered data points . Data points for all the proteins were normalized to their respective whole lysate values . * * p < 0 . 05 , * * p < 0 . 01 , n ≥ 7 animals for each group . One - sample t - test .

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(I) Immunoblots depicting levels of PSD 95, GFAP, Synapsin 1 and tubulin in the whole cortical lysate and the synaptoneurosome preparations. Quantification representing the average fold enrichments of PSD 95, Synapsin 1 and GFAP in the synaptoneurosome preparations compared to the whole cortical lysate. Data presented as mean ± SEM with scattered data points. Data points for all the proteins were normalized to their respective whole lysate values. **p<0.05, **p<0.01, n ≥ 7 animals for each group. One-sample t-test.


( J ) Bar graph representing the normalized average value of synaptic ATP level on the basal condition , on DHPG treatment for 5 minutes , on DHPG treatment with MPEP , on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide ( 350μM ) . Data presented as mean ± SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . * p < 0 . 05 , n = 8 animals .

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(J) Bar graph representing the normalized average value of synaptic ATP level on the basal condition, on DHPG treatment for 5 minutes, on DHPG treatment with MPEP, on DHPG treatment with anisomycin and on DHPG treatment with cycloheximide (350μM). Data presented as mean ± SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. *p<0.05, n=8 animals.


( K ) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition , on NMDA treatment for 5 minutes and on NMDA treatment with D - AP5 . Data presented as mean + / - SEM with scattered data points . Values in all other groups were normalized to the basal group for the corresponding experiment . n = 8 independent platings .

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(K) Bar graph representing the normalized average value of the synaptic ATP level on the basal condition, on NMDA treatment for 5 minutes and on NMDA treatment with D-AP5. Data presented as mean +/- SEM with scattered data points. Values in all other groups were normalized to the basal group for the corresponding experiment. n=8 independent platings.


( B ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in somatic compartments on the bath application of DHPG and NMDA . Scale bar 10μm . ( C ) Representative neurons showing changes in the PercevalHR fluorescence ratio ( ~ ATP / ADP ratio ) in dendritic compartments on the bath application of DHPG and NMDA . Scale bar 10μm .

(B) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in somatic compartments on the bath application of DHPG and NMDA. Scale bar 10μm. (C) Representative neurons showing changes in the PercevalHR fluorescence ratio (~ATP/ADP ratio) in dendritic compartments on the bath application of DHPG and NMDA. Scale bar 10μm.


( D ) Average traces depicting the time course of the normalized somatic ATP / ADP ratio on the basal conditions , on DHPG treatment , and on NMDA treatment . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 5 - 6 cells from ≥ 3 independent platings .

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(D) Average traces depicting the time course of the normalized somatic ATP/ADP ratio on the basal conditions, on DHPG treatment, and on NMDA treatment. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 5-6 cells from ≥3 independent platings.


( E ) Average traces depicting the time course of normalized dendritic ATP / ADP ratio on the basal condition , on the basal condition in the presence of anisomycin , on DHPG treatment , on NMDA treatment , on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin . The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n = 6 - 9 cells from ≥ 3 independent platings .

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(E) Average traces depicting the time course of normalized dendritic ATP/ADP ratio on the basal condition, on the basal condition in the presence of anisomycin, on DHPG treatment, on NMDA treatment, on DHPG treatment in the presence of anisomycin and on NMDA treatment in the presence of anisomycin. The curved arrow on the top indicates the time when the agonists were added and kept in the imaging media. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n= 6-9 cells from ≥3 independent platings.


( G ) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 for comparison with 0 minute . # # p < 0 . 01 and # # # # p < 0 . 0001 for comparison between DHPG + anisomycin . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 5 animals per group .

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(G) Line graph showing the average normalized synaptic ATP level at various time points after DHPG treatment until 5 minutes of recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, *p<0.05, **p<0.01, ****p<0.0001 for comparison with 0 minute. ##p<0.01 and ####p<0.0001 for comparison between DHPG + anisomycin. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 5 animals per group.


( H ) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin . Data presented as mean + / - SEM , * * p < 0 . 01 for comparison with 0 minutes . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . n ≥ 4 animals per group .

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(H) Line graph showing the average normalized synaptic ATP level at various time points after NMDA treatment until 5 minutes after recovery in the presence or absence of anisomycin. Data presented as mean +/- SEM, **p<0.01 for comparison with 0 minutes. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. n≥ 4 animals per group.


( B ) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(B) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( C ) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling ( pseudo - colored ) in cortical neurons at various time points following DHPG and NMDA treatment . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( D ) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments . Data presented as mean + / - SEM . Data points in all groups were normalized to the average of the basal group . * * * p < 0 . 001 n = 21 - 54 neurons per group from 3 independent platings .

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(D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings.


( E ) Representative images showing phospho - eEF2 immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( F ) Box plot showing phospho - eEF2 normalized intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 31 - 49 cells per group from 3 independent platings .

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(F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings.


( G ) Representative immunoblots describing changes in the phospho - eEF2 and total - eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes . Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho / total undefined ratio .

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(G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total undefined ratio.


( H ) Line graph showing the average value of the synaptic phospho / total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery . Data presented as mean + / - SEM , * * p < 0 . 01 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * * p < 0 . 001 , * * * * p < 0 . 0001 , n ≥ 5 animals per group .

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(H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group.


( A ) Representative images showing phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

(A) Representative images showing phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( B ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition , on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes . Data points in all groups were normalized to the average of the basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , * * p < 0 . 01 , n = 30 - 46 cells per group from 3 independent platings .

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(B) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=30-46 cells per group from 3 independent platings.


( C ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total ratio of AMPK .

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(C) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total ratio of AMPK.


( D ) Line graph showing the normalized average value of the synaptic phospho / total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 5 animals per group .

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(D) Line graph showing the normalized average value of the synaptic phospho/total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. *p<0.05, **p<0.01, ****p<0.0001, n= 5 animals per group.


( E ) Representative immunoblots depicting the changes in the α - SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin .

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(E) Representative immunoblots depicting the changes in the α-SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin.


( F ) Line graph showing the normalized average value of the synaptic α - SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin . Data presented as mean + / - SEM . * p < 0 . 05 . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , n ≥ 4 animals per group .

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(F) Line graph showing the normalized average value of the synaptic α-SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. *p<0.05. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, n≥ 4 animals per group.


( G ) Representative images showing the α - SNAP ( cyan ) and AMPK ( magenta ) immunolabeling in low - density cortical neurons . Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes . Scale bar 10μm . Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions . Scale bar 5μm .

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(G) Representative images showing the α-SNAP (cyan) and AMPK (magenta) immunolabeling in low-density cortical neurons. Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes. Scale bar 10μm. Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions. Scale bar 5μm.


( H ) Box plot depicting the quantification of co - localization through Pearson ' s correlation coefficient between α - SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 01 , n ≥ 35 cells per group from 4 independent platings . Unpaired - sample t - test .

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(H) Box plot depicting the quantification of co-localization through Pearson's correlation coefficient between α-SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.01, n≥35 cells per group from 4 independent platings. Unpaired-sample t-test.


( I ) Representative images showing the phospho - AMPK immunolabeling ( Pseudo - colored ) in low - density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α - SNAP siRNA . MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization . Scale bar 10μm .

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(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.


( J ) Box plot showing the normalized phospho - AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA , on DHPG treatment for 5 minutes in the presence of scrambled siRNA , on the basal condition in the presence of α - SNAP siRNA , on DHPG treatment for 5 minutes in the presence of α - SNAP siRNA . Data points in all groups were normalized to the average of the scrambled siRNA basal group . The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset . Whiskers range from minimum to maximum data point . * p < 0 . 05 , n = 29 - 35 cells per group from 3 independent platings .

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(J) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA, on DHPG treatment for 5 minutes in the presence of scrambled siRNA, on the basal condition in the presence of α-SNAP siRNA, on DHPG treatment for 5 minutes in the presence of α-SNAP siRNA. Data points in all groups were normalized to the average of the scrambled siRNA basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, n=29-35 cells per group from 3 independent platings.


( A ) Representative immunoblots describing changes in the phospho - AMPK and total - AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2 + . Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho / total AMPK ratio .

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(A) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after NMDA treatment in cortical synaptoneurosomes in the presence or absence of extracellular Ca2+. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total AMPK ratio.


( B ) Line graph showing the normalized average value for the synaptic phospho / total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2 + . Data presented as mean + / - SEM . Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point . * * p < 0 . 01 , * * * * p < 0 . 0001 , n = 6 animals per group .

(B) Line graph showing the normalized average value for the synaptic phospho/total ratio of AMPK at various time points after NMDA treatment until 5 minutes of recovery in the presence or absence of extracellular Ca2+. Data presented as mean +/- SEM. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. **p<0.01, ****p<0.0001, n= 6 animals per group.


( C ) Representative images depicting intracellular Ca2 + levels through Fluo8 fluorescence ( Pseudo - colored ) of a cortical neuron plated at low - density before stimulation , 15 sec ( immediate ) after NMDA treatment , 300 sec ( delayed ) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2 + . Scale bar 20μm .

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(C) Representative images depicting intracellular Ca2+ levels through Fluo8 fluorescence (Pseudo-colored) of a cortical neuron plated at low-density before stimulation, 15 sec (immediate) after NMDA treatment, 300 sec (delayed) after NMDA treatment and after ionomycin treatment in the presence of 10mM Ca2+. Scale bar 20μm.


( D ) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2 + and D - AP5 . Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point . Ionomycin treatment in the presence of 10mM Ca2 + was used to calculate fluorescence maximum for a particular cell .

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(D) Representative time trace showing the normalized change in Fluo8 fluorescence on NMDA treatment in the presence or absence of extracellular Ca2+ and D-AP5. Values in all other groups were normalized to the unsimulated condition for the corresponding experiment and all the data points are represented as a fraction of the initial time point. Ionomycin treatment in the presence of 10mM Ca2+ was used to calculate fluorescence maximum for a particular cell.