( 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 ) .

O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O 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-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O

(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 ) 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 .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O

(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 ) 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 .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, 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 the OSC lysates . n . i . : non - immune IgG .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O

(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 and its ∆ SAM mutant ( green ) . Both proteins were localized to the nuclei ( DAPI : blue ) . Scale bar : 5 μm .

O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O

(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 the OSC lysates . n . i . : non - immune IgG .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O

(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 and its ∆ SAM mutant ( green ) . Both proteins were localized to the nuclei ( DAPI : blue ) . Scale bar : 5 μm .

O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O

(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 ) 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 .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, 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 and its 8CA mutant , but not with ∆ PHD mutant An asterisk shows background .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

(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 and its 8CA and ∆ PHD mutants ( green ) . All proteins were localized to the nuclei ( DAPI : blue ) . Scale bar : 5 μm .

O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O

(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 ) 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 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O

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

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O O O O

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


( 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 .

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

(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 , 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE I-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O

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

O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O

(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 ) 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 ) .

O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 ) 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 .

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O

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 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 .

B-EXP_ASSAY O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O

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

O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O B-GENEPROD O O O O B-ORGANISM O

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


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 ) .

O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 - 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 .

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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 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 .

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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 ( 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 .

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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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O

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 cells using anti - Ago1x antibody and 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 and HeLa cells 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 cells transfected with three AGO1 - specific siRNAs ( 1 , 2 , 3 ) . qRT - PCR analysis of AGO1 transcript expression in these cells 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 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 . ) .

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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.).


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Expression of Ago1x in mouse organs.


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 ) .

O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O

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 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 .

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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 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 .

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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 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 .

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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 . Assay was done as described above in HeLa cells . Mutated sequence 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 the stop codon . Readthrough assay was done as described above in HeLa cells . * * * , 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 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 .

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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 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 .

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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 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 .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

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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 and MYC mRNAs co - immunoprecipitated with Ago1 or Ago1x in HeLa cells .

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


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

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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 cells 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 images of HeLa cells show co - localization of GW182 with Ago1 , but not with Ago1x . White 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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

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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 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 .

O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_LINE O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O 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-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, 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 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

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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 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

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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 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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-SUBCELLULAR O O O B-GENEPROD O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O O

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 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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_LINE O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O B-CELL_LINE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O B-GENEPROD O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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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 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 .

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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 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 .

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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 - 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 .

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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 - 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 .

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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 - 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 .

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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 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 .

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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. 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 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 .

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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 images 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.


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 .

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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.


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 .

O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR 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 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 .

O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O B-SUBCELLULAR 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 - 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 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O O O 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-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O

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 - 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 ) .

O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O

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).


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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O 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-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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.