( A ) Wild ‐ type HDAC6 KO MEFs were treated with MG132 immunostained with antibodies to Lamp ‐ 1 ( a lysosome marker , red ) ubiquitin ( green ) as indicated . F ‐ actin was detected by phalloidin ( blue ) . arrows indicate ubiquitin ‐ positive that are surrounded by F ‐ actin Lamp ‐ 1 .

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(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies to Lamp‐1 (a lysosome marker, red) and ubiquitin (green) as indicated. F‐actin was detected by phalloidin (blue). The arrows indicate ubiquitin‐positive aggregates that are surrounded by F‐actin and Lamp‐1.


( B , C ) Wild ‐ type HDAC6 KO MEFs were transfected with mCherry ‐ GFP ‐ LC3 , followed by treatment with LatA ( 100 nM ) or nocodazole ( 250 nM ) for 6 h analysed as described in Figure 3A .

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(B, C) Wild‐type and HDAC6 KO MEFs were transfected with mCherry‐GFP‐LC3, followed by treatment with LatA (100 nM) or nocodazole (250 nM) for 6 h and analysed as described in Figure 3A.


( D ) Autophagosomes ( APGs ) lysosomes ( Lys ) isolated from fed mouse hepatocytes were treated or not with latrunculin ( LatA ) as indicated , extensively washed to remove traces of inhibitor , then labelled with antibody subjected to in vitro fusion in presence or absence of purified actin . number of total fusion events / total number of vesicles for each condition was as follows : 176 / 880 ; 233 / 1110 ; 84 / 930 ; 180 / 950 . differences with untreated samples were significant at * * P0 . 01 .

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(D) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed mouse hepatocytes were treated or not with latrunculin (LatA) as indicated, extensively washed to remove traces of the inhibitor, and then labelled with the antibody and subjected to in vitro fusion assay in the presence or absence of purified actin. The number of total fusion events/total number of vesicles for each condition was as follows: 176/880; 233/1110; 84/930; and 180/950. The differences with untreated samples were significant at **P0.01.


( E ) Autophagosomes ( APGs ) lysosomes ( Lys ) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin ( LatA ) as labelled subjected to in vitro fusion . differences with untreated samples were significant at * P0 . 05 . number of total fusion events / total number of vesicles for each condition was as follows : 169 / 1250 ; 87 / 972 ; 190 / 1120 ; 175 / 1165 .

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(E) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed or starved mouse hepatocytes were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at *P0.05. The number of total fusion events/total number of vesicles for each condition was as follows: 169/1250; 87/972; 190/1120; and 175/1165.


( F ) Autophagosomes ( APGs ) lysosomes ( Lys ) isolated from HDAC6 KO MEFs were treated or not with latrunculin ( LatA ) as indicated subjected to in vitro fusion .

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(F) Autophagosomes (APGs) and lysosomes (Lys) isolated from HDAC6 KO MEFs were treated or not with latrunculin (LatA) as indicated and subjected to in vitro fusion assay.


( A ) Biochemical characterization of autophagic compartments isolated from HDAC6 KO . Different subcellular fractions ( 75 μg ) isolated from wild type ( WT ) were subjected to SDS - PAGE immunoblotting for indicated . Hom , homogenate ; APG , autophagosomes ; APL , autophagolysosomes ; Lys , lysosomes .

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(A) Biochemical characterization of autophagic compartments isolated from HDAC6 KO cells. Different subcellular fractions (75 μg protein) isolated from the wild type (WT) were subjected to SDS-PAGE and immunoblotting for the indicated proteins. Hom, homogenate; APG, autophagosomes; APL, autophagolysosomes; Lys, lysosomes.


( B ) Autophagosomes ( APGs ) lysosomes ( Lys ) isolated from fed were treated or not with latrunculin ( LatA ) as labelled subjected to in vitro fusion . differences with untreated samples were significant at * * P0 . 01 .

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(B) Autophagosomes (APGs) and lysosomes (Lys) isolated from fed cells were treated or not with latrunculin (LatA) as labelled and subjected to in vitro fusion assay. The differences with untreated samples were significant at **P0.01.


( C ) WT ATG5 KO MEFs were treated with MG132 immunostained with antibodies to ubiquitin ( green ) as indicated . F ‐ actin was detected by phalloidin ( red ) . arrows indicated ubiquitin ‐ positive .

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(C) WT and ATG5 KO MEFs were treated with MG132 and immunostained with antibodies to ubiquitin (green) as indicated. F‐actin was detected by phalloidin (red). The arrows indicated ubiquitin‐positive protein aggregates.


( A ) Wild ‐ type HDAC6 KO MEFs were treated with MG132 immunostained with antibodies against cortactin ( red ) , ubiquitin ( green ) , phalloidin for F ‐ actin ( blue ) as indicated . arrows indicated ubiquitin ‐ positive that were colocalized with F ‐ actin cortactin .

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(A) Wild‐type and HDAC6 KO MEFs were treated with MG132 and immunostained with antibodies against cortactin (red), ubiquitin (green), and phalloidin for F‐actin (blue) as indicated. The arrows indicated ubiquitin‐positive aggregates that were colocalized with F‐actin and cortactin.


( B ) Wild ‐ type MEFs were transfected with control or cortactin siRNA , treated with MG132 , stained with antibodies for Lap ‐ 1 ( red , to label lysosome ) , or ubiquitin ( green ) phalloidin for actin ( blue ) . Note that F ‐ actin at was lost , but lysosomes remained concentrated in cortactin knockdown ( arrow ) .

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(B) Wild‐type MEFs were transfected with control or cortactin siRNA, treated with MG132, and stained with antibodies for Lap‐1 (red, to label lysosome), or ubiquitin (green) and phalloidin for actin (blue). Note that F‐actin staining at protein aggregates was lost, but lysosomes remained concentrated in cortactin knockdown cells (arrow).


( C ) Wild ‐ type MEFs were transfected with control or cortactin siRNA , treated MG132 2 . 5 μM for 18 h , subjected to filter ‐ trap using a ubiquitin antibody . knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin GAPDH in right panel .

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(C) Wild‐type MEFs were transfected with control or cortactin siRNA, treated MG132 2.5 μM for 18 h, and subjected to filter‐trap assay using a ubiquitin antibody. The knockdown level of endogenous cortactin was confirmed by immunoblotting using an antibody against cortactin and GAPDH in the right panel.


( D ) U2OS were transfected with control siRNA cortactin siRNA . Autophagosome - lysosome fusion was analysed with or without starvation ( 6 h ) using mCherry ‐ GFP ‐ LC3 reporter as described in Figure 3A .

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(D) U2OS cells were transfected with control siRNA and cortactin siRNA. Autophagosome-lysosome fusion was analysed with or without starvation (6 h) using the mCherry‐GFP‐LC3 reporter as described in Figure 3A.


( E ) mCherry ‐ GFP ‐ LC3 plasmid was cotransfected with wild type , 9KQ ( acetylation ‐ mimic ) , or 9KR ( deacetylation ‐ mimic ) cortactin ‐ expressing plasmids into wild ‐ type MEFs . Autophagosome - lysosome fusion was analysed as described in Figure 3A .

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(E) The mCherry‐GFP‐LC3 plasmid was cotransfected with wild type, 9KQ (acetylation‐mimic), or 9KR (deacetylation‐mimic) cortactin‐expressing plasmids into wild‐type MEFs. Autophagosome-lysosome fusion was analysed as described in Figure 3A.


( A ) HDAC6 KO mice accumulate ubiquitin ‐ positive in brain . hippocampus cerebral cortex regions from 6 ‐ month ‐ old wild ‐ type ubiquitin KO were subjected to immunostaining with a ubiquitin antibody counterstained with hematoxylin . red arrows indicate ubiquitin ‐ positive neuritic black arrows indicate . These ubiquitin ‐ positive structures were rarely observed in control . Scale bar , 50

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

(A) HDAC6 KO mice accumulate ubiquitin‐positive protein aggregates in the brain. The hippocampus and cerebral cortex regions from 6‐month‐old wild‐type and ubiquitin KO littermates were subjected to immunostaining with a ubiquitin antibody and counterstained with hematoxylin. The red arrows indicate ubiquitin‐positive neuritic aggregates and black arrows indicate cytoplasmic aggregates. These ubiquitin‐positive structures were rarely observed in control littermates. Scale bar, 50 μm


( B ) Apoptotic in cortex hippocampus region of HDAC6 KO mice as determined by TUNEL . Apoptotic were only observed in HDAC6 KO mice . Scale bar , 100 μm .

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(B) Apoptotic cell death in the cortex and hippocampus region of HDAC6 KO mice as determined by TUNEL staining. Apoptotic cells were only observed in HDAC6 KO mice. Scale bar, 100 μm.


( C ) HDAC6 depletion in Drosophila eye leads to ubiquitin ‐ positive pathology . Immunostaining for ubiquitin ( green ) in frontal eye sections of 1 ‐ day ‐ old ( d1 ) 30 ‐ day ‐ old ( d30 ) fly eyes . eyes of HDAC6 ‐ depleted flies ( GMR : GAL4 / UAS ‐ HDAC6RNAi ) developed ubiquitin ‐ positive inclusions that become more prominent at day 30 ( d30 ) . Blue , DAPI .

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(C) HDAC6 depletion in the Drosophila eye leads to ubiquitin‐positive pathology. Immunostaining for ubiquitin (green) in frontal eye sections of 1‐day‐old (d1) and 30‐day‐old (d30) fly eyes. The eyes of HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) developed ubiquitin‐ positive cytoplasmic inclusions that become more prominent at day 30 (d30). Blue, DAPI.


( D ) Depletion of HDAC6 in Drosophila eye leads to ‐ dependent degeneration . ( left ) corresponding Richardson ‐ stained frontal eye sections ( right ) of 1 ‐ day ‐ old 30 ‐ day ‐ old fly eyes . eyes of control flies ( GMR : GAL4 / + ) HDAC6 ‐ depleted flies ( GMR : GAL4 / UAS ‐ HDAC6RNAi ) show normal highly organized ommatidial array at day 1 ; 30 ‐ day ‐ old control also show no defects , but 30 ‐ day ‐ old HDAC6 ‐ depleted flies show degeneration with disorganization of ommatidial array loss of normal eye architecture ( × 40 × 80 ) .

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(D) Depletion of HDAC6 in the Drosophila eye leads to age‐dependent degeneration. Light micrographs (left) and corresponding Richardson‐stained frontal eye sections (right) of 1‐day‐old and 30‐day‐old fly eyes. The eyes of control flies (GMR:GAL4/+) and HDAC6‐depleted flies (GMR:GAL4/UAS‐HDAC6RNAi) show normal highly organized ommatidial array at day 1; 30‐day‐old control animals also show no defects, but 30‐day‐old HDAC6‐depleted flies show degeneration with disorganization of the ommatidial array and loss of normal eye architecture ( × 40 and × 80).


( A , B ) mRNA of aub ( A ) vasa ( B ) were quantified upon depletion ( RNAi ) of L ( 3 ) mbt , Lint - 1 , CoRest , E2F2 , Myb , Mip120 , Mip130 , were compared with those in normal OSCs ( control ) . Data represent means ± SE ( n = 3 biological replicates ) . p values were calculated with t - . * p : < 0 . 05 , * * p : < 0 . 01 . t - were performed against samples with ⎾ symbol .

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

(A, B) The mRNA levels of aub (A) and vasa (B) were quantified upon the depletion (RNAi) of L(3)mbt, Lint-1, CoRest, E2F2, Myb, Mip120, and Mip130, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( C ) immunoprecipitated with anti - L ( 3 ) mbt antibody from OSC lysates before ( control ) after L ( 3 ) mbt knockdown ( KD ) were silver - stained . bands corresponding to L ( 3 ) mbt - L L ( 3 ) mbt - S are indicated .

(C) Proteins immunoprecipitated with the anti-L(3)mbt antibody from the OSC lysates before (control) and after L(3)mbt knockdown (KD) were silver-stained. The bands corresponding to L(3)mbt-L and L(3)mbt-S are indicated.


( F , G ) mRNA of vasa ( F ) aub ( G ) were quantified upon depletion ( RNAi ) of L ( 3 ) mbt , Atac3 , Prod , CG2662 / Lint - O , CG2199 , were compared with those in normal OSCs ( control ) . Data represent means ± SE ( n = 3 biological replicates ) . p values were calculated with t - . * * p : < 0 . 01 . t - were performed against samples with ⎾ symbol .

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(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon the depletion (RNAi) of L(3)mbt, Atac3, Prod, CG2662/Lint-O, and CG2199, and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( B ) Immunoprecipitation ( IP ) / western blotting shows that WT Lint - O , but not its ∆ SAM mutant co - immunoprecipitated with L ( 3 ) mbt from OSC lysates . n . i . : non - immune IgG .

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(B) Immunoprecipitation (IP)/western blotting shows that WT Lint-O, but not its ∆SAM mutant co-immunoprecipitated with L(3)mbt from the OSC lysates. n.i.: non-immune IgG.


( C ) Subcellular localization of WT Lint - O its ∆ SAM mutant ( green ) . Both were localized to nuclei ( DAPI : blue ) . Scale bar : 5 μm .

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(C) Subcellular localization of WT Lint-O and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.


( D ) IP / western blotting shows that WT L ( 3 ) mbt - L , but not its ∆ SAM mutant co - immunoprecipitated with Lint - O from OSC lysates . n . i . : non - immune IgG .

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(D) IP/western blotting shows that WT L(3)mbt-L, but not its ∆SAM mutant co-immunoprecipitated with Lint-O from the OSC lysates. n.i.: non-immune IgG.


( E ) Subcellular localization of WT L ( 3 ) mbt - L its ∆ SAM mutant ( green ) . Both were localized to nuclei ( DAPI : blue ) . Scale bar : 5 μm .

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(E) Subcellular localization of WT L(3)mbt-L and its ∆SAM mutant (green). Both proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.


( F , G ) mRNA of vasa ( F ) aub ( G ) were quantified upon ectopic expression of EGFP , as well as WT Lint - O its ∆ SAM 8CA mutants ( Fig EV3A ) , in Lint - O - lacking OSCs ( Lint - O RNAi ) were compared with those in normal OSCs ( control ) . Data represent means ± SE ( n = 3 biological replicates ) . p values were calculated with t - . * p : < 0 . 05 . t - were performed against samples with ⎾ symbol .

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(F, G) The mRNA levels of vasa (F) and aub (G) were quantified upon ectopic expression of EGFP, as well as WT Lint-O and its ∆SAM and 8CA mutants (Fig EV3A), in Lint-O-lacking OSCs (Lint-O RNAi) and were compared with those in normal OSCs (control). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05. All t-tests were performed against samples with⎾ symbol.


( H ) IP / western blotting shows that L ( 3 ) mbt co - immunoprecipitated with WT Lint - O its 8CA mutant , but not with ∆ PHD mutant An asterisk shows background .

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(H) IP/western blotting shows that L(3)mbt co-immunoprecipitated with WT Lint-O and its 8CA mutant, but not with ∆PHD mutant An asterisk shows background.


( I ) Subcellular localization of WT Lint - O its 8CA ∆ PHD mutants ( green ) . were localized to nuclei ( DAPI : blue ) . Scale bar : 5 μm .

(I) Subcellular localization of WT Lint-O and its 8CA and ∆PHD mutants (green). All proteins were localized to the nuclei (DAPI: blue). Scale bar: 5 μm.


( J ) ChIP - qPCR shows that L ( 3 ) mbt binding to vasa promoter was weakened after loss of Lint - O . Data represent means ± SE ( n = 3 biological replicates ) . p values were calculated with t - . * * p : < 0 . 01 . t - were performed against samples with ⎾ symbol .

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(J) ChIP-qPCR shows that L(3)mbt binding to the vasa promoter was weakened after the loss of Lint-O. Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( K ) Western blotting showing amounts of L ( 3 ) mbt , Lint - O , histone H3 ( H3 ) in normal ( control ) , L ( 3 ) mbt - depleted ( RNAi ) , Lint - O - depleted ( RNAi ) OSCs .

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(K) Western blotting showing the amounts of L(3)mbt, Lint-O, and histone H3 (H3) in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs.


( L ) lint - O mRNA in normal ( control ) , L ( 3 ) mbt - depleted ( RNAi ) , Lint - O - depleted ( RNAi ) OSCs are shown by fragments per kilobase million ( FPKM ) . Data represent means ± SE ( n = 3 biological replicates ) . p values were calculated with t - . * p : < 0 . 05 , * * p : < 0 . 01 . t - were performed against samples with ⎾ symbol .

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(L) The lint-O mRNA levels in normal (control), L(3)mbt-depleted (RNAi), and Lint-O-depleted (RNAi) OSCs are shown by fragments per kilobase million (FPKM). Data represent the means ± SE (n = 3 biological replicates). The p values were calculated with the t-test. *p: < 0.05, **p: < 0.01. All t-tests were performed against samples with⎾ symbol.


( B ) RT - qPCR shows mRNA of lint - O in y w Lint - OKO ovaries . Data are expressed as mean error bars represent SD . n = 2 biological replicates .

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(B) RT-qPCR analysis shows the mRNA levels of lint-O in y w and Lint-OKO ovaries. Data are expressed as mean and error bars represent SD. n = 2 biological replicates.


( D ) Confocal of y w Lint - OKO ovaries immunostained for Vasa . Scale bar : 50 μm .

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(D) Confocal images of y w and Lint-OKO ovaries immunostained for Vasa. Scale bar: 50 μm.


( E ) Confocal of y w Lint - OKO ovarioles immunostained for Vasa . Vasa was ectopically expressed in follicle ( arrowheads ) . Scale bar : 50 μm .

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(E) Confocal images of y w and Lint-OKO ovarioles immunostained for Vasa. Vasa was ectopically expressed in follicle cells (white arrowheads). Scale bar: 50 μm.


( A ) Western blotting shows that Vasa , Piwi , Aub were ectopically expressed in Lint - OKO L ( 3 ) mbtts1 larval brains at 29ºC . Anti - Lint - O , anti - L ( 3 ) mbt , anti - Vasa , andi - Piwi , anti - Aub , anti - Tubulin antibodies were used .

(A) Western blotting shows that Vasa, Piwi, and Aub were ectopically expressed in Lint-OKO and L(3)mbtts1 larval brains at 29ºC. Anti-Lint-O, anti-L(3)mbt, anti-Vasa, andi-Piwi, anti-Aub, and anti-Tubulin antibodies were used.


( B ) Confocal of y w , Lint - OKO , L ( 3 ) mbtts1 immunostained for Vasa ( magenta ) , MIRA ( green ) , ELAV ( green ) . Scale bar : 100 μm .

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(B) Confocal images of y w, Lint-OKO, and L(3)mbtts1 immunostained for Vasa (magenta), MIRA (green), and ELAV (green). Scale bar: 100 μm.


( C ) of brain lobe volume for following genotypes : Lint - OKO - / + ( n = 42 biological replicates ) , Lint - OKO - / - ( n = 36 biological replicates ) , L ( 3 ) mbtts1 - / - ( n = 34 biological replicates ) . Boxplot central bands , upper edges of boxes , lower edges of boxes , upper whiskers lower whiskers show median , third quartile , first quartile , maxima , minima , respectively . P - values were calculated by Student ' s t - . ( * * * P - value < 0 . 001 ) .

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(C) Quantification of brain lobe volume for the following genotypes: Lint-OKO -/+ (n = 42 biological replicates), Lint-OKO -/- (n = 36 biological replicates), and L(3)mbtts1 -/- (n = 34 biological replicates). Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively. P-values were calculated by the Student's t-test. (***P-value < 0.001).


( D , E ) genomic regions harboring vasa ( D ) aub ( E ) genes . - seq reads in y w , L ( 3 ) mbtts1 , Lint - OKO brains are shown . shading in gray corresponds to exons . y - axis shows number of RPM . - seq samples were biological triplicates .

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(D, E) The genomic regions harboring the vasa (D) and aub (E) genes. The RNA-seq reads in y w, L(3)mbtts1, and Lint-OKO brains are shown. The shading in gray corresponds to exons. The y-axis shows the number of RPM. RNA-seq samples were biological triplicates.


RT ‐ PCR performed on cDNA from isolated from splenic B derived from Tam ‐ treated mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / flmice ( right lane ) ; Syk ‐ Hprt ‐ specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control .

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RT‐PCR performed on cDNA from RNA isolated from splenic B cells derived from Tam‐treated mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/flmice (right lane); the Syk‐ and Hprt‐specific amplification products were identified by agarose gel electrophoresis with Hprt used as an endogenous loading control.


Immunoblot of isolated from B derived from spleens of mb1 ‐ CreERT2 ( left lane ) or mb1 ‐ CreERT2 ; Sykfl / fl ( right lane ) mice both treated with Tam as described ; blots were probed with anti ‐ Syk anti ‐ GAPDH Ab , GAPDH being used as a loading control .

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Immunoblot analysis of proteins isolated from B cells derived from spleens of mb1‐CreERT2 (left lane) or mb1‐CreERT2;Sykfl/fl (right lane) mice both treated with Tam as described; blots were probed with anti‐Syk and anti‐GAPDH Ab, GAPDH being used as a loading control.


Intracellular flow cytometric for Syk expression in B derived from LN of Syk ‐ deficient control mice .

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Intracellular flow cytometric analysis for Syk expression in B cells derived from the LN of Syk‐deficient and control mice.


Genomic of Tam ‐ treated mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / flmice ( as indicated ) . Upper row : amplification of floxed ( fl ) wt ( + ) alleles . Middle row : amplification of deleted ( d ) allele . Lower row : loading control ( TC21 , a gene unaffected by deletion of Syk ) . Established control was used to visualize bands ( as indicated ) .

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Genomic DNA analysis of Tam‐treated mb1‐CreERT2 or mb1‐CreERT2;Sykfl/flmice (as indicated). Upper row: amplification of floxed (fl) and wt (+) alleles. Middle row: amplification of the deleted (d) allele. Lower row: loading control (TC21, a gene unaffected by the deletion of Syk). Established control DNA was used to visualize the individual bands (as indicated).


A , B Flow cytometric of B from ( A ) BM ( B ) SP of mb1 ‐ CreERT2 control ( left ) mb1 ‐ CreERT2 ; Sykfl / flmice ( right ) treated with Tam as described in Materials Methods section . BM were stained with anti ‐ IgM anti ‐ B220 , SP with anti ‐ CD19 anti ‐ CD93 or anti ‐ CD23 anti ‐ CD21 . gated regions in dot blots correspond to B ‐ populations : ( A ) Bone marrow : gate P ( B220 + IgM − ) pro ‐ / pre ‐ B , gate I ( B220loIgM + ) immature B , gate M ( B220hiIgM + ) mature B ; ( B ) spleen : gate T ( CD19 + CD93 + ) transitional B ( T ) , gate M ( CD19 + CD93 − ) mature B ; in CD93 − splenic B , gate M ( CD23hiCD21int CD93 − ) mature follicular B gate MZ ( CD23lo CD21hi CD93 − ) marginal zone B . numbers in dot plots indicate mean relative frequency of in gate . C - E Statistical of absolute numbers per Tam ‐ treated mouse : filled circles indicating obtained from mb1 ‐ CreERT2 control mice open circles from mb1 ‐ CreERT2 ; Sykfl / flmice . ( C ) Statistical of absolute numbers of pro ‐ / pre ‐ , immature mature recirculating B in BM . ( D ) Absolute numbers of transitional , M MZ B found in SP . ( E ) Statistical of absolute numbers of peritoneal B subsets B1 ‐ a B1 ‐ b . An asterisk ( * ) marks statistically significant differences ( P 0 . 05 ) , two asterisks ( * * ) indicate P 0 . 01 , P ‐ values were obtained using two ‐ Student ' s t ‐ . numbers of four to five mice per group are shown , with each dot representing an .

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A, B Flow cytometric analysis of B cells from (A) the BM and (B) the SP of mb1‐CreERT2 control (left) and mb1‐CreERT2;Sykfl/flmice (right) treated with Tam as described in the Materials and Methods section. The BM cells were stained with anti‐IgM and anti‐B220, and the SP cells with anti‐CD19 and anti‐CD93 or anti‐CD23 and anti‐CD21. The gated regions in the dot blots correspond to individual B‐cell populations: (A) Bone marrow: gate P (B220+IgM−) pro‐/pre‐B cells, gate I (B220loIgM+) immature B cells, gate M (B220hiIgM+) mature B cells; (B) spleen: gate T (CD19+ CD93+) transitional B cells (T), gate M (CD19+ CD93−) mature B cells; in CD93−splenic B cells, gate M (CD23hiCD21int CD93−) mature follicular B cells and gate MZ (CD23lo CD21hi CD93−) marginal zone B cells. The numbers in the dot plots indicate the mean relative frequency of cells in the gate.C-E Statistical analysis of absolute cell numbers per Tam‐treated mouse: filled circles indicating cells obtained from mb1‐CreERT2 control mice and open circles from mb1‐CreERT2;Sykfl/flmice. (C) Statistical analysis of the absolute cell numbers of pro‐/pre‐, immature and mature recirculating B cells in the BM. (D) Absolute cell numbers of transitional, M and MZ B cells found in the SP. (E) Statistical analysis of the absolute cells numbers of peritoneal B cell subsets B1‐a and B1‐b. An asterisk (*) marks statistically significant differences (P 0.05), two asterisks (**) indicate P 0.01, P‐values were obtained using two‐tailed Student's t‐test. Cell numbers of four to five mice per group are shown, with each dot representing an individual animal.


A Flow cytometric of IgM , CD19 CD23 expression of splenic B from Tam ‐ treated mb1 ‐ CreERT2 control ( dashed ) mb1 ‐ CreERT2 ; Sykfl / flmice ( solid ) .

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A Flow cytometric analysis of IgM, CD19 and CD23 expression of splenic B cells from Tam‐treated mb1‐CreERT2 control (dashed line) and mb1‐CreERT2;Sykfl/flmice (solid line).


B Flow cytometric of tyrosine phosphorylation ( pY ) in splenic B from Tam ‐ treated mb1 ‐ CreERT2 mb1 ‐ CreERT2 ; Sykfl / flmice stimulated with medium ( dotted ) or 50 μM pervanadate ( dashed solid lines ) .

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B Flow cytometric analysis of tyrosine phosphorylation (pY) in splenic B cells from Tam‐treated mb1‐CreERT2 and mb1‐CreERT2;Sykfl/flmice stimulated with medium (dotted line) or 50 μM pervanadate (dashed and solid lines).


C , D Flow cytometric of intracellular Ca2 + influx in purified splenic B derived from Tam ‐ treated mice . mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl were treated with ( C ) 10 μg / ml anti ‐ Kappa F ( ab ′ ) 2 fragments or ( D ) 1 μM ionomycin .

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C, D Flow cytometric analysis of the intracellular Ca2+ influx in purified splenic B cells derived from Tam‐treated mice. mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl were treated with (C) 10 μg/ml anti‐Kappa F(ab′)2 fragments or (D) 1 μM ionomycin.


Ex vivo activation of splenic mature B from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . Purified splenic B were either left unstimulated ( medium ) or were stimulated with indicated stimuli . were analyzed by flow cytometry after 24 h . Shown are CD19 versus CD86 dot plots . 7 ‐ AAD was included to distinguish dead from viable . numbers in quadrants indicate relative frequencies of in gate .

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Ex vivo activation assay of splenic mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). Purified splenic B cells were either left unstimulated (medium) or were stimulated with the indicated stimuli. The cells were analyzed by flow cytometry after 24 h. Shown are CD19 versus CD86 dot plots. 7‐AAD was included to distinguish dead from viable cells. The numbers in the quadrants indicate the relative frequencies of cells in the gate.


Ex vivo proliferation with mature B from Tam ‐ treated mb1 ‐ CreERT2 ( upper row ) or Tam ‐ treated mb1 ‐ CreERT2 ; Sykfl / flmice ( lower row ) . were incubated with indicated stimuli for 90 h .

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Ex vivo proliferation assay with mature B cells from Tam‐treated mb1‐CreERT2 (upper row) or Tam‐treated mb1‐CreERT2;Sykfl/flmice (lower row). The cells were incubated with the indicated stimuli for 90 h.


A Flow cytometric of splenic B from Rag2 − / − ; γc − / − mice . CD19 versus CD3ε dot plots are shown . mice were injected i . v . with 5 × 106 splenic ( CD19 + CD93 − ) B ( CD3ε + ) T from Tam ‐ untreated mb1 ‐ CreERT2 control or mb1 ‐ CreERT2 ; Sykfl / fl mice . recipient mice were treated with Tam beginning 1 day after transfer as described in Materials Methods section . B Quantitative of B T from Rag2 − / − ; γc − / − mice repopulated with mb1 ‐ CreERT2 or mb1 ‐ CreERT2 ; Sykfl / fl splenocytes . Each symbol represents an mouse . Filled circles squares represent Syk + / + B T , respectively ; open circles squares represent Sykfl / fl B T .

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A Flow cytometric analysis of splenic B cells from Rag2−/−;γc−/− mice. CD19 versus CD3ε dot plots are shown. The mice were injected i.v. with 5 × 106 splenic (CD19+ CD93−) B cells and (CD3ε+) T cells from Tam‐untreated mb1‐CreERT2 control or mb1‐CreERT2;Sykfl/fl mice. The recipient mice were treated with Tam beginning 1 day after transfer as described in the Materials and Methods section.B Quantitative analysis of B and T cells from Rag2−/−;γc−/− mice repopulated with mb1‐CreERT2 or mb1‐CreERT2;Sykfl/fl splenocytes. Each symbol represents an individual mouse. Filled circles and squares represent Syk+/+ B and T cells, respectively; open circles and squares represent Sykfl/fl B and T cells.


Demonstration of translational readthrough of AGO1 using luciferase - reporter . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - FLuc were transfected in HEK293 translational readthrough was detected as FLuc activity normalized to activity of co - transfected renilla luciferase ( RLuc ) . FLuc mRNA determined by RT - PCR are shown below . * * * , P = 0 . 0001 .

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Demonstration of translational readthrough of AGO1 using luciferase-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-FLuc were transfected in HEK293 cells and translational readthrough was detected as FLuc activity normalized to the activity of co-transfected renilla luciferase (RLuc). FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0001.


Significance of identity of canonical stop codon in AGO1 translational readthrough . AGO1 - ISR - FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 translational readthrough was quantified as described above . FLuc mRNA determined by RT - PCR are shown below . * * * , P = 0 . 0002 ; * * , P = 0 . 0004 .

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Significance of the identity of the canonical stop codon in AGO1 translational readthrough. AGO1-ISR-FLuc constructs containing TGA or TAA or TAG were transfected in HEK293 cells and translational readthrough was quantified as described above. FLuc mRNA levels determined by RT-PCR are shown below. ***, P = 0.0002; **, P = 0.0004.


First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough . AGO1 - TGA - FLuc constructs with different lengths of ISR ( in - frame with AGO1 FLuc ) were in vitro transcribed in vitro translated using rabbit reticulocyte lysate . FLuc activity reflects readthrough activity . * * , P = 0 . 012 ; * , P = 0 . 017 .

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First 57 nucleotides of ISR are sufficient to drive AGO1 translational readthrough. AGO1-TGA-FLuc constructs with different lengths of ISR (all in-frame with AGO1 and FLuc) were in vitro transcribed and in vitro translated using rabbit reticulocyte lysate. FLuc activity reflects readthrough activity. **, P = 0.012; *, P = 0.017.


Demonstration of translational readthrough of AGO1 using - reporter . Plasmid containing in - frame AGO1 - TGA ( or GCA ) - ISR - GFP were transfected in HEK293 translational readthrough was detected as . Scale bar , 50 um . bar graph shows mean intensities in transfected with indicated constructs . was measured by flowcytometry . * * * , P = 0 . 007 .

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Demonstration of translational readthrough of AGO1 using fluorescence-based reporter assay. Plasmid containing in-frame AGO1-TGA (or GCA)-ISR-GFP were transfected in HEK293 cells and translational readthrough was detected as fluorescence. Scale bar, 50 um. The bar graph shows mean fluorescence intensities in cells transfected with indicated constructs. Fluorescence intensity was measured by flowcytometry. ***, P = 0.007.


Detection of endogenous Ago1x by Western blot in HEK293 using anti - Ago1x antibody pre - immune serum .

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Detection of endogenous Ago1x by Western blot in HEK293 cells using anti-Ago1x antibody and pre-immune serum.


Western blot of lysates from HEK293 HeLa using anti - Ago1x antibody pre - treated with Ago1x - specific peptide .

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Western blot of lysates from HEK293 and HeLa cells using anti-Ago1x antibody pre-treated with Ago1x-specific peptide.


Expression of Ago1x in HEK293 transfected with three AGO1 - specific siRNAs ( 1 , 2 , 3 ) . qRT - PCR of AGO1 transcript expression in these is shown below .

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Expression of Ago1x in HEK293 cells transfected with three AGO1-specific siRNAs (1,2,3). qRT-PCR analysis of AGO1 transcript expression in these cells is shown below.


Proportion of Ago1x isoform in HEK293 . To separate Ago1x Ago1 ( ~ 3 kDa difference ) , lysate was electrophoresed for 3 . 5 h in 10 % polyacrylamide gel . Proportion of Ago1x canonical isoform ( Ago1 ) was calculated by densitometric . Results from three independent experiments are shown as a bar graph ( mean ± S . E . ) .

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Proportion of Ago1x isoform in HEK293 cells. To separate Ago1x and Ago1 (~ 3 kDa difference), the cell lysate was electrophoresed for 3.5 h in 10% polyacrylamide gel. Proportion of Ago1x and canonical isoform (Ago1) was calculated by densitometric analysis. Results from three independent experiments are shown as a bar graph (mean ± S.E.).


Expression of Ago1x in mouse organs.


Presence of ribosome footprint in Inter stop codon region of AGO1 transcript in U2 - OS . Ribosome profiling data was taken from NCBI ' s read archive ( SRA Accession no . SRR1257257 ) ribosome footprints on AGO1 transcript were analyzed as described in Methods section . Zoomed near inter - stop codon region is shown in inset . Position of canonical stop codon downstream in - frame stop codon are shown . Detection of Ago1x by Western blot in U2 - OS is shown ( right ) .

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Presence of ribosome footprint in the Inter stop codon region of AGO1 transcript in U2-OS cells. Ribosome profiling data was taken from NCBI's sequence read archive (SRA Accession no. SRR1257257) and ribosome footprints on AGO1 transcript were analyzed as described in Methods section. Zoomed area near the inter-stop codon region is shown in the inset. Position of canonical stop codon and the downstream in-frame stop codon are shown. Detection of Ago1x by Western blot in U2-OS cells is shown (right).


Effect of let - 7a inhibitor on translational readthrough of AGO1 . let - 7a inhibitor or control inhibitor was transfected in HeLa expression of Ago1x was determined by Western blot after 48 h . Densitometric of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 01 .

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Effect of let-7a inhibitor on translational readthrough of AGO1. let-7a inhibitor or control inhibitor was transfected in HeLa cells and the expression of Ago1x was determined by Western blot after 48 h. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.01.


Effect of let - 7a overexpression on translational readthrough of AGO1 . HeLa were stably transfected with pri - let - 7a overexpression construct . Ago1x expression was determined by Western blot . Densitometric of three independent experiments is shown as a bar diagram ( mean ± S . E . ) . * , P = 0 . 007 .

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Effect of let-7a overexpression on translational readthrough of AGO1. HeLa cells were stably transfected with pri-let-7a overexpression construct. Ago1x expression was determined by Western blot. Densitometric analysis of three independent experiments is shown as a bar diagram (mean ± S.E.). *, P = 0.007.


let - 7a inhibitor reduces AGO1 readthrough efficiency . HeLa were transfected with let - 7a inhibitor or control inhibitor along with AGO1 - TGA ( or GCA ) - ISR - FLuc construct . Readthrough was quantified by measuring FLuc activity , which was normalized to co - transfected renilla luciferase activity . FLuc mRNA expression in HeLa treated with miRNA inhibitors is shown ( Right ) . * , P = 0 . 027 .

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let-7a inhibitor reduces the AGO1 readthrough efficiency. HeLa cells were transfected with let-7a inhibitor or control inhibitor along with AGO1-TGA (or GCA)-ISR-FLuc construct. Readthrough was quantified by measuring FLuc activity, which was normalized to co-transfected renilla luciferase activity. FLuc mRNA expression in HeLa cells treated with miRNA inhibitors is shown (Right). *, P = 0.027.


AGO1 ISR with mutated let - 7a binding site shows reduced translational readthrough activity . was done as described above in HeLa . Mutated is shown ( top ) . * * * , P = 0 . 008 .

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AGO1 ISR with mutated let-7a binding site shows reduced translational readthrough activity. Assay was done as described above in HeLa cells. Mutated sequence is shown (top). ***, P = 0.008.


AGO1 ISR with displaced let - 7a binding site ( AGO1 - TGA - disISR - FLuc ) shows reduced translational readthrough activity . let - 7a binding site was moved 18 nucleotides downstream of stop codon . Readthrough was done as described above in HeLa . * * * , P < 0 . 0001 .

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AGO1 ISR with displaced let-7a binding site (AGO1-TGA-disISR-FLuc) shows reduced translational readthrough activity. let-7a binding site was moved 18 nucleotides downstream of the stop codon. Readthrough assay was done as described above in HeLa cells. ***, P < 0.0001.


AGO1 ISR can drive translational readthrough in a heterologous context of RHOA . AGO1 coding in AGO1 - TGA - ISR - FLuc construct was replaced by RHOA coding ( 582 nucleotides ) readthrough was done as described above in HeLa . 3 ′ UTRRho , 159 nucleotides in proximal 3 ′ UTR of RHOA ( NM _ 001664 ) ; ISRAgo1 , Inter - stop codon region of AGO1 . * * * , P < 0 . 0001 .

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AGO1 ISR can drive translational readthrough in a heterologous context of RHOA. AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RHOA coding sequence (582 nucleotides) and the readthrough assay was done as described above in HeLa cells. 3′UTRRho, 159 nucleotides in the proximal 3′UTR of RHOA (NM_001664); ISRAgo1, Inter-stop codon region of AGO1. ***, P < 0.0001.


AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase ( RLuc ) . AGO1 coding in AGO1 - TGA - ISR - FLuc construct was replaced by RLuc coding readthrough was done as described above in HeLa ( left ) in vitro using rabbit reticulocyte lysate system ( right ) . * * , P = 0 . 006 ; * * * , P = 0 . 0001 .

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AGO1 ISR can drive translational readthrough in a heterologous context of renilla luciferase (RLuc). AGO1 coding sequence in AGO1-TGA-ISR-FLuc construct was replaced by RLuc coding sequence and the readthrough assay was done as described above in HeLa cells (left) and in vitro using rabbit reticulocyte lysate system (right). **, P = 0.006; ***, P = 0.0001.


Tethering of N - peptide - tagged Ago1 or Ago1x can induce readthrough of AGO1 . Two BoxB elements were cloned downstream of AGO1 upstream of in - frame FLuc as shown in schematic . This construct was transfected in HeLa expressing N - peptide - tagged or FLAG - tagged Ago as indicated . Readthrough was measured as described above . * , P = 0 . 035 ( with Welch correction ) ; * * , P = 0 . 016 .

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Tethering of N-peptide-tagged Ago1 or Ago1x can induce readthrough of AGO1. Two BoxB elements were cloned downstream of the AGO1 and upstream of the in-frame FLuc as shown in the schematic. This construct was transfected in HeLa cells expressing N-peptide-tagged or FLAG-tagged Ago proteins as indicated. Readthrough was measured as described above. *, P = 0.035 (with Welch correction); **, P = 0.016.


Linear regression of miRNAs co - immunoprecipiated with Ago1 those co - immunoprecipiated with Ago1x in HeLa . Reads per million ( RPM ) values of 395 miRNAs , which were common to Ago1 Ago1x are shown .

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Linear regression analysis of miRNAs co-immunoprecipiated with Ago1 and those co-immunoprecipiated with Ago1x in HeLa cells. Reads per million (RPM) values of 395 miRNAs, which were common to Ago1 and Ago1x are shown.


qRT - PCR results showing enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa . Bars show mean of three experiments ± S . E .

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qRT-PCR results showing the enrichment of seven miRNAs in Ago1x immunoprecipitate relative to that in Ago1 immunoprecipitate in HeLa cells. Bars show mean of three experiments ± S.E.


RT - PCR results showing PTEN MYC mRNAs co - immunoprecipitated with Ago1 or Ago1x in HeLa .

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RT-PCR results showing PTEN and MYC mRNAs co-immunoprecipitated with Ago1 or Ago1x in HeLa cells.


Result of co - immunoprecipitation experiments demonstrating interaction of Dicer with Ago1 Ago1x in HeLa .

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Result of co-immunoprecipitation experiments demonstrating the interaction of Dicer protein with Ago1 and Ago1x in HeLa cells.


Result of co - immunoprecipitation experiment in HeLa shows that GW182 is co - immunoprecipitated with Ago1 , but not with Ago1x .

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Result of co-immunoprecipitation experiment in HeLa cells shows that GW182 is co-immunoprecipitated with Ago1, but not with Ago1x.


Confocal immunofluoresence of HeLa show co - localization of GW182 with Ago1 , but not with Ago1x . circles indicate zoomed areas .

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Confocal immunofluoresence images of HeLa cells show co-localization of GW182 with Ago1, but not with Ago1x. White circles indicate zoomed areas.


Confocal microscopy of proximity ligation ( PLA ) in HeLa showing interaction of GW182 with Ago1 , but not with Ago1x . Rabbit anti - GW182 Mouse anti - FLAG antibodies were used to achieve proximity ligation . Interaction is indicated by red dots that results from proximity ligation . of dots per is shown ( right ) . Each black circle represents a microscopic field . Mean ± S . E . are shown as horizontal lines ( n = 10 fields for vector control ; n = 12 fields for Ago1 ; n = 11 fields for Ago1x ) . At least 100 were counted for dots in each category . * * * , P < 0 . 0001 ( Mann - Whitney ) .

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Confocal microscopy images of proximity ligation assay (PLA) in HeLa cells showing interaction of GW182 with Ago1, but not with Ago1x. Rabbit anti-GW182 and Mouse anti-FLAG antibodies were used to achieve proximity ligation. Interaction is indicated by red dots that results from proximity ligation. Quantification of dots per cell is shown (right). Each black circle represents a microscopic field. Mean ± S.E. are shown as horizontal lines (n=10 fields for vector control; n=12 fields for Ago1; n=11 fields for Ago1x). At least 100 cells were counted for dots in each category. ***, P < 0.0001 (Mann-Whitney test).


Activity of RLuc relative to co - transfected FLuc in HeLa expressing N - HA - tagged or untagged Ago as indicated . Bar graph ( mean ± S . E . ) is a representative of four independent experiments done in triplicates . Expression of HA - tagged Ago was confirmed by Western blot ( bottom ) . * , P = 0 . 018 ; # , P = 0 . 041 ; $ , P = 0 . 013 . Numbers on bars represent % change in relative luciferase activity .

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Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Bar graph (mean ± S.E.) is a representative of four independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). *, P = 0.018; #, P = 0.041; $, P = 0.013. Numbers on the bars represent % change in relative luciferase activity.


Expression of RLuc in HeLa expressing N - HA - tagged or untagged Ago as indicated . Densitometric of three independent experiments is shown as bar graph ( mean ± S . E . ) . RT - PCR for RLuc mRNA level is shown ( bottom ) . * * , P = 0 . 001 .

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Expression of RLuc protein in HeLa cells expressing N-HA-tagged or untagged Ago proteins as indicated. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). RT-PCR analysis for RLuc mRNA level is shown (bottom). **, P = 0.001.


Activity of RLuc relative to co - transfected FLuc in HeLa expressing N - HA - tagged Ago2 ( N - HA - Ago2 ) or N - HA - tagged chimeric Ago2 appended with ISR region of Ago1x ( N - HA - Ago2 - ISRAgo1 ) as indicated . Bar graph ( mean ± S . E . ) is a representative of three independent experiments done in triplicates . Expression of HA - tagged Ago was confirmed by Western blot ( bottom ) . * * * , P < 0 . 0001 ; * * , P = 0 . 0004 .

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Activity of RLuc relative to co-transfected FLuc in HeLa cells expressing N-HA-tagged Ago2 (N-HA-Ago2) or N-HA-tagged chimeric Ago2 appended with the ISR region of Ago1x (N-HA-Ago2-ISRAgo1) as indicated. Bar graph (mean ± S.E.) is a representative of three independent experiments done in triplicates. Expression of HA-tagged Ago proteins was confirmed by Western blot (bottom). ***, P < 0.0001; **, P = 0.0004.


Ribopuromycylation showing global translation profile of HeLa overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric of four independent experiments is shown as bar graph ( mean ± S . E . ) . Puromycin densities were normalized to ponceau S stain densities . * , P < 0 . 029 ( Mann - Whitney ) .

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Ribopuromycylation assay showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of four independent experiments is shown as bar graph (mean ± S.E.). Puromycin densities were normalized to ponceau S stain densities. *, P < 0.029 (Mann-Whitney test).


[ 35S ] - methionine autoradiogram showing global translation profile of HeLa overexpressing FLAG - HA - Ago1 or FLAG - HA - Ago1x . Densitometric of three independent experiments is shown as bar graph ( mean ± S . E . ) . Autoradiogram densities were normalized to Coomassie stain densities . * , P < 0 . 029 ( Mann - Whitney ) .

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[35S]-methionine autoradiogram showing global translation profile of HeLa cells overexpressing FLAG-HA-Ago1 or FLAG-HA-Ago1x. Densitometric analysis of three independent experiments is shown as bar graph (mean ± S.E.). Autoradiogram densities were normalized to Coomassie stain densities. *, P < 0.029 (Mann-Whitney test).


A Pancreatic sections ( 20X magnification ) from 10 - months old WT Glo1KD mice stained with hematoxylin eosin ( H & E ) , anti - insulin , anti - glucagon anti - F4 / 80 antibodies , are representative of sections from 7 mice each group .

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A Pancreatic sections (20X magnification) from 10-months old WT and Glo1KD mice stained with hematoxylin and eosin (H&E), anti-insulin, anti-glucagon and anti-F4/80 antibodies, are representative of sections from 7 mice each group.


H , I J Insulin glucagon IS , calculated as score multiplied with of positive ( H , I ) of islets stained with F4 / 80 ( J ) ( n = 7 mice for each genotype ) . Data information : box plots show minimum maximum values ( ends of whiskers ) , interquartile range ( of box ) , median ( through box ) mean ( + ) values . * p ≤ 0 . 05 , * * p ≤ 0 . 01 ( Mann - Whitney ) .

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H, I and J Insulin and glucagon IS, calculated as the intensity score multiplied with percentage of positive cells (H, I) and the percentage of islets area stained with F4/80 (J) (n=7 mice for each genotype). Data information: box plots show the minimum and maximum values (ends of the whiskers), interquartile range (length of the box), median (line through the box) and mean (+) values. *p≤0.05, **p≤0.01 (Mann-Whitney test).


C Whole lysates of INS - 1 832 / 13 β - analysed by Western Blot with anti - p53 anti - p21 antibodies ; anti - vinculin antibody was used as loading control ( n = 9 biological replicates for each treatment group ) . Data information : Data in bar graphs are presented as mean ± . In C D , blots show representative image of replicate experiments ; were quantified by densitometric of independent experiments showed in bar graphs as over not treated control . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 : MGO vs CTR ; # p ≤ 0 . 05 , # # p ≤ 0 . 01 , # # # p ≤ 0 . 001 : D / Q vs MGO ( Mann - Whitney

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C Whole cell protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-p53 and anti-p21 antibodies; anti-vinculin antibody was used as loading control (n=9 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test


D lysates of INS - 1 832 / 13 β - analysed by Western Blot with anti - HMGB1 antibody ; anti - histone H3 antibody was used as loading control ( n = 5 biological replicates for each treatment group ) . Data information : Data in bar graphs are presented as mean ± . In C D , blots show representative image of replicate experiments ; were quantified by densitometric of independent experiments showed in bar graphs as over not treated control . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 : MGO vs CTR ; # p ≤ 0 . 05 , # # p ≤ 0 . 01 , # # # p ≤ 0 . 001 : D / Q vs MGO ( Mann - Whitney

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D Nuclear protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-HMGB1 antibody; anti-histone H3 antibody was used as loading control (n=5 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . of IMM in living HeLa , L6 , H1975 , stained with 10 - N - nonyl acridine orange ( NAO ) . NAO preferentially binds phospholipids in IMM , such as cardiolipin . Arrowheads indicate cristae in IMM . Scale bar = 500 nm . N ≥ 3 independent experiments for each type .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . Mitochondrion cropped from HeLa shown in A ( dashed ) zoomed in to show cristae , inner boundary ( IBM ) , matrix ( arrows ) . Scale bar = 100 nm .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondrion cropped from HeLa cell shown in A (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . H1975 transduced with matrix - targeted - DsRed stained with NAO . Matrix - targeted Ds - Red differentiates matrix from cristae stained with NAO . Arrowheads point to cristae . Scale bar = 500 nm . N = 1 independent experiment .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . structure of IMM in Mic13 - KO ( HeLa ) , stained with NAO . number of cristae is decreased in Mic13 - KO compared to control shown in panel A , labeled with arrowheads . Scale bar = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . Live - imaging of IMM in control PTPMT1 KD H1975 , a model of cardiolipin deficiency . A gallery of mitochondria from various control H1975 expressing scrambled shRNA stained with NAO , showing cristae ( arrowheads ) . Scale bars = 500 nm . N = 3 independent experiments . A gallery of mitochondria from various H1975 expressing PTPMT1 shRNA stained with NAO . Note derangement of ultrastructure ( arrowheads ) . Scale bars = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . Mitochondria from HeLa , co - stained with NAO TMRE . from dashed box , zoomed to right , shows red green intensities of TMRE NAO colocalized . Scale bars = 500 nm . N = 3 independent experiments . Mitochondria in L6 myoblast , co - stained with NAO TMRE . from dashed box , zoomed to right , shows colocalizing NAO TMRE at cristae ( arrowheads ) . Scale bars = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria from HeLa cell, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows the red and green intensities of TMRE and NAO colocalized. Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in L6 myoblast, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows colocalizing NAO and TMRE at the cristae membrane (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.


High - resolution imaging of mitochondria in live using Airyscan module of Zeiss LSM880 confocal microscope . Mitochondria in HeLa , stained with NAO alone ( top row ) vs . NAO + TMRE ( bottom row ) , simultaneously excited with 488 - 561 - nm lasers . Note that mitochondria stained with NAO alone do not emit noticeable in red channel ; only after adding TMRE does strong signal appear in red channel , showing negligible bleed - through . Scale bars = 500 nm . N = 2 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488- and 561-nm lasers. Note that mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel; only after adding TMRE does strong signal appear in the red channel, showing negligible bleed-through. Scale bars = 500 nm. N = 2 independent experiments.


Time - lapse Airyscan imaging of ΔΨm in living HeLa , co - stained with MTG ( ΔΨm - insensitive after loading ) TMRE ( ΔΨm sensitive ) . Arrowhead points to a flickering event where a mitochondrion depolarizes ( ~ 9 sec ) repolarizes ( ~ 16 sec ) , showing that heterogeneous signal from TMRE ( but not MTG ) disappears reappears . Scale bar = 500 nm . N = 4 independent experiments . of A . Plot shows average TMRE ( FI ) of cristae ( red ) vs . matrix ( red ) during time series . drop in TMRE FI during depolarization phase of flickering is ΔΨm - sensitive component of TMRE signal . remaining TMRE FI during depolarization can be considered as ΔΨm - insensitive portion of TMRE signal . Note that remaining TMRE FI after depolarization at cristae matrix are approximately identical , indicating that differences in TMRE FI between cristae matrix prior to depolarization are derived from differences in ΔΨm . N = 4 independent experiments .

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Time-lapse Airyscan imaging of ΔΨm in living HeLa cell, co-stained with MTG (ΔΨm-insensitive after loading) and TMRE (ΔΨm sensitive). Arrowhead points to a flickering event where a mitochondrion depolarizes (~9 sec) and repolarizes (~16 sec), showing that heterogeneous signal from TMRE (but not MTG) disappears and reappears. Scale bar = 500 nm. N = 4 independent experiments. Quantification of A. Plot shows average TMRE fluorescence intensity (FI) of cristae (dark red line) vs. matrix (light red line) during the time series. The drop in TMRE FI during the depolarization phase of the flickering is the ΔΨm-sensitive component of the TMRE signal. The remaining TMRE FI during depolarization can be considered as the ΔΨm-insensitive portion of TMRE signal. Note that the remaining TMRE FI after depolarization at the cristae and matrix are approximately identical, indicating that differences in TMRE FI between the cristae and matrix prior to depolarization are derived from differences in ΔΨm. N = 4 independent experiments.


Live - Airyscan image of mitochondrion in HeLa , showing different potentials in different mitochondrial regions . potentials were calculated on TMRE FI differences between compartments ( FIcomp ) . Regions of interest from cristae IBM used for ΔΨm calculations are labeled with arrows on right - side of mitochondrion , using cytosol as reference compartment . Labeled with forks on left - side of mitochondrion are calculations of ΔΨm between cristae their neighboring IBM . Nernst equation used to calculate different voltages . Scale bar = 500 nm . LUT of mitochondrion in HeLa shown in A , color - coding TMRE FIs on scale of ( most intense ) to blue ( least intense ) : LUT scale shown in lower left - corner . Arrowheads indicate cristae . Scale bar = 500 nm . Note that most - intense pixels ( ) only associate with cristae . of ΔΨm ( mV ) at cristae IBM relative to average ΔΨm of whole mitochondrion , calculated as in A . Note : ΔΨm at cristae is significantly higher than ΔΨm at IBM , indicating electrochemical boundaries separate these two regions of IMM . N = 3 independent experiments .

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

Live-cell Airyscan image of mitochondrion in HeLa cell, showing different membrane potentials in different mitochondrial regions. Membrane potentials were calculated based on TMRE FI differences between compartments (FIcomp). Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right-hand side of the mitochondrion, using the cytosol as the reference compartment. Labeled with forks on the left-hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM. Nernst equation used to calculate different voltages. Scale bar = 500 nm. LUT of mitochondrion in HeLa cell shown in A, color-coding TMRE FIs on scale of white (most intense) to blue (least intense): LUT scale shown in lower left-hand corner. Arrowheads indicate cristae. Scale bar = 500 nm. Note that most-intense pixels (white) only associate with cristae. Quantification of ΔΨm (mV) at cristae and IBM relative to the average ΔΨm of the whole mitochondrion, calculated as in A. Note: ΔΨm at cristae is significantly higher than ΔΨm at IBM, indicating electrochemical boundaries separate these two regions of the IMM. N = 3 independent experiments.


Live - Airyscan imaging of mitochondria in cultured mouse hepatocytes stained with TMRE . Arrowheads denote cristae . D . Image showing TMRE - labeled mitochondria . E . LUT color - coding of TMRE FI . F . of ΔΨm differences using Nernst equation as shown in A . Scale bar = 500 nm . N = 2 independent experiments .

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Live-cell Airyscan imaging of mitochondria in cultured primary mouse hepatocytes stained with TMRE. Arrowheads denote cristae. D. Image showing TMRE-labeled mitochondria. E. LUT color-coding of TMRE FI. F. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 2 independent experiments.


Live - STED imaging of mitochondria in HeLa stained with TMRM . Arrowheads show cristae . Scale bar = 500 nm . G . Image showing TMRM - labeled mitochondrion . H . LUT color - coding of TMRM FI . I . of ΔΨm differences using Nernst equation as shown in A . Scale bar = 500 nm . N = 4 independent experiments . Note that calculation of ΔΨm differences between compartments on captured by STED by Airyscan were very similar .

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Live-cell STED imaging of mitochondria in HeLa cell stained with TMRM. Arrowheads show cristae. Scale bar = 500 nm. G. Image showing TMRM-labeled mitochondrion. H. LUT color-coding of TMRM FI. I. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 4 independent experiments. Note that calculation of ΔΨm differences between the compartments based on images captured by STED and by Airyscan were very similar.


Response of ΔΨm to F1Fo ATP Synthase inhibitor , oligomycin ( 10 µM ) , compared to uncoupler , FCCP ( 10 µM ) , determined using live - Airyscan imaging of HeLa co - stained with MTG TMRE . of total mitochondrial TMRE FI . N = 3 independent experiments . of cristae ΔΨm relative to IBM ( ΔΨCr - IBM ) in response to oligomycin compared to FCCP . F1Fo ATP Synthase is exclusively localized to cristae significant increase in ΔΨCr - IBM in response to blocking of F1Fo ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae IBM are driven by oxidative phosphorylation . N = 3 independent experiments .

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

Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Quantification of total mitochondrial TMRE FI. N = 3 independent experiments. Quantification of cristae ΔΨm relative to IBM (ΔΨCr-IBM) in response to oligomycin compared to FCCP. F1Fo ATP Synthase is exclusively localized to the cristae and significant increase in ΔΨCr-IBM in response to blocking of F1Fo ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae and IBM are driven by oxidative phosphorylation. N = 3 independent experiments.


Response of ΔΨm to F1Fo ATP Synthase inhibitor , oligomycin ( 10 µM ) , compared to uncoupler , FCCP ( 10 µM ) , determined using live - Airyscan imaging of HeLa co - stained with MTG TMRE . Representative Airyscan showing mitochondria from living HeLa stained with MTG TMRE . were first stained for one hour only then treated with oligomycin or FCCP . show hyperpolarization of cristae under oligomycin ( middle row ; arrowheads ) depolarization of cristae under FCCP ( bottom row ; arrowheads ) compared to control ( top row ; arrowheads ) . Scale bar = 500 nm . N = 3 independent experiments . Note that ΔΨm - independent fraction of TMRE is less than 5 % of signal in control conditions .

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Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Representative Airyscan images showing mitochondria from living HeLa cells stained with MTG and TMRE. Cells were first stained for one hour and only then treated with oligomycin or FCCP. Images show hyperpolarization of cristae under oligomycin (middle row; arrowheads) and depolarization of cristae under FCCP (bottom row; arrowheads) compared to control (top row; arrowheads). Scale bar = 500 nm. N = 3 independent experiments. Note that the ΔΨm-independent fraction of TMRE staining is less than 5% of the signal in control conditions.


Zoomed - in region of FCCP - treated mitochondria in C . Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP . These show diminished heterogeneity of ΔΨCr - IBM values .

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Zoomed-in region of FCCP-treated mitochondria in C. Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP. These images show diminished heterogeneity of ΔΨCr-IBM values.


role of cristae structure as well as CJ formation sealing on generation of difference in ΔΨm between cristae IBM were studied Representative of mitochondria in control ( top row ) vs . Mic13 - KO ( bottom row ) HeLa . Note that TMRE FI in Mic13 - KO mitochondria is distributed more evenly along IMM , so that ΔΨCr - IBM is decreased . Scale bar = 500 nm . N = 3 independent experiments . of A , showing deletion of Mic13 in HeLa results in significant decrease in ΔΨCr - IBM . While Mic13 - KO show a substantial decrease in cristae number , loss of cristae does not appear absolute , making it feasible to compare TMRE FI at cristae relative to IBM . N = 3 independent experiments .

O O O O O O O O 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 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 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

The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ΔΨCr-IBM is decreased. Scale bar = 500 nm. N = 3 independent experiments. Quantification of A, showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.


role of cristae structure as well as CJ formation sealing on generation of difference in ΔΨm between cristae IBM were studied by disrupting cristae using Mic10 - Mic60 - deficient . Mic60 support cristae formation . Mic10 is essential for CJ formation , , in its absence , cristae tend to remain as vesicles detached from IBM . Live - Airyscan imaging of TMRE was used in figure panels models . Representative of Hap1 control ( top row ) vs . Mic60 KO ( middle row ) Mic10 KO ( bottom row ) ; Mic60 - KO mitochondria show very few cristae structures . TMRE along IMM is relatively homogeneous , indicating decreased ΔΨCr - IBM . In general , Mic10 KO in Hap1 results in decreased TMRE signal at cristae relative to IBM ( arrowheads ) as compared to control . Deletion of Mic10 induces detachment of cristae from IBM ( see Figure 8 ) . Except for detached cristae vesicles , TMRE is homogeneously distributed n Mic10 - KO , indicating decreased ΔΨCr - IBM . Scale bar = 500 nm . N = 3 independent experiments . of C , showing Mic60 KO in Hap1 results in significant decrease in ΔΨCr - IBM . While Mic60 - KO show a marked loss of normal cristae structures , depletion of cristae does not appear absolute , making it possible to compare TMRE FI at cristae relative to IBM . N = 3 independent experiments . of C , showing Mic10 KO in Hap1 results in a significant decrease in ΔΨCr - IBM . Note : of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM . N = 3 independent experiments .

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

The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10- and Mic60-deficient cells. Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models. Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ΔΨCr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (see Figure 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed n Mic10-KO cells, indicating decreased ΔΨCr-IBM. Scale bar = 500 nm. N = 3 independent experiments. Quantification of C, showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments. Quantification of C, showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr-IBM. Note: quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.


Studies show that Opa1 interacts with MICOS , promoting closure of CJs . Thus , we tested whether Opa1 was required to maintain difference in ΔΨm between cristae IBM . Live - Airyscan of MEF control ( top row ) vs . Opa1 KO ( bottom row ) , co - stained with NAO TMRE . Arrowheads indicate decreased of TMRE FI at cristae compared to IBM . Note more even distribution of TMRE , indicating cristae IBM are relatively equipotential . Scale bar = 500 nm . of G , showing Opa1 - KO MEFs have significantly decreased ΔΨCr - IBM . N = 3 independent experiments .

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

Studies show that Opa1 interacts with MICOS complex, promoting closure of CJs. Thus, we tested whether Opa1 was required to maintain the difference in ΔΨm between cristae and IBM. Live-cell Airyscan images of MEF control (top row) vs. Opa1 KO (bottom row), co-stained with NAO and TMRE. Arrowheads indicate decreased intensity of TMRE FI at cristae compared to IBM. Note the more even distribution of TMRE staining, indicating the cristae and IBM are relatively equipotential. Scale bar = 500 nm. Quantification of G, showing Opa1-KO MEFs have significantly decreased ΔΨCr-IBM. N = 3 independent experiments.


Live - Airyscan of - induced depolarization time series of Mic10 KO Hap1 stained with TMRE . Mitochondrion exposed to rapid , high 2 - photon power in region of box , arrow ( ~ 0 . 3 sec ) ; large , hyperpolarized vesicle ( arrowhead ) remains polarized after of mitochondrion loses ΔΨm , indicating electrochemical discontinuity between hyperpolarized vesicles of mitochondrion , including IBM . Scale bar = 500 nm . N = 2 independent experiments .

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Live-cell Airyscan images of laser-induced depolarization time series of Mic10 KO Hap1 cell stained with TMRE. Mitochondrion exposed to rapid, high 2-photon laser power in region of white box, arrow (~0.3 sec); large, hyperpolarized vesicle (arrowhead) remains polarized after the rest of the mitochondrion loses ΔΨm, indicating electrochemical discontinuity between hyperpolarized vesicles and the rest of the mitochondrion, including the IBM. Scale bar = 500 nm. N = 2 independent experiments.


Live - Airyscan image of time series showing hyperpolarized vesicles ( arrowheads ; region cropped from dashed box ) inside mitochondria lacking both Opa1 Drp1 . Scale bar = 500 nm . N = 2 independent experiments . Free movement of hyperpolarized vesicles within matrix indicate their detachment from IBM , representing an extreme case of cristae independency within one mitochondrion ( see Movie EV 1 for time series ) .

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Live-cell Airyscan image of time series showing hyperpolarized vesicles (arrowheads; region cropped from dashed white box) inside mitochondria lacking both Opa1 and Drp1. Scale bar = 500 nm. N = 2 independent experiments. Free movement of hyperpolarized vesicles within the matrix indicate their detachment from the IBM, representing an extreme case of cristae independency within one mitochondrion (see Movie EV 1 for time series).


- induced mitochondrial depolarization in L6 myoblasts shows gradual depolarization of elements along IMM , visualized using live - Airyscan microscopy . Representative of - induced depolarization time series from L6 myoblast , stained with ΔΨm - dependent dye , Rho123 . show LUT color - coded for Rho123 FI . blue pixels represent most least intense ΔΨm , respectively . See legend of LUT colors on right . Note that at first time point ( ~ 0 . 15 sec ) arrow points to box marking of mitochondrion exposed to phototoxic of 2 - photon ( ≤ 5 msec ) , inducing depolarization . Ensuing frames show wavelike depolarization away from site of perturbation ( arrowheads ) . Scale bar = 500 nm . N = 3 independent experiments . ( See Movie EV 2 for time series . ) Profile plot showing Rho123 pixel as function of along mitochondrion in A . Note that zero value in X axis corresponds to top of mitochondrion shown in A , near site of 2 - photon exposure ( box ) . Red corresponds to frame immediately before depolarization ( ~ 0 . 15 sec ) ; green , blue , purple , purple lines correspond to ~ 0 . 30 , ~ 0 . 60 , ~ 1 . 05 , ~ 2 . 10 sec , respectively , after initial depolarization , showing wavelike dissipation of ΔΨm over time .

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

Laser-induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM, visualized using live-cell Airyscan microscopy. Representative images of laser-induced depolarization time series from L6 myoblast, stained with ΔΨm-dependent dye, Rho123. Images show LUT color-coded for Rho123 FI. White and blue pixels represent most and least intense ΔΨm, respectively. See legend of LUT colors on the right. Note that at the first time point (~0.15 sec) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Ensuing frames show wavelike depolarization away from site of perturbation (arrowheads). Scale bar = 500 nm. N = 3 independent experiments. (See Movie EV 2 for time series.) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A. Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A, near site of 2-photon laser exposure (box). Red line corresponds to frame immediately before depolarization (~0.15 sec); green, blue, purple, and dark purple lines correspond to ~0.30, ~0.60, ~1.05, and ~2.10 sec, respectively, after initial depolarization, showing wavelike dissipation of ΔΨm over time.


- induced mitochondrial depolarization in living human skin fibroblasts co - stained with TMRE MTG imaged with Airyscan , showing gradual depolarization of elements along IMM . Time - lapse of merged green ( MTG ) red ( TMRE ) channels following - induced mitochondrial depolarization . Note that , at first time point ( 0 . 3 sec ) , arrow points to box marking of mitochondrion exposed to phototoxic of 2 - photon ( ≤ 5 msec ) , inducing depolarization . Depolarization is marked by dissipation of ΔΨm - dependent dye ( TMRE ) while green ( MTG ) signal persists . Scale bar = 1 µm . N = 4 independent experiments . TMRE channel from C , showing gradual of loss of TMRE along IMM . Note that although depolarization has largely completed at near phototoxic stimulus already at 0 . 9 sec , some specific regions at very top maintain ΔΨm while others depolarize . of ΔΨm using TMRE FI from C - D . Measurement of TMRE pixel intensities immediately after - induced depolarization at site distal ( ≥ 10 µm ) vs . proximal ( ≤ 1 µm ) to box . N = 4 independent experiments ; see specific p values in panel . of C - D . of mitochondria that depolarize in a wavelike ( i . e . , gradual ) vs . instantaneous manner after - induced depolarization . Note : imaging at high temporal resolution ( ~ 100 - 500 msec / frame ) reveals wavelike depolarizations predominate , suggesting ΔΨm is composed of multiple , disparate electrochemical domains along IMM . time scale of propagation of depolarization is slower than propagation of electrical phenomena . N = 4 independent experiments .

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Laser-induced mitochondrial membrane depolarization in living human skin fibroblasts co-stained with TMRE and MTG and imaged with Airyscan, showing gradual depolarization of elements along the IMM. Time-lapse images of merged green (MTG) and red (TMRE) channels following laser-induced mitochondrial membrane depolarization. Note that, at the first time point (0.3 sec), white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Depolarization is marked by the dissipation of the ΔΨm-dependent dye (TMRE) while the green (MTG) signal persists. Scale bar = 1 µm. N = 4 independent experiments. TMRE channel from C, showing gradual of the loss of TMRE along the IMM. Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0.9 sec, some specific regions at the very top maintain ΔΨm while others depolarize. Quantification of ΔΨm using TMRE FI from C-D. Measurement of TMRE pixel intensities immediately after laser-induced depolarization at site distal (≥ 10 µm) vs. proximal (≤ 1 µm) to box. N = 4 independent experiments; see specific p values in panel. Quantification of C-D. Percentage of mitochondria that depolarize in a wavelike (i.e., gradual) vs. instantaneous manner after laser-induced depolarization. Note: imaging at high temporal resolution (~100-500 msec/frame) reveals wavelike depolarizations predominate, suggesting the ΔΨm is composed of multiple, disparate electrochemical domains along the IMM. The time scale of propagation of depolarization is slower than the propagation of electrical phenomena. N = 4 independent experiments.