( D ) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - test .

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(D) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


( E ) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2 .

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(E) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2.


( F ) Cells transfected with TMEM135 siRNA were subjected to qPCR . Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - test .

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(F) Cells transfected with TMEM135 siRNA were subjected to qPCR. Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


( A ) RPE1 cells were transfected with siRNAs as indicated , followed by serum starvation for 24 h , and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( B ) Quantification of the percentage of ciliated cells shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by serum starvation for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum - starvation media followed by filipin staining . Scale bar , 20 μm .

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(C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.


( D ) Cells were transfected with siRNAs shown in ( C ) and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) Quantification of the percentage of ciliated cells shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(D) Cells were transfected with siRNAs shown in (C) and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of ciliated cells shown in (D). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( F ) Cells were transfected with siRNAs as indicated , incubated with cholesterol - MβCD complex in serum - starvation media , and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) Quantification of the percentage of ciliated cells shown in ( F ) . Data represent mean ± SD ( n = 3experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) RPE1 cells were transfected with siRNA as indicated , and immunostained for Rab8 ( red ) and γ - tubulin ( green ) in either 10 % FBS medium ( upper panel ) or serum - starvation medium ( lower panel ) . Scale bar , 10 μm . Arrowhead indicates Rab8 localized to the centrioles .

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(A) RPE1 cells were transfected with siRNA as indicated, and immunostained for Rab8 (red) and γ-tubulin (green) in either 10% FBS medium (upper panel) or serum-starvation medium (lower panel). Scale bar, 10 μm. Arrowhead indicates Rab8 localized to the centrioles.


( B ) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD ( n = 3 experiments ) , 200 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) Cells were transfected with siRNA and immunostained for IFT20 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm .

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(C) Cells were transfected with siRNA and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.


( D ) Quantification of the percentage of IFT20 intensity at the centrioles Data represent mean ± SD ( n = 3 experiments ) , 150 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(D) Quantification of the percentage of IFT20 intensity at the centrioles Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) RPE1 cells were transfected with siRNAs as indicated , followed by transfection with wild - type pGFP - Rab8a ( WT - Rab8 ) , constitutively active pGFP - Rab8a ( Q67L ) ( CA - Rab8 ) , or DN Rab8 dominant - negative pGFP Rab8a ( T22N ) , incubated in serum - starved media for 12 h , and immunostained for ARL13B ( Red ) , GFP - Rab8 ( green ) , and DAPI ( blue ) . Scale bar , 10 μm .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm.


( B ) Quantification of the percentage of ciliated cells Data represent mean ± SD ( n = 3 experiments ) , 200 GFP - positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(B) Quantification of the percentage of ciliated cells Data represent mean ± SD (n=3 experiments), 200 GFP-positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) ( Upper panel ) Cells were transfected as indicated and the cell lysates were incubated with purified proteins , including GST or GST - JCF1 ( RBD ) . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) Intensity of the bands was quantified by Image J software . The amount of GTP - Rab8 were normalized to the control level . Bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - test .

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(C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.


( D ) Cells were transfected and cell lysates were incubated with purified GST - JCF1 ( RBD ) fusion protein . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody . ( Lower panel ) Cells were transfected and cell lysates were incubated with purified GST - protein . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody .

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(D) Cells were transfected and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.


( A ) RPE1 cells were transfected with siRNAs as indicated , followed by 24 h incubation in serum - starved media . Cells were then and subjected to fractionation , and Western blot for IFT20 , the Golgi marker GM130 , membrane marker UBXD8 , and nuclear marker CREB .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by 24 h incubation in serum-starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB.


( B ) Efficiency of IFT20 knockdown by western blot .

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(B) Efficiency of IFT20 knockdown by western blot.


( C ) Cells were transfected with siRNAs as indicated , followed by incubation in serum - starved media for 24 h , and immunostained for ARL13B ( red ) . Scale bar , 10 μm . The bar graph represents the quantification of the percentage of ciliated cells . Data represent mean ± SD ( n = 3 experiments ) , 250 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( D ) Cells were transfected and immunostained for Rab8 and γ - tubulin , followed by quantification of the percentage of cells with Rab8 localized to the centriole . Data represent average ( n = 2 experiments ) .

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(D) Cells were transfected and immunostained for Rab8 and γ-tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n=2 experiments).


( E ) Cells were transfected with siRNAs as indicated , followed by transfection with Flag - IFT20 , incubation in serum - starvation media for 12 h , and immunostained for ARL13B . Representative fluorescent images of Flag - IFT20 ( green ) , ARL13B ( red ) , and DAPI ( blue ) are shown . Scale bar , 10 μm . ( F ) Quantification of the percentage of ciliated cells with both the Flag - IFT20 and ARL13B localized in the cilium . Data represent mean ± SD ( n = 3 experiments ) , 150 Flag positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. (F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( G ) Cells were transfected with siRNAs as indicated , followed by further transfection with CA - Rab8 , incubation in serum - starvation media for 12 h , and immunostained for acetylated - tubulin . Representative fluorescent images of GFP - Rab8 Q67L ( green ) , acetylated - tubulin ( red ) , and DNA ( blue ) are shown . Scale bar , 10 μm . ( H ) Quantification of the percentage of GFP - positive ciliated cells ( only those cilia having both GFP - Rab8 and acetylated - tubulin on cilium were considered for quantification ) . Data represent mean ± SD ( n = 3 experiments ) , 200 GFP positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. (H) Quantification of the percentage of GFP -positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells .

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(A) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells.


( B ) RPE1 cells were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , and immunostained for ARL13B ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm .

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(B) RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm.


( C ) Quantification of the percentage of ciliated cells Data represent mean ± SD ( n = 3 experiments ) , 250 GFP positive cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(C) Quantification of the percentage of ciliated cells Data represent mean ± SD (n = 3 experiments), 250 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( D ) Cells were transfected by siRNa as indicated followed by incubation in serum - starvation media for 24 h , and immunostained for EHD1 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 150 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(D) Cells were transfected by siRNa as indicated followed by incubation in serum-starvation media for 24 h, and immunostained for EHD1 (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( F ) Cells were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , and immunostained for IFT20 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in ( F ) . Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(F) Cells were transfected by siRNAs as indicated, followed by incubation in serum-starvation media for 24 h, and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD (n=3 experiments), 150 were scored per condition per experiment, *P < 0.05, Student's t-test.


( H , I , J ) Cells were transfected by siRNAs as indicated , followed by transfection with GFP - Rab8 WT , GFP - Rab8 Q67L , or GFP - Rab8 T22N , and further incubated in serum - starvation media for 12 h . Cell lysate was subjected to immunoprecipitation with anti - GFP antibody , followed by Western blot with antibody against GFP .

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(H, I, J) Cells were transfected by siRNAs as indicated, followed by transfection with GFP-Rab8 WT, GFP-Rab8 Q67L, or GFP-Rab8 T22N, and further incubated in serum-starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti-GFP antibody, followed by Western blot with antibody against GFP.


( A ) RPE1 cells were transfected as indicated , followed by incubation in serum - starvation media in the presence or absence of cholesterol / MCD for 24 h , and then immunostained for Rab8 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm . Representative magnified images are shown from cells labeled with white asterisks .

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(A) RPE1 cells were transfected as indicated, followed by incubation in serum-starvation media in the presence or absence of cholesterol/MCD for 24 h, and then immunostained for Rab8 (red) and γ-tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks.


( B ) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD ( n = 3 experiments ) , 200 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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

(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) ( Upper panel ) Cells were transfected and cell lysate was incubated with purified GST - JCF1 ( RBD ) fusion protein . The amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) The intensity of bands was quantified by Image J software . The amount of GTP - Rab8 were normalized to the control level . The bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - test .

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(C) (Upper panel) Cells were transfected and cell lysate was incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) The intensity of bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. The bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.


( D ) Cells were transfected and immunostained for IFT20 ( red ) and γ - tubulin ( green ) . Scale bar , 10 μm .

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(D) Cells were transfected and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.


( E ) Quantification of IFT20 intensity at the centriole Data represent mean ± SD ( n = 3 experiments ) , 150 cells were scored per condition per experiment , * P < 0 . 05 , Student ' s t - test .

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(E) Quantification of IFT20 intensity at the centriole Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


Morphology of wild - type ( Tefm + / + ) and Tefm homozygous knockout ( Tefm - / - ) embryos at embryonic day 8 . 5 ( analyzed embryos , n = 43 ) . Scale bar , 500 μm .

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Morphology of wild-type (Tefm+/+) and Tefm homozygous knockout (Tefm-/-) embryos at embryonic day 8.5 (analyzed embryos, n= 43). Scale bar, 500 μm.


Western blot analyses of the TEFM protein level in heart mitochondrial extracts from 8 - week - old control ( L / L ) and tissue - specific Tefm knockout ( Tefm knockout ; L / L , cre ) mice ( n = 12 mice for each group ) . VDAC was used as loading control .

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Western blot analyses of the TEFM protein level in heart mitochondrial extracts from 8-week-old control (L/L) and tissue-specific Tefm knockout (Tefm knockout; L/L, cre) mice (n = 12 mice for each group). VDAC was used as loading control.


Survival curve for control ( L / L ; n = 60 ) and Tefm knockout ( L / L , cre ; n = 22 ) mice .

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Survival curve for control (L/L; n= 60) and Tefm knockout (L/L, cre; n= 22) mice.


Heart weight to body weight ratio in 4 - and 8 - week - old control ( L / L ) and Tefm knockout mice ( L / L , cre ) . At 4 weeks : L / L n = 22 , L / L , cre n = 15 ; 8 weeks : L / L n = 41 , L / L , cre n = 39 .

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Heart weight to body weight ratio in 4- and 8-week-old control (L/L) and Tefm knockout mice (L/L, cre). At 4 weeks: L/L n= 22, L/L, cre n= 15; 8 weeks: L/L n= 41, L/L, cre n= 39.


Transmission electron micrographs of heart tissue in 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . Scale bar , 2 μm ( upper panel ) and 1 μm ( lower panel ) .

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Transmission electron micrographs of heart tissue in 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Scale bar, 2 μm (upper panel) and 1 μm (lower panel).


Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks ( Upper 2 blots ; n = 6 mice at each time - point for each group ) . 18S rDNA was used as loading control . DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls . Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks ( Bottom 2 blots ; ; n = 6 mice at each time - point for each group ) . VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA .

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Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Upper 2 blots; n= 6 mice at each time-point for each group). 18S rDNA was used as loading control. DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls. Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Bottom 2 blots; ; n= 6 mice at each time-point for each group). VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA.


Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . The analyzed enzyme activities are NADH coenzyme Q reductase ( complex I ) , NADH cytochrome c reductase ( complex I / III ) , succinate dehydrogenase ( complex II ) and cytochrome c oxidase ( complex IV ) .

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Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The analyzed enzyme activities are NADH coenzyme Q reductase (complex I), NADH cytochrome c reductase (complex I/III), succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV).


Barplot of the significantly changed proteins ( Benjamini - Hochberg adjusted P < 0 . 05 ) in Tefm knockout mice compared to control mice ( n = 5 mice for each group ) , organized by individual OXPHOS complexes . Proteins are organized in alphabetic order based on gene name . Bars represent mean log2 [ L / L , cre / L / L ] ± 95 % confidence interval .

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Barplot of the significantly changed proteins (Benjamini-Hochberg adjusted P <0.05) in Tefm knockout mice compared to control mice (n= 5 mice for each group), organized by individual OXPHOS complexes. Proteins are organized in alphabetic order based on gene name. Bars represent mean log2 [L/L, cre / L/L] ± 95% confidence interval.


Boxplots of the protein intensity ( LFQ ) of TEFM ( top ) and POLRMT ( bottom ) . Red dot : mean ; blue dots : intensity value for each biological replicate ( n = 5 mice for each group ) .

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Boxplots of the protein intensity (LFQ) of TEFM (top) and POLRMT (bottom). Red dot: mean; blue dots: intensity value for each biological replicate (n= 5 mice for each group).


Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks ( n = 12 mice for each group ) . VDAC was used as loading control .

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Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks (n= 12 mice for each group). VDAC was used as loading control.


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Northern blot analyses and relative quantification of mtRNA transcripts in control and Tefm knockout mouse hearts at the age of 4 and 8 weeks (n= 12 mice at each time-point for each group). 18S rRNA and 5.8S rRNA were used as loading controls, respectively. HSP transcripts: 12S rRNA 4 weeks P= 0.005966044, 8 weeks P= 0.0806819; 16S rRNA 4 weeks P= 0.000223004, 8 weeks P= 0.00243443; Nd1 4 weeks P= 0.0352088, 8 weeks P= 0.000344158; Cox1 4 weeks P= 0.000353895, 8 weeks P= 0.00145773; Cox2 4 weeks P= 0.0145193, 8 weeks P= 0.00578484; Cox3 4 weeks P= 0.00834317, 8 weeks P= 0.000392364; Nd5/Cyb 4 weeks P= 0.0010185, 8 weeks P= 0.00153049; Nd5 4 weeks P= 0.024621, 8 weeks P= 7.38074E-05; Cyb 4 weeks P= 3.46175E-06, 8 weeks P= 0.000991942; LSP transcript: Nd6 4 weeks P= 0.000162704, 8 weeks P= 0.000187855. (E) HSP transcripts: Tf 4 weeks P= 0.0297685, 8 weeks P= 0. 000212544; Tv 4 weeks P= 0.842023, 8 weeks P= 0.269231; Tl1 4 weeks P= 0.00921072, 8 weeks P= 0.000430068; Tm 4 weeks P= 0.0084742, 8 weeks P= 0.000142524; Tk 4 weeks P= 0.00297015, 8 weeks P= 0.00010425; Ts2 4 weeks P= 0.0128829, 8 weeks P= 2.27321E-05; Tl2 4 weeks P= 0.00701324, 8 weeks P= 0.000411549; Tt 4 weeks P= 0.00572118, 8 weeks P= 0.000807943; LSP transcript: Tp 4 weeks P= 0.0427191, 8 weeks P= 0.0483328; Te 4 weeks P= 0.464031, 8 weeks P= 0.13412; Ts1 4 weeks P= 0.0382215, 8 weeks P= 0.000478237; Tc 4 weeks P= 0.00418795, 8 weeks P= 0.000287525; Tn 4 weeks P= 0.00531668, 8 weeks P= 0.000374367; Ta 4 weeks P= 0.00247257, 8 weeks P= 0.000564597; Tq 4 weeks P= 0.00385524, 8 weeks P= 0.000727981.


In organello transcription analyses using heart mitochondria isolated from 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . The steady - state levels of 12S rRNA and tRNAV were used as endogenous controls , due to their unchanged levels . VDAC was used as loading control for the equal input of mitochondrial extracts . This experiment has performed five times with either two or three replicates .

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In organello transcription analyses using heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The steady-state levels of 12S rRNA and tRNAV were used as endogenous controls, due to their unchanged levels. VDAC was used as loading control for the equal input of mitochondrial extracts. This experiment has performed five times with either two or three replicates.


Northern blot analyses of 7S RNA level in 4 - and 8 - week - old control and Tefm knockout mouse hearts ( n = 12 mice at each time - point for each group ) . 18S rRNA was used as loading control . RNA from tissue - specific Mterf4 knockout mouse hearts and tissue - specific Polrmt knockout mouse hearts were loaded as positive and negative control , respectively .

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Northern blot analyses of 7S RNA level in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice at each time-point for each group). 18S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively.


Southern blot analyses of mtDNA to obtain 7S DNA in 8 - week - old control and Tefm knockout mice . Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA . Equal amounts of mtDNA shown on the top of the gel , were loaded as the endogenous control . This experiment has performed three times .

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Southern blot analyses of mtDNA to obtain 7S DNA in 8-week-old control and Tefm knockout mice. Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA. Equal amounts of mtDNA shown on the top of the gel, were loaded as the endogenous control. This experiment has performed three times.


In organello replication assays were performed on heart mitochondria isolated from 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . VDAC was used as loading control .

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In organello replication assays were performed on heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). VDAC was used as loading control.


Northern blot analyses of the 7S RNA levels in 4 - and 8 - week - old control and Tefm knockout mouse hearts ( n = 12 mice for each group ) . 5S rRNA was used as loading control . RNA from tissue - specific Mterf4 knockout mouse hearts and tissue - specific Polrmt knockout mouse hearts were loaded as positive and negative control , respectively .

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Northern blot analyses of the 7S RNA levels in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice for each group). 5S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively.


Strand - specific RT - qPCR detection of the mitochondrial precursor transcripts containing the antisense of mt - tRNAA and antisense of mt - tRNAC in control and Tefm knockout mice ( n = 12 mice for each group ) . The mt - mRNAs are shown in light blue and mt - tRNAs in grey . Arrows show the binding position of RT - qPCR primers . Actin transcript was used as endogenous control .

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Strand-specific RT-qPCR detection of the mitochondrial precursor transcripts containing the antisense of mt-tRNAA and antisense of mt-tRNAC in control and Tefm knockout mice (n= 12 mice for each group). The mt-mRNAs are shown in light blue and mt-tRNAs in grey. Arrows show the binding position of RT-qPCR primers. Actin transcript was used as endogenous control.


Strand - specific RT - qPCR detection of the mitochondrial precursor transcripts spanning the junction of Nd5 and antisense of Nd6 in control and Tefm knockout mice ( n = 25 mice for each group ) . The mt - mRNAs are shown in light blue . Arrows show the binding position of RT - qPCR primers . Actin transcript was used as endogenous control .

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Strand-specific RT-qPCR detection of the mitochondrial precursor transcripts spanning the junction of Nd5 and antisense of Nd6 in control and Tefm knockout mice (n= 25 mice for each group). The mt-mRNAs are shown in light blue. Arrows show the binding position of RT-qPCR primers. Actin transcript was used as endogenous control.


Northern blot analyses of the precursor transcripts containing the antisense of mt - tRNAA in 4 - and 8 - week - old control and Tefm knockout mouse hearts ( n = 6 mice for each group ) .

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Northern blot analyses of the precursor transcripts containing the antisense of mt-tRNAA in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 6 mice for each group).


Volcano plot showing TEFM - BioID . TEFM - BirA * - HA was stably expressed in HEK 293 cells , followed by streptavidin affinity purification and mass spectrometry analysis to detect the associated proteins . Mitochondrial targeted BirA * ( MTS - BirA * ) was used as a control . The significantly enriched proteins are presented above the horizontal dashed line with 5 % false discovery rate ( FDR ) significance . The blue dots highlight proteins involved in mtDNA transcription and the green dots represent proteins involved in RNA metabolism . All other proteins are in grey .

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Volcano plot showing TEFM-BioID. TEFM-BirA*-HA was stably expressed in HEK 293 cells, followed by streptavidin affinity purification and mass spectrometry analysis to detect the associated proteins. Mitochondrial targeted BirA* (MTS-BirA*) was used as a control. The significantly enriched proteins are presented above the horizontal dashed line with 5% false discovery rate (FDR) significance. The blue dots highlight proteins involved in mtDNA transcription and the green dots represent proteins involved in RNA metabolism. All other proteins are in grey.


( A - C ) Individuals in the neuropathology cohort were divided in groups of no significant primary pathology ( N = 31 ) , AD ( N = 36 ) , PD ( N = 13 ) , and others ( N = 8 ) , and analyzed together with N = 9 additional subjects with primary non - AD tauopathies ( PSP / CBD ) . Data is shown for plasma P - tau217 ( panel A ) , plaque density ( panel B ) , and tangle density ( panel C ) . Plasma P - tau217 was not elevated in PSP / CBD compared to subjects without significant primary pathology . AD , Alzheimer ' s disease ; CBD , corticobasal degeneration ; PSP , progressive supranuclear palsy ; PD , Parkinson ' s disease . The central band of the boxes are medians and the boxes show inter - quartile ranges . The whiskers are defined as the smallest ( largest ) observation greater ( less ) than or equal to the first ( third ) quartile minus ( plus ) 1 . 5 times the inter - quartile range .

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(A-C) Individuals in the neuropathology cohort were divided in groups of no significant primary pathology (N=31), AD (N=36), PD (N=13), and others (N=8), and analyzed together with N=9 additional subjects with primary non-AD tauopathies (PSP/CBD). Data is shown for plasma P-tau217 (panel A), plaque density (panel B), and tangle density (panel C). Plasma P-tau217 was not elevated in PSP/CBD compared to subjects without significant primary pathology. AD, Alzheimer's disease; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; PD, Parkinson's disease. The central band of the boxes are medians and the boxes show inter-quartile ranges. The whiskers are defined as the smallest (largest) observation greater (less) than or equal to the first (third) quartile minus (plus) 1.5 times the inter-quartile range.


Panel B shows associations between plasma P - tau217 and Aβ PET stratified by tertiles ( T ) of tau PET ( T1 : SUVR ≤ 1 . 05 , T2 : 1 . 05 < SUVR ≤ 1 . 11 , T3 : 1 . 11 < SUVR ≤ 3 . 03 ) . Panel C shows associations between plasma P - tau217 and tau PET stratified by tertiles ( T ) of Aβ PET ( T1 : SUVR ≤ 0 . 468 , T2 : 0 . 468 < SUVR ≤ 0 . 579 , T3 : 0 . 579 < SUVR ≤ 1 . 08 ) .

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Panel B shows associations between plasma P-tau217 and Aβ PET stratified by tertiles (T) of tau PET (T1: SUVR ≤ 1.05, T2: 1.05 < SUVR ≤ 1.11, T3: 1.11 < SUVR ≤ 3.03).   Panel C shows associations between plasma P-tau217 and tau PET stratified by tertiles (T) of Aβ PET (T1: SUVR ≤ 0.468, T2: 0.468 < SUVR ≤ 0.579, T3: 0.579 < SUVR ≤ 1.08).


G . Purified recombinant FLAG - FAM111A proteins were incubated at indicated temperatures for 4 h , and FAM111A auto - proteolytic activity was analyzed by immunoblotting with FLAG antibody . H . As in ( G ) , using recombinant human FLAG - FAM111B proteins .

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G. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting with FLAG antibody. H. As in (G), using recombinant human FLAG-FAM111B proteins.


I . U2OS cell lines conditionally expressing indicated GFP - FAM111A alleles were fixed at the indicated times after treatment with Doxycycline ( DOX ) to induce expression of the transgenes and stained with crystal violet .

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I. U2OS cell lines conditionally expressing indicated GFP-FAM111A alleles were fixed at the indicated times after treatment with Doxycycline (DOX) to induce expression of the transgenes and stained with crystal violet.


A . Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP - FAM111A .

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A. Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP-FAM111A.


B . DNA replication rates in U2OS / GFP - FAM111A cells treated with DOX for the indicated times , pulse - labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image - based cytometry ( QIBC ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) . See also Appendix Figure S6A .

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B. DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6A.


C . Cells treated as in ( B ) were pre - extracted , fixed and stained with PCNA or MCM2 antibody , and subsequently analyzed by QIBC ( n > 2000 cells per condition ) .

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C. Cells treated as in (B) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC (n>2000 cells per condition).


D - F . Quantification of data in ( C ) ( red bars , mean ) . Cells in S phase were identified based on EdU positivity . See also Appendix Figure S6B - D .

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D-F. Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Figure S6B-D.


G . Analysis of FAM111A interactors . U2OS / GFP - FAM111A WT cells were treated or not with DOX for 4 h , subjected to GFP immunoprecipitation ( IP ) and analyzed by mass spectrometry . Volcano plot shows enrichment of individual proteins ( + DOX / - DOX ratio ) plotted against the P value . Dashed lines indicate the significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

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G. Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


H . U2OS or U2OS / ∆ FAM111A cells were subjected to IP with IgG ( control ) or RFC1 antibody followed by immunoblotting with indicated antibodies .

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H. U2OS or U2OS/∆FAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies.


I . U2OS cells transfected with empty vector ( EV ) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies .

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I. U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies.


J . As in ( C ) , except that cells were stained with RFC1 antibody ( n > 2000 cells per condition ) .

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J. As in (C), except that cells were stained with RFC1 antibody (n>2000 cells per condition).


K . Quantification of data in ( J ) for S phase ( EdU - positive ) cells ( red bars , mean ) .

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K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).


A . Immunoblot analysis of U2OS / GFP - FAM111A WT cells treated with DOX for the indicated times .

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A. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated with DOX for the indicated times.


B . Representative images of U2OS / GFP - FAM111A cell lines that were treated or not with DOX , fixed and stained with γ - H2AX antibody and DAPI .

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B. Representative images of U2OS/GFP-FAM111A cell lines that were treated or not with DOX, fixed and stained with γ-H2AX antibody and DAPI.


C . Cells in ( B ) were subjected to QIBC analysis of γ - H2AX signal intensity ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

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C. Cells in (B) were subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


D . Immunoblot analysis of U2OS / GFP - FAM111A WT cells treated or not with DOX and the pan - Caspase inhibitor Z - VAD - FMK as indicated .

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D. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated or not with DOX and the pan-Caspase inhibitor Z-VAD-FMK as indicated.


Transcriptional activity in U2OS / GFP - FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC ( red bars , mean ; n > 2000 cells per condition ) .

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Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


Transcriptional activity in U2OS / GFP - FAM111A cells treated or not with DOX pulse - labeled with EU were analyzed by QIBC ( red bars , mean ; n > 2000 cells per condition ) .

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Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


Transcriptional activity in U2OS / GFP - FAM111A cells treated or not with DOX pulse - labeled with EU and stained with DAPI were analyzed by QIBC ( red bars , mean ; n > 2000 cells per condition ) .

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Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU and stained with DAPI were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


H . U2OS / GFP - FAM111A cell lines treated or not with DOX were pre - extracted , fixed and stained with RPB1 antibody , and analyzed by QIBC ( n > 2000 cells per condition ) . I . Quantification of data in ( H ) ( red bars , mean ) .

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H. U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, and analyzed by QIBC (n>2000 cells per condition). I. Quantification of data in (H) (red bars, mean).


U2OS / GFP - FAM111A cell lines treated or not with DOX were pre - extracted , fixed and stained with RPB1 antibody , J . Representative images from the experiment in ( H ) . Scale bars , 10 μm .

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U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, J. Representative images from the experiment in (H). Scale bars, 10 μm.


B . Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP - FAM111A alleles . U2OS / GFP - FAM111A WT ( low ) cells express the transgene at a lower level than U2OS / GFP - FAM111A WT cells used in Figure 1 and 2 ( see Figure EV4A ) .

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B. Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP-FAM111A alleles. U2OS/GFP-FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP-FAM111A WT cells used in Figure 1 and 2 (see Figure EV4A).


C . U2OS / GFP - FAM111A cell lines treated or not with DOX , pulse - labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC .

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C. U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC.


U2OS / GFP - FAM111A cell lines treated or not with DOX , pulse - labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC . D . Quantification of data in ( C ) for S phase ( EdU - positive ) cells ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

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U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. D. Quantification of data in (C) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


U2OS / GFP - FAM111A cell lines treated or not with DOX , and stained with DAPI were analyzed for DAPI signal intensity using QIBC . cells were pulse - labeled with EU .

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U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. cells were pulse-labeled with EU.


U2OS / GFP - FAM111A cell lines treated or not with DOX , and stained with DAPI were analyzed for DAPI signal intensity using QIBC . Quantification of EU incorporation in cells in ( E ) ( red bars , mean ; n > 2000 cells per condition ) .

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U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. Quantification of EU incorporation in cells in (E) (red bars, mean; n>2000 cells per condition).


G . U2OS / GFP - FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody , pre - extracted and fixed , and stained with DAPI . RFC1 and DAPI signal intensities were analyzed by QIBC .

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G. U2OS/GFP-FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC.


U2OS / GFP - FAM111A cell lines that were treated or not with DOX were pre - extracted and fixed , and stained with DAPI . DAPI signal intensities were analyzed by QIBC . H . cells were stained with RPB1 antibody .

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U2OS/GFP-FAM111A cell lines that were treated or not with DOX were pre-extracted and fixed, and stained with DAPI. DAPI signal intensities were analyzed by QIBC. H. cells were stained with RPB1 antibody.


I . Immunoblot analysis of U2OS / GFP - FAM111A cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK .

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I. Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK . J . using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles .

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Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK. J. using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles.


K . Purified recombinant FLAG - FAM111A proteins were incubated at indicated temperatures for 4 h , and FAM111A auto - proteolytic activity was analyzed by immunoblotting .

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K. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting.


L . GFP IPs from U2OS cell lines expressing GFP - FAM111A WT or D528G mutant were analyzed by mass spectrometry . Volcano plot shows enrichment of individual proteins ( WT / D528G ratio ) plotted against the P value . Dashed lines indicate the significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

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L. GFP IPs from U2OS cell lines expressing GFP-FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


B . U2OS / GFP - FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies . C . As in ( B ) , using U2OS / GFP - FAM111B WT cells .

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B. U2OS/GFP-FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies. C. As in (B), using U2OS/GFP-FAM111B WT cells.


D . Purified recombinant FLAG - FAM111A and FLAG - FAM111B proteins were incubated separately or mixed , subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody .

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D. Purified recombinant FLAG-FAM111A and FLAG-FAM111B proteins were incubated separately or mixed, subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody.


E . Immunoblot analysis of U2OS / GFP - FAM111B cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK .

E. Immunoblot analysis of U2OS/GFP-FAM111B cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


F . Cells in ( E ) were pulse - labeled with EdU , stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC .

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F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.


Cells analyzed using QIBC . G . Quantification of data in ( F ) for S phase ( EdU - positive ) cells ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

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Cells analyzed using QIBC. G. Quantification of data in (F) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC . H . cells were pulse - labeled with EU .

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Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC. H. cells were pulse-labeled with EU.


Cells analyzed using QIBC . I . Quantification of EU incorporation in cells in ( H ) ( red bars , mean ; n > 2000 cells per condition ) .

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Cells analyzed using QIBC. I. Quantification of EU incorporation in cells in (H) (red bars, mean; n>2000 cells per condition).


J . U2OS / GFP - FAM111B cell lines ( Figure 5E ) were fixed at the indicated times after DOX treatment and stained with crystal violet .

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J. U2OS/GFP-FAM111B cell lines (Figure 5E) were fixed at the indicated times after DOX treatment and stained with crystal violet.


K . Cells in ( E ) were fixed , immunostained with γ - H2AX antibody and subjected to QIBC analysis of γ - H2AX signal intensity ( red bars , mean ; n > 2000 cells per condition ) .

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K. Cells in (E) were fixed, immunostained with γ-H2AX antibody and subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean; n>2000 cells per condition).


L . Immunoblot analysis of purified recombinant FLAG - FAM111B proteins and their auto - cleavage products .

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L. Immunoblot analysis of purified recombinant FLAG-FAM111B proteins and their auto-cleavage products.


C . Aliquots of the supernatants of the indicated HEK293T transfectants ( all expressing λ chains ) were resolved under non - reducing conditions and blotted with anti - µ antibodies . Wild type µs chains are secreted only as pentamers and hexamers , whilst mutants lacking C575 release mostly µs2L2 subunits ( indicated with a red arrow ) as does the double C414A - C575A . L566A releases instead mainly µs2L2 subunits of faster migration ( blue arrow ) . C414 mutants release no hexamers , but smaller assemblies , including fast migrating µs2L2 subunits .

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C. Aliquots of the supernatants of the indicated HEK293T transfectants (all expressing λ chains) were resolved under non-reducing conditions and blotted with anti-µ antibodies. Wild type µs chains are secreted only as pentamers and hexamers, whilst mutants lacking C575 release mostly µs2L2 subunits (indicated with a red arrow) as does the double C414A-C575A. L566A releases instead mainly µs2L2 subunits of faster migration (blue arrow). C414 mutants release no hexamers, but smaller assemblies, including fast migrating µs2L2 subunits.


E . Aliquots of the supernatants were treated with or without Endo - H and electrophoresed under reducing conditions . The blot was decorated with anti - µ antibodies . N563 - glycans acquire Endo - H resistance in µsL566A and µsC575A , but remain largely sensitive in wild type µs .

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E. Aliquots of the supernatants were treated with or without Endo-H and electrophoresed under reducing conditions. The blot was decorated with anti-µ antibodies. N563-glycans acquire Endo-H resistance in µsL566A and µsC575A, but remain largely sensitive in wild type µs.


A . Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non - reducing conditions before or after removal of N - glycans by PNGaseF . Blots were decorated with anti - µ antibodies . Blue and red arrows point to the closed ( oxidized ) and open µs2L2 species respectively .

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A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.


Intracellular µs2L2 species purified from the murine hybridoma B1 . 8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS / MS spectrum Y and B ions are shown in blue and red , respectively .

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Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.


A . I . 29µ + B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion . At the indicated times , cells were lysed in TritonX114 to separate soluble and integral membrane proteins ( aqueous and detergent phases , respectively ) . Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti - µ antibodies . The blue arrow points at a band likely corresponding to µs2L2C575OX , the intensity of which increases upon LPS stimulation .

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A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation.


C . µ - chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM .

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C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM.


D . The bar graphs show the levels of C575 and C414 oxidation , expressed as the ratio between IAA and NEM modified peptides .

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D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.


ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . A . Aliquots from the spent media ( Extracellular ) were blotted under non - reducing conditions and decorated with anti - µ antibodies . The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575 - dependent retention , and wild type µs are secreted only as polymers ( black arrow ) . Traces of covalent HaloERp44 - µ2L2 complexes ( black asterisks ) are detected in the media of cells expressing HaloERp44 ∆ RDEL and µs or L566A . The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44 ∆ RDEL suggest that the latter inhibits processing of the N563 glycan , likely hindering it to the Golgi enzymes upon non - covalent binding . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition .

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

ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . B . The corresponding cell lysates are shown in this panel . The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44 . The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2 . Traces of intracellular µs2L2 - ERp44 ∆ RDEL complexes are visible in µs and L566A but absent from C575A expressing cells . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition .

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

ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


C . Lysates of the ERp44KO transfectants were precipitated with NP - sepharose , resolved under non reducing conditions and the blot decorated sequentially with anti - µ , anti - ERp44 or anti - Halo . The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo - tagged ERp44 .

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C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.


B . To selectively label intracellular HC575A , cells were treated with 1 nM Halo - tag TMR Direct Ligand for 24 hours . The left panel shows aliquots of the lysates of the indicated transfectants , resolved under non - reducing conditions and decorated with anti - λ antibodies . In the two right panels , samples were precipitated with NP - Sepharose and electrophoresed under non reducing conditions . The signal for the Halo - tag TMR was checked directly in the gel . The arrows point at the various assemblies between µ , HC575A and λ chains .

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B. To selectively label intracellular HC575A, cells were treated with 1 nM Halo-tag TMR Direct Ligand for 24 hours. The left panel shows aliquots of the lysates of the indicated transfectants, resolved under non-reducing conditions and decorated with anti-λ antibodies. In the two right panels, samples were precipitated with NP-Sepharose and electrophoresed under non reducing conditions. The signal for the Halo-tag TMR was checked directly in the gel. The arrows point at the various assemblies between µ, HC575A and λ chains.


C . The experiment shown aims to determine whether ERp44 can bind also to a single C575 , or rather to non - native bonds . Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP - Sepharose , resolved under non reducing conditions and the blot decorated with anti - λ antibodies or anti - ERp44 as indicated . Black asterisks point to covalent complexes of Halo - ERp44 with µs2L2 .

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C. The experiment shown aims to determine whether ERp44 can bind also to a single C575, or rather to non-native bonds. Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP-Sepharose, resolved under non reducing conditions and the blot decorated with anti-λ antibodies or anti-ERp44 as indicated. Black asterisks point to covalent complexes of Halo-ERp44 with µs2L2.


A Western blots of acid - extracted histones from activated CD4 + T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells . One of three similar experiments is shown .

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A Western blots of acid-extracted histones from activated CD4+ T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells. One of three similar experiments is shown.


C , D Western blots of acid - extracted histones from activated CD4 + T cells in the presence of glycolysis inhibitor 2 - DG and PDK inhibitor DCA , respectively . Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti - CD3 ( 5 µg / ml ) .

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C, D Western blots of acid-extracted histones from activated CD4+ T cells in the presence of glycolysis inhibitor 2-DG and PDK inhibitor DCA, respectively. Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti-CD3 (5 µg/ml).


E Immunoblotting of acid - extracted lactylated histones from anti - CD3 - activated CD4 + T cells in the presence of extracellular lactate ( 25 mM ) at 24 hours of cell culture . One representative out of three experiments is shown . For all immunoblotting experiments , ex vivo purified , non - activated CD4 + T cells were used as control lymphocytes .

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E Immunoblotting of acid-extracted lactylated histones from anti-CD3-activated CD4+ T cells in the presence of extracellular lactate (25 mM) at 24 hours of cell culture. One representative out of three experiments is shown. For all immunoblotting experiments, ex vivo purified, non-activated CD4+ T cells were used as control lymphocytes.


F ChIP - sequncing showing H3K18 lactylation signals in control Th17 cells and lactate - treated Th17 lymphocytes ( 25 mM ) . Biological duplicates are shown .

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F ChIP-sequncing showing H3K18 lactylation signals in control Th17 cells and lactate-treated Th17 lymphocytes (25 mM). Biological duplicates are shown.


A - I . Correlative light electron microscopy ( CLEM ) showing Prom1 at the plasma membrane of primary hepatocytes . Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) or KO ( Prom1cre - ert2 - nlacz ) mice . The boxed areas in panel ( A , D , G ) and ( B , E , H ) were magnified and presented as panel ( B , E , H ) and ( C , F , I ) , respectively . Scale bar ; 5 μm ( A , D and G ) , 1 μm ( B , E and H ) , and 0 . 2 μm ( C , F and I ) .

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A-I. Correlative light electron microscopy (CLEM) showing Prom1 at the plasma membrane of primary hepatocytes. Primary hepatocytes were isolated from 12-week-old male WT (Prom1+/+) or KO ( Prom1cre-ert2-nlacz) mice. The boxed areas in panel (A, D, G) and (B, E, H) were magnified and presented as panel (B, E, H) and (C, F, I), respectively. Scale bar; 5 μm (A, D and G), 1 μm (B, E and H), and 0.2 μm (C, F and I).


J . Immunofluorescent staining of Prom1 , CK19 and HNF4α in two serial cryosections of mouse liver . The boxed area in the top panel was magnified and presented as the bottom panel . Asterisks ( * ) in the top panel indicate cholangiocytes of the bile bucts and triangles ( Γ ) in the bottom panel indicate hepatocytes . Scale bar , 100 μm .

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J. Immunofluorescent staining of Prom1, CK19 and HNF4α in two serial cryosections of mouse liver. The boxed area in the top panel was magnified and presented as the bottom panel. Asterisks (*) in the top panel indicate cholangiocytes of the bile bucts and triangles (Γ) in the bottom panel indicate hepatocytes. Scale bar, 100 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( K ) Differentially expressed genes showing more than a 2 - fold change with P < 0 . 05 ( 118 DEGs ) induced ( n = 55 ) or repressed ( n = 63 ) by Prom1 knockout ( n = 2 / group ) . G6pc , glucose - 6 - phosphatase catalytic subunit ; Pck1 , phosphoenolpyruvate carboxykinase 1 .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (K) Differentially expressed genes showing more than a 2-fold change with P < 0.05 (118 DEGs) induced (n=55) or repressed (n=63) by Prom1 knockout (n = 2/group). G6pc, glucose-6-phosphatase catalytic subunit; Pck1, phosphoenolpyruvate carboxykinase 1.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( L ) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT - PCR ( n = 3 / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (L) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT-PCR (n = 3/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( M ) Levels of the Prom1 , PCK , G6PC and GAPDH proteins were determined by immunoblotting .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (M) Levels of the Prom1, PCK, G6PC and GAPDH proteins were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice and serum - starved . ( N ) Prom1 + / + or Prom1 - / - hepatocytes serum - starved for 8 hours were incubated with 2 - [ 3H ] deoxyglucose for 10 minutes . Glucose uptake was assessed by calculating the amount of cell - associated radioactivity normalized to the amount of protein ( n = 3 mice / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (N) Prom1+/+ or Prom1-/- hepatocytes serum-starved for 8 hours were incubated with 2-[3H]deoxyglucose for 10 minutes. Glucose uptake was assessed by calculating the amount of cell-associated radioactivity normalized to the amount of protein (n = 3 mice/group).


A . Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT - PCR . The level of each mRNA was normalized to the level of 18s rRNA ( n = 3 / fed , 7 or 8 / fasted ) .

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A. Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT-PCR. The level of each mRNA was normalized to the level of 18s rRNA (n = 3/fed, 7 or 8/fasted).


B . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , G6pc Pck , Prom1 and GAPDH were determined after 18 hours fasting . Band intensity for p - CREB was quantified using Photoshop software .

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B. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, G6pc Pck, Prom1 and GAPDH were determined after 18 hours fasting. Band intensity for p-CREB was quantified using Photoshop software.


C . Blood glucose levels ( mg / dL ) of mice after fasted for 24 hours ( n = 12 or 14 / group ) .

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C. Blood glucose levels (mg/dL) of mice after fasted for 24 hours (n = 12 or 14/group).


D . Serum glucagon level in the fed or fasted mice ( n = 3 / fed , 7 or 8 / fasted ) .

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D. Serum glucagon level in the fed or fasted mice (n = 3/fed, 7 or 8/fasted).


E . Glycogen contents in the liver of mouse in each condition was shown by PAS ( periodic acid schiff ) staining . Scale bar , 500 μm .

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E. Glycogen contents in the liver of mouse in each condition was shown by PAS (periodic acid schiff) staining. Scale bar, 500 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . F . Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation ( 10 nM ) were determined by qRT - PCR . The level of each mRNA was normalized to the level of the 18S rRNA . For these experiments , primary hepatocytes were cultured under 3D conditions using a Matrigel matrix ( n = 3 mice / group ) .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. F. Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation (10 nM) were determined by qRT-PCR. The level of each mRNA was normalized to the level of the 18S rRNA. For these experiments, primary hepatocytes were cultured under 3D conditions using a Matrigel matrix (n = 3 mice/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . G . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , phospho - Inositol trisphosphate receptor ( p - IP3R ) , IP3R , phospho - Hormone - Sensitive Lipase ( p - HSL ) , HSL , Prom1 and Gapdh 10 minutes after glucagon stimulation ( 10 nM ) were determined by immunoblotting .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. G. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, phospho-Inositol trisphosphate receptor (p-IP3R), IP3R, phospho-Hormone-Sensitive Lipase (p-HSL), HSL, Prom1 and Gapdh 10 minutes after glucagon stimulation (10 nM) were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . H . The nuclear localization of p - CREB 10 minutes after glucagon stimulation ( 10 nM ) was determined by immunofluorescence staining . Scale bar , 20 μm .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. H. The nuclear localization of p-CREB 10 minutes after glucagon stimulation (10 nM) was determined by immunofluorescence staining. Scale bar, 20 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . I . Relative PKA activities 10 minutes after glucagon stimulation ( 10 nM ) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. I. Relative PKA activities 10 minutes after glucagon stimulation (10 nM) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . J . The cAMP concentration 10 minutes after glucagon stimulation ( 10 nM ) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. J. The cAMP concentration 10 minutes after glucagon stimulation (10 nM) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).