( B ) RPE1 were transfected by siRNAs as indicated , followed by incubation in serum - starvation media for 24 h , immunostained for ARL13B ( red ) 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 ) of of ciliated Data represent mean ± SD ( n = 3 experiments ) , 250 GFP positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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

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

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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 ) were transfected by siRNAs as indicated , followed by transfection with GFP - Rab8 WT , GFP - Rab8 Q67L , or GFP - Rab8 T22N , further incubated in serum - starvation media for 12 h . 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 were transfected as indicated , followed by incubation in serum - starvation media in presence or absence of cholesterol / MCD for 24 h , then immunostained for Rab8 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . Representative magnified are shown from labeled with 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 ) of of with Rab8 localized to centriole Data represent mean ± SD ( n = 3 experiments ) , 200 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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

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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 ) were transfected immunostained for IFT20 ( red ) 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 ) of IFT20 at centriole Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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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.


of wild - type ( Tefm + / + ) Tefm homozygous knockout ( Tefm - / - ) at embryonic day 8 . 5 ( analyzed , 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 TEFM level in heart mitochondrial extracts from 8 - week - old control ( L / L ) 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 ) 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 to in 4 - 8 - week - old control ( L / L ) Tefm knockout mice ( L / L , cre ) . At 4 : L / L n = 22 , L / L , cre n = 15 ; 8 : 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 of heart tissue in 8 - week - old control Tefm knockout mice ( n = 5 mice for each group ) . Scale bar , 2 μm ( upper panel ) 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 in control Tefm knockout hearts at of 4 8 ( Upper 2 blots ; n = 6 mice at each time - point for each group ) . 18S rDNA was used as loading control . isolated from Lrpprc Mterf4 knockout hearts used as controls . Western blot analyses of VDAC Histone H3 in control Tefm knockout hearts at of 4 8 ( Bottom 2 blots ; ; n = 6 mice at each time - point for each group ) . VDAC represents mitochondria loading Histone H3 was used as a loading control for nucleus .

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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 activities were measured spectrophotometrically normalized to citrate synthase activity in heart mitochondria from 8 - week - old control Tefm knockout mice ( n = 5 mice for each group ) . analyzed enzyme activities are NADH coenzyme Q reductase ( I ) , NADH cytochrome c reductase ( I / III ) , succinate dehydrogenase ( II ) cytochrome c oxidase ( 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 significantly changed ( Benjamini - Hochberg adjusted P < 0 . 05 ) in Tefm knockout mice compared to control mice ( n = 5 mice for each group ) , organized by OXPHOS complexes . are organized in alphabetic order 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 ( LFQ ) of TEFM ( top ) POLRMT ( bottom ) . Red dot : mean ; blue dots : 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 in control Tefm knockout heart mitochondria at of 8 ( 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 Tefm knockout mice ( n = 5 mice for each group ) . steady - state of 12S rRNA tRNAV were used as endogenous controls , due to their unchanged . VDAC was used as loading control for 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 level in 4 - 8 - week - old control Tefm knockout mouse hearts ( n = 12 mice at each time - point for each group ) . 18S rRNA was used as loading control . from tissue - specific Mterf4 knockout mouse hearts tissue - specific Polrmt knockout mouse hearts were loaded as positive 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 in 8 - week - old control Tefm knockout mice . Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA . Equal amounts of mtDNA shown on top of gel , were loaded as 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 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 7S in 4 - 8 - week - old control Tefm knockout mouse hearts ( n = 12 mice for each group ) . 5S rRNA was used as loading control . from tissue - specific Mterf4 knockout mouse hearts tissue - specific Polrmt knockout mouse hearts were loaded as positive 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 mitochondrial precursor transcripts containing antisense of mt - tRNAA antisense of mt - tRNAC in control Tefm knockout mice ( n = 12 mice for each group ) . mt - mRNAs are shown in blue mt - tRNAs in grey . Arrows show 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 mitochondrial precursor transcripts spanning junction of Nd5 antisense of Nd6 in control Tefm knockout mice ( n = 25 mice for each group ) . mt - mRNAs are shown in blue . Arrows show 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 precursor transcripts containing antisense of mt - tRNAA in 4 - 8 - week - old control 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 , followed by streptavidin affinity purification mass spectrometry to detect associated . Mitochondrial targeted BirA * ( MTS - BirA * ) was used as a control . significantly enriched are presented above horizontal dashed with 5 % false discovery rate ( FDR ) significance . blue dots highlight involved in mtDNA transcription green dots represent involved in metabolism . other 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 neuropathology cohort were divided in groups of no significant pathology ( N = 31 ) , AD ( N = 36 ) , PD ( N = 13 ) , others ( N = 8 ) , analyzed together with N = 9 additional subjects with non - AD tauopathies ( PSP / CBD ) . Data is shown for plasma P - tau217 ( panel A ) , plaque density ( panel B ) , tangle density ( panel C ) . Plasma P - tau217 was not elevated in PSP / CBD compared to subjects without significant pathology . AD , Alzheimer ' s disease ; CBD , corticobasal degeneration ; PSP , progressive supranuclear palsy ; PD , Parkinson ' s disease . central band of boxes are medians boxes show inter - quartile ranges . whiskers are defined as smallest ( largest ) observation greater ( less ) than or equal to first ( third ) quartile minus ( plus ) 1 . 5 times 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 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 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 were incubated at indicated temperatures for 4 h , FAM111A auto - proteolytic activity was analyzed by immunoblotting with FLAG antibody . H . As in ( G ) , using recombinant human FLAG - FAM111B .

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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 lines conditionally expressing indicated GFP - FAM111A alleles were fixed at indicated times after treatment with Doxycycline ( DOX ) to induce expression of transgenes 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 of stable U2OS lines left untreated or incubated in 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 . replication rates in U2OS / GFP - FAM111A treated with DOX for indicated times , - labeled with EdU stained with DAPI were analyzed by quantifying EdU signal in S phase using quantitative image - cytometry ( QIBC ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 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 . treated as in ( B ) were pre - extracted , fixed stained with PCNA or MCM2 antibody , subsequently analyzed by QIBC ( n > 2000 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 . of data in ( C ) ( red bars , mean ) . in S phase were identified 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 . of FAM111A interactors . U2OS / GFP - FAM111A WT were treated or not with DOX for 4 h , subjected to GFP immunoprecipitation ( IP ) analyzed by mass spectrometry . Volcano plot shows enrichment of ( + DOX / - DOX ) plotted against P value . Dashed lines indicate 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 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 transfected with empty vector ( EV ) or indicated RFC subunit expression plasmids were subjected to FLAG IP 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 were stained with RFC1 antibody ( n > 2000 per condition ) .

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


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

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


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

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


B . Representative of U2OS / GFP - FAM111A lines that were treated or not with DOX , fixed stained with γ - H2AX antibody 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 . in ( B ) were subjected to QIBC of γ - H2AX signal ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 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 of U2OS / GFP - FAM111A WT treated or not with DOX 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 treated or not with DOX for indicated times were analyzed by QIBC ( red bars , mean ; n > 2000 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 treated or not with DOX - labeled with EU were analyzed by QIBC ( red bars , mean ; n > 2000 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 treated or not with DOX - labeled with EU stained with DAPI were analyzed by QIBC ( red bars , mean ; n > 2000 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 lines treated or not with DOX were pre - extracted , fixed stained with RPB1 antibody , analyzed by QIBC ( n > 2000 per condition ) . I . 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 lines treated or not with DOX were pre - extracted , fixed stained with RPB1 antibody , J . Representative from 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 of U2OS lines left untreated or incubated in presence of DOX to induce expression of indicated GFP - FAM111A alleles . U2OS / GFP - FAM111A WT ( low ) express transgene at a lower level than U2OS / GFP - FAM111A WT used in Figure 1 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 lines treated or not with DOX , - labeled with EdU stained with DAPI were analyzed for DAPI EdU signal 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 lines treated or not with DOX , - labeled with EdU stained with DAPI were analyzed for DAPI EdU signal using QIBC . D . of data in ( C ) for S phase ( EdU - positive ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 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 lines treated or not with DOX , stained with DAPI were analyzed for DAPI signal using QIBC . were - 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 lines treated or not with DOX , stained with DAPI were analyzed for DAPI signal using QIBC . of EU incorporation in in ( E ) ( red bars , mean ; n > 2000 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 lines that were treated or not with DOX were stained with RFC1 antibody , pre - extracted fixed , stained with DAPI . RFC1 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 lines that were treated or not with DOX were pre - extracted fixed , stained with DAPI . DAPI signal intensities were analyzed by QIBC . H . 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 of U2OS / GFP - FAM111A lines treated or not with DOX in 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 of lines treated or not with DOX in absence or presence of Z - VAD - FMK . J . using U2OS 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 were incubated at indicated temperatures for 4 h , 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 lines expressing GFP - FAM111A WT or D528G mutant were analyzed by mass spectrometry . Volcano plot shows enrichment of ( WT / D528G ) plotted against P value . Dashed lines indicate 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 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 .

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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 FLAG - FAM111B were incubated separately or mixed , subjected to IP with FAM111A antibody 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 of U2OS / GFP - FAM111B lines treated or not with DOX in 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 . in ( E ) were - labeled with EdU , stained with DAPI analyzed for DAPI EdU signal 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.


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


stained with DAPI analyzed for DAPI signal using QIBC . H . were - labeled with EU .

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


analyzed using QIBC . I . of EU incorporation in in ( H ) ( red bars , mean ; n > 2000 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 lines ( Figure 5E ) were fixed at indicated times after DOX treatment 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 . in ( E ) were fixed , immunostained with γ - H2AX antibody subjected to QIBC of γ - H2AX signal ( red bars , mean ; n > 2000 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 of purified recombinant FLAG - FAM111B 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 supernatants of indicated HEK293T transfectants ( expressing λ chains ) were resolved under non - reducing conditions blotted with anti - µ antibodies . Wild type µs chains are secreted only as pentamers hexamers , whilst mutants lacking C575 release mostly µs2L2 subunits ( indicated with a red arrow ) as does 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 supernatants were treated with or without Endo - H electrophoresed under reducing conditions . blot was decorated with anti - µ antibodies . N563 - glycans acquire Endo - H resistance in µsL566A 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 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 red arrows point to closed ( oxidized ) 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 murine hybridoma B1 . 8 were subjected to sequential alkylation analyzed by mass spectrometry In MS / MS spectrum Y B ions are shown in blue 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 were treated with LPS to induce differentiation towards IgM secretion . At indicated times , were lysed in TritonX114 to separate soluble integral ( aqueous detergent phases , respectively ) . Aliquots from two phases were resolved electrophoretically blot was decorated by anti - µ antibodies . blue arrow points at a band likely corresponding to µs2L2C575OX , 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 migrate more slowly than those which were reduced at time of lysis 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 . bar graphs show of C575 C414 oxidation , expressed as between IAA NEM modified peptides .

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


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

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

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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 ERp44KO transfectants were precipitated with NP - sepharose , resolved under non reducing conditions blot decorated sequentially with anti - µ , anti - ERp44 or anti - Halo . two black asterisks point at bands corresponding to covalent complexes containing µs2L2 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 , were treated with 1 nM Halo - tag TMR Direct Ligand for 24 hours . left panel shows aliquots of lysates of indicated transfectants , resolved under non - reducing conditions decorated with anti - λ antibodies . In two right panels , samples were precipitated with NP - Sepharose electrophoresed under non reducing conditions . signal for Halo - tag TMR was checked directly in gel . arrows point at various assemblies between µ , HC575A 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 . experiment shown aims to determine whether ERp44 can bind also to a single C575 , or rather to non - native bonds . Aliquots of lysates from indicated ERp44KO transfectants were precipitated with NP - Sepharose , resolved under non reducing conditions 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 showing global histone lactylation H3K18 lactylation in presence absence of glucose at 24 hours after stimulation of . 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 in presence of glycolysis inhibitor 2 - DG PDK inhibitor DCA , respectively . were analysed for global lactylation 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 in presence of extracellular lactate ( 25 mM ) at 24 hours of culture . One representative out of three experiments is shown . For immunoblotting experiments , ex vivo purified , non - activated CD4 + T 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 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 electron microscopy ( CLEM ) showing Prom1 at plasma of hepatocytes . hepatocytes were isolated from 12 - week - old WT ( Prom1 + / + ) or KO ( Prom1cre - ert2 - nlacz ) mice . boxed areas in panel ( A , D , G ) ( B , E , H ) were magnified presented as panel ( B , E , H ) ( C , F , I ) , respectively . Scale bar ; 5 μm ( A , D G ) , 1 μm ( B , E H ) , 0 . 2 μm ( C , F 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 of Prom1 , CK19 HNF4α in two serial cryosections of mouse liver . boxed in top panel was magnified presented as bottom panel . Asterisks ( * ) in top panel indicate cholangiocytes of bile bucts triangles ( Γ ) in 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.


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice 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.


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice serum - starved . ( L ) Relative expression of G6pc 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).


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice serum - starved . ( M ) of Prom1 , PCK , G6PC GAPDH 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.


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice 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 amount of - associated radioactivity normalized to amount of ( 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 . of G6pc Pck1 mRNAs in liver of mice after 18 hours fasting were determined by qRT - PCR . level of each mRNA was normalized to 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 . of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , G6pc Pck , Prom1 GAPDH were determined after 18 hours fasting . Band 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 ( 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 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 liver of mouse in each condition was shown by PAS ( periodic acid schiff ) . 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.


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

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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).


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved stimulated with glucagon . G . 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 Gapdh 10 minutes after glucagon stimulation ( 10 nM ) were determined by immunoblotting .

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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.


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

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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.


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

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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).


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

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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).


12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with glucagon . ( A ) Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon stimulation ( 200 µg / kg , n = 10 / group ) .

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12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (200 µg/kg body weight, n = 10/group).


12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with glucagon . ( B ) of p - PKA substrates , p - CREB , CREB GAPDH in liver after a 5 minutes glucagon treatment were determined by immunoblotting ( 2 mg / kg ) .

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12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (B) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting (2 mg/kg body weight).


12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with glucagon . ( C ) Relative PKA activities in liver after a 5 minutes glucagon treatment were determined using PKA kit ( 2 mg / kg , n = 3 / group ) .

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12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (C) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group).


12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with glucagon . ( D ) cAMP in liver after a 5 minutes glucagon treatment was determined using cAMP kit ( 2 mg / kg , n = 3 / group ) .

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

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (D) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group).


Glucose disposal rates were measured in 12 - week - old mice using glucose , pyruvate ( E ) tolerance ( n = 10 mice / group ) .

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

Glucose disposal rates were measured in 12-week-old male mice using glucose, pyruvate (E) tolerance tests (n = 10 mice/group).


Glucose disposal rates were measured in 12 - week - old mice using insulin ( F ) tolerance ( n = 10 mice / group ) .

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

Glucose disposal rates were measured in 12-week-old male mice using insulin (F) tolerance tests (n = 10 mice/group).


G . of p - AKT , AKT , p - ERK , ERK , IRβ GAPDH were determined by immunoblotting after insulin stimulation ( 10 nM ) .

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

G. Levels of p-AKT, AKT, p-ERK, ERK, IRβ and GAPDH were determined by immunoblotting after insulin stimulation (10 nM).


hepatocytes were isolated from 12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice , serum - starved for 16 hours stimulated with isoprenaline . ( H ) localization of p - CREB after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined by immunofluorescence . Scale bar , 20 μm .

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

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (H) The nuclear localization of p-CREB after isoprenaline stimulation (10 μM) for 10 minutes was determined by immunofluorescence staining. Scale bar, 20 μm.


hepatocytes were isolated from 12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice , serum - starved for 16 hours stimulated with isoprenaline . ( I ) of p - PKA substrates , p - CREB , CREB , Prom1 GAPDH after isoprenaline stimulation ( 10 μM ) for 0 , 10 or 20 minutes were determined by immunoblotting .

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

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (I) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH after isoprenaline stimulation (10 μM) for 0, 10 or 20 minutes were determined by immunoblotting.


hepatocytes were isolated from 12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice , serum - starved for 16 hours stimulated with isoprenaline . ( J ) cAMP after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined using cAMP kit in presence of 10 μM IBMX ( n = 3 / group ) .

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

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).


WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( K ) Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after epinephrine

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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulation


WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( L ) of p - PKA substrates , p - CREB , CREB GAPDH in liver 15 minutes after epinephrine treatment were determined by immunoblotting .

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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (L) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting.


M - O . WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours subjected to immobilization . ( M ) Blood glucose ( mg / dL ) were measured 0 , 60 120 minutes after immobilization ( n = 10 / group ) .

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

M-O. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60 and 120 minutes after immobilization (n = 10/group).


WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours subjected to immobilization . ( N ) of p - PKA substrates , p - CREB , CREB GAPDH in liver after a 30 minutes - immobilization were determined by immunoblotting .

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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (N) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 30 minutes-immobilization were determined by immunoblotting.


WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours subjected to immobilization . ( O ) Serum epinephrine level of mice after a 30 minutes - immobilization was determined ( n = 3 / control group , 7 or 8 / immobilized group ) .

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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (O) Serum epinephrine level of mice after a 30 minutes-immobilization was determined (n = 3/control group, 7 or 8/immobilized group).


A . expression of Prom1 , radixin , AKAP7 , AKAP8 , AKAP8I , AKAP9 , AKAP10 , AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs . Hepatocytes were then serum - starved for 18 hours stimulated with glucagon ( 10 nM ) for 10 minutes . phosphorylation of PKA substrates was determined by immunoblotting .

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

A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting.


Prom1 radixin expression were silenced in WT ( Prom1 + / + ) KO ( Prom1 - / - ) hepatocytes by infection with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) or sh - Prom1 for 24 hours . Hepatocytes were further serum - starved for 18 hours stimulated with glucagon ( 10 nM ) for 10 minutes . ( B ) localization of p - CREB after glucagon stimulation was determined by immunofluorescence . Blue ; DAPI , Red ; p - CREB . of with p - CREB among more than 300 in each group was also statistically analyzed ( Figure EV3G ) . Scale bar , 20 μm .

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

Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (B) The nuclear localization of p-CREB after glucagon stimulation was determined by immunofluorescence staining. Blue; DAPI, Red; p-CREB. The percentage of cells with nuclear p-CREB staining among more than 300 cells in each group was also statistically analyzed (Figure EV3G). Scale bar, 20 μm.


Prom1 radixin expression were silenced in WT ( Prom1 + / + ) KO ( Prom1 - / - ) hepatocytes by infection with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) or sh - Prom1 for 24 hours . Hepatocytes were further serum - starved for 18 hours stimulated with glucagon ( 10 nM ) for 10 minutes . ( C ) of p - PKA substrates , p - CREB , CREB , p - IP3R , IP3R , Prom1 , radixin GAPDH after glucagon stimulation ( 10 minutes ) were determined by immunoblotting .

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

Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (C) Levels of p-PKA substrates, p-CREB, CREB, p-IP3R, IP3R, Prom1, radixin and GAPDH after glucagon stimulation (10 minutes) were determined by immunoblotting.


D . Immunofluorescence of Prom1 in mouse liver tranduced with adenoviral particles . Confocal microscope of WT mouse liver infected with adenovirus harboring sh - Con or sh - Rdx linked with IRES - GFP . Scale bar , 20 μm .

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D. Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles. Confocal microscope images of WT mouse liver infected with adenovirus harboring sh-Con or sh-Rdx linked with IRES-GFP. Scale bar, 20 μm.


12 - week - old wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( E ) Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon administration ( 200 µg / kg , n = 10 mice / group ) .

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

12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (E) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon administration (200 µg/kg body weight, n = 10 mice/ group).


12 - week - old wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( F ) of p - PKA , p - CREB , CREB , radixin GAPDH in liver after a 10 - minutes glucagon stimulation ( 2 mg / kg ) were determined by immunoblotting .

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

12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (F) Levels of p-PKA, p-CREB, CREB, radixin and GAPDH in the liver after a 10-minutes glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting.


12 - week - old wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( G ) Glucose disposal rates in sh - Con or sh - Rdx ( radixin ) mice were measured using glucose , insulin pyruvate tolerance ( n = 10 mice / group ) .

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

12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Glucose disposal rates in sh-Con or sh-Rdx (radixin) mice were measured using glucose, insulin and pyruvate tolerance tests (n = 10 mice/group).


hepatocytes were obtained from 12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice or KO ( Prom1 - / - ) hepatocytes infected with an adenovirus harboring LacZ ( Ad - LacZ ) or Prom1 ( Ad - Prom1 ) for 24 hours . molecular interaction between Prom1 radixin ( A ) in these hepatocytes was determined by a proximity ligation using anti - Prom1 anti - radixin antibodies ( A )

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

Primary hepatocytes were obtained from 12-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice or KO (Prom1-/-) hepatocytes infected with an adenovirus harboring LacZ (Ad-LacZ) or Prom1 (Ad-Prom1) for 24 hours. The molecular interaction between Prom1 and radixin (A) in these hepatocytes was determined by a proximity ligation assay using anti-Prom1 and anti-radixin antibodies (A)


hepatocytes were obtained from 12 - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice or KO ( Prom1 - / - ) hepatocytes infected with an adenovirus harboring LacZ ( Ad - LacZ ) or Prom1 ( Ad - Prom1 ) for 24 hours . molecular interaction between Prom1 PKA regulatory subunits ( B ) in these hepatocytes was determined by a proximity ligation using anti - Prom1 anti - PKA regulatory subunits ( B ) , respectively . Scale bar , 20 μm .

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

Primary hepatocytes were obtained from 12-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice or KO (Prom1-/-) hepatocytes infected with an adenovirus harboring LacZ (Ad-LacZ) or Prom1 (Ad-Prom1) for 24 hours. The molecular interaction between Prom1 and PKA regulatory subunits (B) in these hepatocytes was determined by a proximity ligation assay using anti-Prom1 and anti-PKA regulatory subunits (B), respectively. Scale bar, 20 μm.


C . Human liver specimens were analyzed by double immunostaining for PROM1 ( red ) radixin ( green ) . lower panels represent magnification of square frame in upper panels . Scale bar , 20 μm .

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

C. Human liver specimens were analyzed by double immunostaining for PROM1 (red) and radixin (green). The lower panels represent the magnification of the square frame in the upper panels. Scale bar, 20 μm.


D . molecular interaction between Prom1 , radixin ( RDX ) actin in WT mouse hepatocytes was determined by co - immunoprecipitation with an anti - Prom1 antibody or isotype control IgG . WCL ; whole lysate .

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

D. The molecular interaction between Prom1, radixin (RDX) and actin in WT mouse primary hepatocytes was determined by co-immunoprecipitation with an anti-Prom1 antibody or isotype control IgG. WCL; whole cell lysate.


HEK293 were transiently transfected with combination of Prom1 - FLAG Myc - radixin plasmids for 24 hours . molecular interaction between Prom1 - FLAG Myc - radixin was determined by co - immunoprecipitation using isotype control IgG , anti - Myc ( E ) antibodies .

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

HEK293 cells were transiently transfected with the combination of Prom1-FLAG and Myc-radixin plasmids for 24 hours. The molecular interaction between Prom1-FLAG and Myc-radixin was determined by co-immunoprecipitation using isotype control IgG, anti-Myc (E) antibodies.


HEK293 were transiently transfected with combination of Prom1 - FLAG Myc - radixin plasmids for 24 hours . molecular interaction between Prom1 - FLAG Myc - radixin was determined by co - immunoprecipitation using isotype control IgG , anti - FLAG ( F ) antibodies .

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

HEK293 cells were transiently transfected with the combination of Prom1-FLAG and Myc-radixin plasmids for 24 hours. The molecular interaction between Prom1-FLAG and Myc-radixin was determined by co-immunoprecipitation using isotype control IgG, anti-FLAG (F) antibodies.


G . localization of radixin actin in WT ( Prom1 + / + ) KO ( Prom1 - / - ) hepatocytes was determined by immunofluorescence for radixin phalloidin . Scale bar , 20 μm .

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

G. The cellular localization of radixin and actin in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes was determined by immunofluorescence staining for radixin and phalloidin staining. Scale bar, 20 μm.


H . Detergent - resistant lipid rafts were isolated from WT ( Prom1 + / + ) KO ( Prom1 - / - ) mouse liver samples . of Prom1 , radixin ( RDX ) , flotillin transferrin receptor ( TfR ) were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting .

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

H. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse liver samples. Levels of the Prom1, radixin (RDX), flotillin and transferrin receptor (TfR) proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.


I . Detergent - resistant lipid rafts were isolated from WT ( Prom1 + / + ) KO ( Prom1 - / - ) mouse hepatocytes after cholesterol depletion by methyl - β - cyclodextrin ( mβCD , 30 mM ) for 30 minutes at 4 oC . of Prom1 , radixin , flotillin transferrin receptor were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting .

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

I. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse primary hepatocytes after cholesterol depletion by methyl-β-cyclodextrin (mβCD, 30 mM) for 30 minutes at 4 oC. Levels of the Prom1, radixin, flotillin and transferrin receptor proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.


molecular interactions between PROM1 radixin mutants were determined by co - immunoprecipitation with an anti - FLAG antibody . HEK293 were co - transfected with plasmids expressing Myc - radixin various deletion mutants of PROM1 - FLAG ( A ) for 24 hours .

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

The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing Myc-radixin and various deletion mutants of PROM1-FLAG (A) for 24 hours.


molecular interactions between PROM1 radixin mutants were determined by co - immunoprecipitation with an anti - FLAG antibody . HEK293 were co - transfected with plasmids expressing PROM1 - FLAG various deletion mutants of Myc - radixin ( B ) for 24 hours .

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

The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing PROM1-FLAG and various deletion mutants of Myc-radixin (B) for 24 hours.


C . molecular interaction between PROM1 - FLAG Myc - radixin in presence of increasing amounts of Myc - FERM was determined by co - immunoprecipitation with an anti - FLAG antibody .

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

C. The molecular interaction between PROM1-FLAG and Myc-radixin in the presence of increasing amounts of Myc-FERM was determined by co-immunoprecipitation with an anti-FLAG antibody.


WT hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . localization of radixin was determined by immunofluorescence . Scale bar , 20 μm .

O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O

WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. The cellular localization of radixin was determined by immunofluorescence staining. Scale bar, 20 μm.


WT hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . localization of p - CREB after glucagon stimulation for 10 minutes ( E ) was determined by immunofluorescence . Scale bar , 20 μm .

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

WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. the nuclear localization of p-CREB after glucagon stimulation for 10 minutes (E) was determined by immunofluorescence staining. Scale bar, 20 μm.


WT hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . ( F ) Ad - FERM - Myc - or Ad - LacZ - expressing WT hepatocytes were serum - starved for 16 hours stimulated with glucagon ( 10 nM ) for 10 minutes . of p - PKA substrates , p - CREB , CREB , FERM - Myc GAPDH were determined by immunoblotting .

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

WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. (F) Ad-FERM-Myc- or Ad-LacZ-expressing WT hepatocytes were serum-starved for 16 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, FERM-Myc and GAPDH were determined by immunoblotting.