K Effects of LAMTOR1 KD treatment with a calcineurin inhibitor , FK506 , on LFS - induced LTD in CA1 . L Means ± of fEPSPs measured 45 min after LFS in different groups ( n = 3 - 4 slices from 3 - 4 mice ) . Data information : Slopes of fEPSPs were normalized to average values recorded during first 10 min baseline Statistical significance was assessed by two - way ANOVA with Tukey ' s post - * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 compared with shSc , Vehicle , or TAT , # P < 0 . 05 , # # P < 0 . 01 , # # # P < 0 . 001 compared with shLAMTOR1 . Insets show representative traces of evoked fEPSPs before 40 min after LFS . Scale bar 0 . 5 mV / 10 ms .

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

K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS-induced LTD in CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups (n = 3-4 slices from 3-4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after LFS. Scale bar 0.5 mV/10 ms.


LAMTOR1 KD significantly reduced of GluA1 phosphorylation total GluA1 as well as of p - GluA1 to GluA1 in hippocampal neurons , this effect was reversed by ML - SI1 treatment . A Representative of CA1 pyramidal neurons stained with anti - p - GluA1 S845 ( magenta ) , anti - GluA1 ( red ) , anti - GFP ( green ) antibodies . Scale bar = 20 µm .

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

LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. A Representative images of CA1 pyramidal neurons stained with anti-p-GluA1 S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm.


E Representative of proximal dendrites of hippocampal neurons stained with anti - GluA1 ( green ) , anti - LAMP2 ( red ) , anti - GFP ( gray ) antibodies . Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false - colored green GFP false - colored gray to better show colocalization of GluA1 with LAMP2 . Arrowheads indicate clearly co - localized . Scale bar , 5 µm .

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

E Representative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2 (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and GFP false-colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly co-localized puncta. Scale bar, 5 µm.


A Mice received 10 min of training in an environment with two identical objects received a retention 24 h later in which one object was replaced with a novel one . LAMTOR1 shRNA - injected mice exhibited a significant deficit 24 h after training , which was reversed by ML - SI1 treatment ( N = 7 - 18 mice ) . B % freezing for different experimental groups in context memory ( N = 7 - 19 mice ) . Data information : Data with error bars are represented as means ± . Statistical significance was assessed by two - way ANOVA with Tukey ' s post - . * P < 0 . 05 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 .

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

A Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7-18 mice). B % freezing for different experimental groups in context memory (N = 7-19 mice). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test. *P < 0.05, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1.


Peptide array dot blot to identify LIR motifs in indicated SNARE . for identified LIRs from positive signals are marked on each SNARE . are denoted as acid single letter codes in blot .

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Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.


Co - immunoprecipitation ( Co - IP ) of interactions between FLAG - tagged Stx16 / Vti1a / Stx6 EGFP - tagged LC3B or GABARAP overexpressed in HEK293T . * indicates mouse IgG heavy chain of precipitated mouse - anti - FLAG antibody .

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Co-immunoprecipitation (Co-IP) analysis of interactions between FLAG-tagged Stx16/Vti1a/Stx6 and EGFP-tagged LC3B or GABARAP overexpressed in HEK293T cells. * indicates mouse IgG heavy chain of precipitated mouse-anti-FLAG antibody.


GST pull - down of interactions between radiolabeled Myc - Stx16 GST - tagged mAtg8 .

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GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.


GST pull - down of interactions between wild type ( WT ) or LIR - mutant ( L219A / V222A ) Stx16 GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP . Data shown as means ± of precipitated Stx16 , n = 3 .

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GST pull-down analysis of interactions between wild type (WT) or LIR-mutant (L219A/V222A) Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


GST pull - down of interactions between WT or different types of LIR - mutant Stx16 GST - tagged LC3C or GABARAP . Lower panel shows percentages of WT or LIR - mutant Stx16 bound to GST - LC3C or GST - GABARAP .

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GST pull-down analysis of interactions between WT or different types of LIR-mutant Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP.


GST pull - down of interactions between radiolabeled Myc - Stx16 WT or LDS - mutant GABARAP ( GABARAP - Y49A - Y49A / F104A ) . Lower panel shows percentages of Stx16 bound to WT or LDS - mutant GABARAP . Data shown as means ± of precipitated Stx16 , n = 3 .

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GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and WT or LDS-mutant GABARAP (GABARAP-Y49A and -Y49A/F104A). Lower panel shows percentages of Stx16 bound to WT or LDS-mutant GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.


WT or STX17 - knockout ( STX17KO ) HeLa were starved with or without presence of bafilomycin A1 ( Baf A1 , 100 nM ) for 2 h , lysates were subjected to Western blot of LC3B p62 . Quantifications of LC3B - II normalized to β - actin from treated as in ( A ) ; Data shown as means ± of LC3B - II β - actin ratios , n = 3 ; * , p < 0 . 05 ( one - way ANOVA ) .

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

WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.   Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).


WT , STX16KO , STX17KO or STX16 / STX17 double KO ( STX16 / STX17DKO ) HeLa were starved with or without presence of Baf A1 ( 100 nM ) for 2 h , lysates were subjected to Western blot of LC3B . Quantifications of LC3B - II normalized to β - actin from ( C ) ; Data shown as means ± of LC3B - II β - actin ratios , n = 3 ; † , not significant ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.   Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).


WT or STX16 / STX17DKO HeLa were starved in EBSS for 2 h , subjected to ultrastructural of autophagic vesicles ( AV ) with electron microscopy . AVi : initial autophagic vacuoles ; AVd : degradative autophagic vacuoles ; G : Golgi apparatus . Image acquisition counting as in Methods . Scale bars , 1 μm 0 . 5 µm ( top sections ) . Quantifications of autophagic vesicles in WT STX16 / STX17DKO HeLa treated as ( E ) ; Data shown as means ± of AV profiles relative to ; † , not significant ; * , p < 0 . 05 ( two - way ANOVA ) . AV profiles from 47 of each samples were counted .

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

WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the autophagic vesicles (AV) with electron microscopy. AVi: initial autophagic vacuoles; AVd: degradative autophagic vacuoles; G: Golgi apparatus. Image acquisition and counting as in Methods. Scale bars, 1 μm and 0.5 µm (top sections).   Quantifications of autophagic vesicles in WT and STX16/STX17DKO HeLa cells treated as (E); Data shown as means ± SEM of AV profiles relative to cytoplasmic area; †, not significant; *, p < 0.05 (two-way ANOVA). AV profiles from 47 images of each samples were counted.


WT or STX16 / STX17DKO HeLa - YFP - Parkin were treated with CCCP ( 10 µM ) or oligomycin A ( 5 µM ) antimycin A ( 10 µM ) ( OA ) for 16 hours , subjected to high - content microscopy ( HCM ) of mitochondria clearance . Masks : blue , nuclei ; red , mitochondria stained with mitochondrial ( mtDNA ) antibody . Scale bar : 20 µm . Quantifications of mtDNA by object or total per in WT or STX16 / STX17DKO HeLa - YFP - Parkin treated as in ( A ) ; Data shown as means ± of mtDNA object ( upper panel ) or object total ( lower panel ) per ; minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO Huh7 were treated with H2O2 ( 0 . 4 mM ) for indicated time points , autophagic clearance of peroxisomes was measured by of PMP70 , PEX14 p62 . Quantifications of peroxisomal PMP70 PEX14 for WT or STX16 / STX17DKO Huh7 treated as in ( C ) ; Data shown as means ± of PEX14 or PMP70 β - actin ratios , n = 3 ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

O 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O 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 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 or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


WT , STX16KO , STX17KO or STX16 / STX17DKO THP - 1 were infected with Mycobacterium tuberculosis ( Mtb ) , followed by starvation to induce autophagy . Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates ; Data shown as means ± of Mtb colonies , n = 4 ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


Validation of STX16 / STX17 DKO by Western blot in HEK293 stably expressing RPS3 - Keima ( left panel ) HCT116 stably expressing RPL28 - Keima ( right panel ) .

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Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel).


WT or STX16 / STX17DKO HEK293 RPS3 - Keima were cultured in full medium or starved for 8 hours , subjected to HCM of Keima accumulated in autolysosomes with excitation wavelength at 560 nm . Masks : , identified on nuclei ; red , Keima detected with excitation emission wavelengths of 560 nm 620 nm , respectively ; lower panel shows Keima without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima in WT or STX16 / STX17DKO HEK293 RPS3 - Keima treated as in ( C ) . Data shown as means ± of Keima per , minimum 1000 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16 /STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO HCT116 RPL28 - Keima were cultured in full medium or starved for 8 hours , subjected to HCM of Keima accumulated in autolysosomes with excitation wavelength at 560 nm . Masks : , identified on nuclei ; red , Keima detected with excitation emission wavelengths of 560 nm 620 nm , respectively ; lower panel shows Keima without masks . Scale bar : 20 µm . Quantifications of autolysosomal Keima in WT or STX16 / STX17DKO HCT116 RPL28 - Keima treated as in ( E ) . Data shown as means ± of Keima per , minimum 1000 were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * * , p < 0 . 01 ( two - way ANOVA ) .

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

WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).


( A ) WT or STX16KO HeLa were starved in EBSS for 2 h subjected to HCM of LAMP2 LC3 . Masks : , identified on nuclei ; red , LC3 ; green , LAMP2 ; yellow , overlap of LC3 LAMP2 . Scale bar : 20 µm .

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(A) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm.


( B - D ) Quantifications of LC3 per ( B ) , LC3 LAMP2 overlap per ( C ) , LAMP2 per ( D ) in WT or STX16KO treated Data shown as means ± of or overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( two - way ANOVA ) .

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(B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA).


( E ) Western blot of LAMP1 LAMP2 in WT separate clones of STX16KO HeLa , Huh7 , U2OS .

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(E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells.


( F ) Western blot of LAMP2 in WT separate clones of STX6KO or VTI1AKO HeLa .

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(F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells.


( G ) Time course of mTOR inactivation during EBSS starvation in WT vs . STX16KO HeLa analyzed by Western blotting for mTOR substrates ULK1 4E - BP1 .

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(G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1.


( H ) WT or STX16KO HeLa were starved in EBSS for 1 h subjected to HCM of mTOR . Masks : , identified on nuclei ; red , mTOR . Scale bar : 20 µm . ( I ) Quantifications of mTOR in WT or STX16KO treated as in ( H ) . Data shown as means ± of mTOR per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

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(H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm. (I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16KO HeLa were starved in EBSS for 1 h , followed by starvation with presence of LysoTracker Red DND - 99 ( LTR ) ( 100 nM ) for additional 30 min , subjected to HCM of LTR . Scale bar : 20 µm . Quantifications of LTR per or total per in WT STX16KO HeLa treated as in ( A ) . Data shown as means ± of LTR per or object total per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ( two - way ANOVA ) .

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

WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.   Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).


WT or STX16KO HeLa were starved in EBSS for 1 h , followed by starvation with presence of LTR ( 100 nM ) for additional 30 min , subjected to HCM of colocalization between LTR TGN46 . Masks : , identified on nuclei ; red , LTR ; green , TGN46 ; yellow , overlap between LTR TGN46 . Scale bar : 20 µm . Quantifications of overlaps between LysoTracker Red TGN46 in WT STX16KO treated as in ( C ) . Data shown as means ± of LTR TGN46 overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * * , p < 0 . 01 ( two - way ANOVA ) .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O 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 O O O O O 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 or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.   Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).


WT or STX16 / STX17DKO HeLa were starved in EBSS for 2 h , subjected to ultrastructural of Golgi apparatus with electron microscopy . VF of Golgi : volume fraction of Golgi relative to total . Scale bar , 2 μm . Red arrows indicate Golgi apparatus .

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WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy. VF of Golgi: volume fraction of Golgi relative to total cytoplasmic area. Scale bar, 2 μm. Red arrows indicate Golgi apparatus.


WT , LC3TKO , GABARAPTKO or HexaKO HeLa were starved in EBSS for 90 min subjected to HCM of overlaps between LAMP2 Stx16 . Masks : , identified on nuclei ; green , LAMP2 ; red , Stx16 ; yellow , overlap of LAMP2 Stx16 . Scale bar : 20 µm .

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

WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm.


Quantifications of overlaps between LAMP2 Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa treated Data shown as means ± of LAMP2 Stx16 overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


Quantifications of overlaps between LBPA Stx16 in WT , LC3TKO , GABARAPTKO or HexaKO HeLa grown in full medium or treated Data shown as means ± of LBPA Stx16 overlap per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


WT , LC3TKO , GABARAPTKO or HexaKO HeLa were starved in EBSS for 1 hour , followed by starvation with presence of LTR ( 100 nM ) for additional 30 min , subjected to HCM of LTR . Scale bar : 20 µm .

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WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.


Quantifications of LTR in WT , LC3TKO , GABARAPTKO or HexaKO HeLa treated Data shown as means ± of LTR per , minimum 500 were counted each well from at least 12 wells , 3 independent experiments ; † , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( one - way ANOVA ) .

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

Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).


a , ( wt , ∆ atg18 or ∆ atg18 / ∆ atg21 / ∆ hsv2 ( ) ) , with without a plasmid expressing wt or a mutant form of Atg18 ) were grown in YPD , vacuoles were stained with FM4 - 64 imaged by confocal microscopy . At least 10 confocal sections spaced at 300 nm were assembled into maximum projections . show before ( 0 min ) 15 , 30 60 min after induction of vacuolar fragmentation by addition of 0 . 4 M NaCl . Red : FM4 - 64 . Gray : DIC . Scale bar 5 µm .

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

a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm.


b , number of vacuoles per was quantified for experiment in a . 150 from three independent experiments were counted for each condition . Values represent mean , error bars .

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b, The number of vacuoles per cell was quantified for the experiment in a. 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.


atg18 expressing plasmids carrying Atg18 - GFP or Atg18FGGG - GFP , a mutant in lipid binding sites , were stained with FM4 - 64 . a , Confocal z - stacks were taken before 2 , 6 10 min after addition of 0 . 4 M NaCl . Stacks were processed into maximum projections using ImageJ . Scale bar : 5 µm .

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atg18 cells expressing plasmids carrying Atg18-GFP or Atg18FGGG-GFP, a mutant in the lipid binding sites, were stained with FM4-64. a, Confocal z-stacks were taken before and 2, 6 and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. Scale bar: 5 µm.


b , Same experiment as in a , but in a fab1 or vps34 background .

b, Same experiment as in a, but in a fab1 or vps34 background.


d , Localisation of Atg18 - GFP 5 min after salt shock . Experiment as in a . Scale bar : 1 µm .

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d, Localisation of Atg18-GFP 5 min after salt shock. Experiment as in a. Scale bar: 1 µm.


b , c Their secondary structure was analyzed by circular dichroism spectroscopy in presence of increasing concentrations of hexafluoro - 2 - propanol ( HFIP ) , in presence of 6 mM small unilamellar vesicles ( SUVs , 70 % egg PC 30 % PS , total phospholipid approx . 6 mM ) , or of or control buffer only ( blue ) . Spectra are shown for b , wildtype

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptide


b , c Their secondary structure was analyzed by circular dichroism spectroscopy in presence of increasing concentrations of hexafluoro - 2 - propanol ( HFIP ) , in presence of 6 mM small unilamellar vesicles ( SUVs , 70 % egg PC 30 % PS , total phospholipid approx . 6 mM ) , or of or control buffer only ( blue ) . Spectra are shown for c , SLoop peptide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for c, the SLoop peptide.


d , Vacuole of expressing Atg18Sloop - GFP , imaged before after osmotic shock as in Fig 1a . Scale bar : 5 µm .

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d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm.


e , Number of vacuoles per in d , assayed as in Figure 1b . n = 3 .

e, Number of vacuoles per cell in d, assayed as in Figure 1b. n=3.


f , Vacuole of expressing Atg18DLoop - GFP , imaged as in d .

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f, Vacuole morphology of cells expressing Atg18DLoop-GFP, imaged as in d.


g , Number of vacuoles per in e , assayed as in Fig . 1b . n = 3 .

g, Number of vacuoles per cell in e, assayed as in Fig. 1b. n=3.


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for a ,

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

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for a, Atg18wt


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for b , Atg18FGGG ;

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

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for b, Atg18FGGG;


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for c , Atg18DLoop ;

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for c, Atg18DLoop;


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of indicated phosphoinositides , or of EPC indicated fraction of PS . Cholesterol had been added to 20 mol % of total phospholipid amount . Liposomes were incubated with purified for 30 min at room ( 25 ° C ) , centrifuged supernatants ( S ) pellets ( P ) analyzed by SDS - PAGE Coomassie . Binding is shown for d , Atg18SLoop .

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for d, Atg18SLoop.


e , bands from a - d were quantified from 3 independent experiments . mean with is shown .

e, The bands from a-d were quantified from 3 independent experiments. The mean with SEM is shown.


a . Autophagy measured with pho8 ∆ 60 in atg18 complemented with wt , FGGG , SLoop or DLoop alleles of ATG18 .

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

a. Autophagy measured with the pho8∆60 assay in atg18 cells complemented with wt, FGGG, SLoop or DLoop alleles of ATG18.


b . Same as in a , but in atg18 atg21 complemented with wt or SLoop versions of ATG18 ATG21 . Atg21SLoop contains K426F F429K substitutions . Helical wheel projections for both Atg21 helices are given . arrows indicate magnitude of hydrophobic moment .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR 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. Same assay as in a, but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given. The arrows indicate the magnitude of the hydrophobic moment.


c . Vacuole fragmentation in atg2 , assayed 15 min after a moderate hypertonic shock as in Fig . 1 .

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c. Vacuole fragmentation in atg2 cells, assayed 15 min after a moderate hypertonic shock as in Fig. 1.


d . Number of vacuoles per in c , assayed as in Figure 1b . n = 3 .

d. Number of vacuoles per cell in c, assayed as in Figure 1b. n=3.


a , Time lapse of Atg18 added to liposomes . 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5 % PI3P 1 % PI ( 3 , 5 ) P2 . sample was imaged on a spinning disc confocal microscope . Most of tubules underwent scission into small vesicles ( See movie EV6 ) . Scale bar 5 µm . Areas in boxes 1 2 of c are shown at higher magnification in b c . Scale bars : 2 . 5 µm .

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

a, Time lapse analysis of Atg18 added to liposomes. 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5% PI3P and 1% PI(3,5)P2. The sample was imaged on a spinning disc confocal microscope. Most of the tubules underwent scission into small vesicles (See movie EV6). Scale bar 5 µm. Areas in boxes 1 and 2 of c are shown at higher magnification in b and c. Scale bars: 2.5 µm.


d , of fission activity . For each movie , whole frame was divided into squares of 10 per 10 µm number of small vesicles per 100 µm2 counted . mean of three independent experiments is shown with .

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

d, Quantification of fission activity. For each movie, the whole frame was divided into squares of 10 per 10 µm and the number of small vesicles per 100 µm2 counted. The mean of three independent experiments is shown with SEM.


a , 1µM Atg18 was added to wells containing sedimented GUVs with 10 % PI3P or PI ( 3 , 5 ) P2 while imaging a single plane by spinning disc microscopy . tension exerted by Atg18 binding suffices to induce liposome collapse , which allows to better judge mobility of tubular or clustered membranes that remain . are extracted from movies EV2 to EV5 . Scale bar 5 µm .

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a, 1µM Atg18 was added to wells containing sedimented GUVs with 10% PI3P or PI(3,5)P2 while imaging a single plane by spinning disc microscopy. Membrane tension exerted by Atg18 binding suffices to induce liposome collapse, which allows to better judge the mobility of the tubular or clustered membranes that remain. Images are extracted from movies EV2 to EV5. Scale bar 5 µm.


b , Atg18 was covalently labeled with green dye Alexa488 TFP ( ThermoFisher ) used in experiments as in a , with rhodamine - phosphatidylethanolamine - labeled ( Rh - PE ) GUVs containing indicated phosphoinositides . Scale bar 5

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b, Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP (ThermoFisher) and used in experiments as in a, with rhodamine-phosphatidylethanolamine-labeled (Rh-PE) GUVs containing the indicated phosphoinositides. Scale bar 5 µm


c , Effect of 1µM of purified recombinant Atg18 variants on GUVs containing 10mol % of indicated phosphoinositides . Vesicles are shown after 1 to 3 min of incubation .

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c, Effect of 1µM of purified recombinant Atg18 variants on GUVs containing 10mol% of the indicated phosphoinositides. Vesicles are shown after 1 to 3 min of incubation.


a , Cross - linking of Atg18 . Purified recombinant Atg18wt or Atg18FGGG ( 1 . 5 µM ) was incubated with liposomes containing 5 % PI3P , PI ( 3 , 5 ) P2 , PI ( 4 , 5 ) P2 or 15 % PS as a control for negative charge . After addition of a cleavable cross - linker or buffer only , liposomes were pelleted , dissolved in sample buffer , incubated in presence or absence of 100 mM dithiothreitol ( DTT ) for 5 min at room analysed by SDS - PAGE Coomassie . Cross - link products migrate at upper limit of separating gel .

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

a, Cross-linking of Atg18. Purified recombinant Atg18wt or Atg18FGGG (1.5 µM) was incubated with liposomes containing 5% PI3P, PI(3,5)P2, PI(4,5)P2 or 15% PS as a control for negative charge. After addition of a cleavable cross-linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature and analysed by SDS-PAGE and Coomassie staining. Cross-link products migrate at the upper limit of the separating gel.


b , Cross - linking experiment with recombinant Atg18wt as in a , performed in presence of increasing concentrations of liposomes containing 5 % PI ( 3 , 5 ) P2 .

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b, Cross-linking experiment with recombinant Atg18wt as in a, performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5)P2.


A ) Distribution curve of replication fork rates for Mybl2 + / + Mybl2Δ / Δ ESCs . Statistical was performed using Mann Whitney U . n = 6 experimental replicates . A minimum of 490 replication forks were counted . ( * * * * p < 0 . 0001 ) .

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A) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs. Statistical analysis was performed using the Mann Whitney U test. n=6 experimental replicates. A minimum of 490 replication forks were counted.( **** p< 0.0001).


B ) Left panel : representative elongating replication forks ( stable unstable ) . of average IdU / CldU in Mybl2 + / + Mybl2Δ / Δ ESCs . of highly unstable forks ( above 2 ) are indicated . Y - axis was cut to change scale display large values . Statistical was performed using Mann Whitney U . ( * * * * p < 0 . 0001 ) . At least 450 ratios were recorded from 6 experimental replicates .

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B) Left panel: representative elongating replication forks (stable and unstable). Quantification of the average IdU/CldU ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Percentage of highly unstable forks (ratio above 2) are indicated. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. (**** p< 0.0001). At least 450 ratios were recorded from 6 experimental replicates.


C ) of both CldU labels surrounding IdU tracks ( newly fired origins ) was measured before calculating a representing symmetry around new origins . of average positive in Mybl2 + / + Mybl2Δ / Δ ESCs . Y - axis was cut to change scale display large values . Statistical was performed using Mann Whitney U . 55 75 ratios were recorded in Mybl2 + / + Mybl2Δ / Δ respectively from 3 replicates ( * * * * p < 0 . 0001 ) .

O O O O O 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 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

C) The length of both CldU labels surrounding IdU tracks (newly fired origins) was measured before calculating a ratio representing symmetry around new origins. Quantification of the average positive ratio in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Y-axis was cut to change scale and display large values. Statistical analysis was performed using the Mann Whitney U test. 55 and 75 ratios were recorded in the Mybl2+/+ and Mybl2Δ/Δ respectively from 3 replicates ( **** p< 0.0001).


D ) Representative image showing presence of UFB by positive Immunostaining for PICH . ( Scale bar 10μm ) . Frequency of anaphases positive for UFBs relative to anaphases in Mybl2 + / + Mybl2Δ / Δ ESCs . At least 75 anaphases were counted per experimental group from 3 independent experiments . Statistical was performed using an unpaired two - t - ( * * * * p < 0 . 0001 ) .

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

D) Representative image showing the presence of UFB by positive Immunostaining for PICH. (Scale bar 10μm). Frequency of anaphases positive for UFBs relative to all anaphases in the Mybl2+/+ and Mybl2Δ/Δ ESCs. At least 75 anaphases were counted per experimental group from 3 independent experiments. Statistical analysis was performed using an unpaired two-tailed t-test (**** p< 0.0001).


E ) Representative for comets in Mybl2 + / + Mybl2Δ / Δ untreated Mybl2 + / + Mybl2Δ / Δ plus CPT ( 20x magnification ) . ( Scale bar 50μm ) .

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

E) Representative images for comets in Mybl2+/+ and Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT (20x magnification). (Scale bar 50μm).


F ) of mean distributions of olive tail moments in Mybl2 + / + Mybl2Δ / Δ untreated CPT - treated . Y - axis was cut to change scale display large values . Statistical using Mann Whitney U . At least 300 comets were measured for each group from 5 experimental replicates ( * * * * p < 0 . 0001 ) .

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

F) Quantification of the mean and distributions of olive tail moments in Mybl2+/+ and Mybl2Δ/Δ untreated and CPT-treated. Y-axis was cut to change scale and display large values. Statistical analysis using Mann Whitney U tests. At least 300 comets were measured for each group from 5 experimental replicates (**** p< 0.0001).


G ) Frequency of EdU positive with over 6 53BP1 foci in Mybl2 + / + , Mybl2Δ / Δ untreated Mybl2 + / + Mybl2Δ / Δ plus CPT . Error bars represent . Statistical using unpaired two - t - . At least 200 EdU positive nuclei were analysed from 4 experimental repeats .

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

G) Frequency of EdU positive cells with over 6 53BP1 foci in Mybl2+/+, Mybl2Δ/Δ untreated and Mybl2+/+ and Mybl2Δ/Δ plus CPT. Error bars represent SEM. Statistical analysis using unpaired two-tailed t-test. At least 200 EdU positive nuclei were analysed from 4 experimental repeats.


A ) were treated for 1 . 5 hours with 5µM CPT , before sequential addition of IdU CldU for 20 minutes each . Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + Mybl2Δ / Δ ESCs treated or not with CPT ( n = 3 independent experiments ; Error bars indicate ) . Statistical using two - unpaired t - .

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

A) Cells were treated for 1.5 hours with 5µM CPT, before sequential addition of IdU and CldU for 20 minutes each. Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with CPT (n=3 independent experiments; Error bars indicate SEM). Statistical analysis using two- tailed unpaired t-test.


( B ) Distribution of replication rate in Mybl2 + / + Mybl2Δ / Δ ESC treated or not with CPT as above . Statistical was carried out using unpaired Mann Whitney U . A minimum of 200 forks were quantified for each of genotypes conditions shown from 3 independent repeats ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC treated or not with CPT as above. Statistical analysis was carried out using unpaired Mann Whitney U tests. A minimum of 200 forks were quantified for each of the genotypes and conditions shown from 3 independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).


C ) ESCs were treated with or without CPT for 4 hours before 0 . 1ug / ml colcemid was added during final 3 . 5 hours of treatment . Representative image of H3 - pS10 positive ( turquoise ) . ( Scale bar 10μm ) . Frequency of H3 - pS10 positive in Mybl2 + / + Mybl2Δ / Δ ESCs . Data from three independent experiments . Error bars represent . Statistical was carried out using a two - unpaired t - ( * p < 0 . 05 ; * * p < 0 . 01 ; ns = no significant ) .

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

C) ESCs were treated with or without CPT for 4 hours before 0.1ug/ml colcemid was added during the final 3.5 hours of treatment. Representative image of H3-pS10 positive cells (turquoise). (Scale bar 10μm). Frequency of H3-pS10 positive cells in Mybl2+/+ and Mybl2Δ/Δ ESCs. Data from three independent experiments. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (*p < 0.05; **p< 0.01; ns= no significant).


D ) P - CDK1 ( Tyr15 ) , CDK1 Beta - actin expression of Mybl2 + / + Mybl2Δ / Δ ESCs with or without CPT treatment analyzed by immunoblotting . Bar graph represents average band density of P - CDK1 in Mybl2Δ / Δ ESCs relative to loading control relative to MYBL2 + / + from 6 repeats . Error bars represent . Statistical was carried out using a two - unpaired t - ( * * p < 0 . 01 ) .

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

D) P-CDK1 (Tyr15), CDK1 and Beta-actin expression levels of Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment analyzed by immunoblotting. Bar graph represents average band density of P-CDK1 in Mybl2Δ/Δ ESCs relative to loading control and relative to the MYBL2+/+ from 6 repeats. Error bars represent SEM. Statistical analysis was carried out using a two-tailed unpaired t-test (**p< 0.01).


A ) Immunoblot showing of phosphorylated CHK1 ( Ser345 ) GAPDH in Mybl2 + / + Mybl2Δ / Δ ESCs with or without CPT treatment ( 2 . 5µM CPT for 4 hours ) .

O O O O O O B-MEASURED_VAR 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

A) Immunoblot showing the levels of phosphorylated CHK1 (Ser345) and GAPDH in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (2.5µM CPT for 4 hours).


B ) Immunoblot showing of P - CHK1 ( Ser345 ) CHK1 in irradiated Mybl2 + / + , Mybl2Δ / Δ ESCs with or without 3 hours inhibitor treatment : ATR ( AZ20 , 5µM ) ATM ( Ku60019 , 10µM ) . were also exposed to 5Gy irradiation before final hour of inhibitor treatment . Bar graph ( lower panel ) represents average band density of P - CHK1 in Mybl2Δ / Δ ESCs relative to CHK1 relative to untreated . Mybl2 + / + from two independent repeats Mybl2Δ / Δ ESCs from three independent repeats . Error bars represent SD . Statistical was carried out using a two - unpaired t - ( * p < 0 . 05 ) .

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

B) Immunoblot showing the levels of P-CHK1 (Ser345) and CHK1 in irradiated Mybl2+/+, and Mybl2Δ/Δ ESCs with or without 3 hours inhibitor treatment: ATR (AZ20, 5µM) and ATM (Ku60019, 10µM). Cells were also exposed to 5Gy irradiation before the final hour of inhibitor treatment. Bar graph (lower panel) represents average band density of P-CHK1 in Mybl2Δ/Δ ESCs relative to CHK1 and relative to untreated cells. Mybl2+/+ from two independent repeats and Mybl2Δ/Δ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (*p< 0.05).


C ) were treated for 1 . 5 hours with 5µM AZ20 , before sequential addition of IdU CldU for 20 minutes each . Dot plot representing effect of ATR inhibition upon replication rate in Mybl2 + / + Mybl2Δ / Δ ESC . Statistical of distributions was carried out using unpaired Mann Whitney U . A minimum of 240 forks were quantified for each of genotypes conditions shown from 2 independent repeats ( * * * * p < 0 . 0001 )

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

C) Cells were treated for 1.5 hours with 5µM AZ20, before sequential addition of IdU and CldU for 20 minutes each. Dot plot representing the effect of ATR inhibition upon replication rate in Mybl2+/+ and Mybl2Δ/Δ ESC. Statistical analysis of distributions was carried out using unpaired Mann Whitney U tests. A minimum of 240 forks were quantified for each of the genotypes and conditions shown from 2 independent repeats (**** p< 0.0001)


D ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + Mybl2Δ / Δ ESCs treated or not with ATR inhibitor AZ20 . At least 500 replication structures were counted per treatment from two independent repeats . Statistical using two - unpaired t - . ( * p < 0 . 05 ; ns = no significant ) .

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

D) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with ATR inhibitor AZ20. At least 500 replication structures were counted per treatment from two independent repeats. Statistical analysis using two- tailed unpaired t-test.(*p< 0.05; ns= no significant).


E ) Distribution average of IdU / CldU fork in Mybl2 + / + Mybl2Δ / Δ ESCs with or without ATR inhibitor treatment . of highly unstable forks ( above 2 ) are indicated . Dotted at 1 indicates positive . Y - axis was cut to change scale display large values . Statistical was carried out using Mann Whitney U . At least 150 ratios were calculated per treatment group from two independent repeats ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

E) Distribution and average of IdU/CldU fork ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATR inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 150 ratios were calculated per treatment group from two independent repeats (*p < 0.05; **p< 0.01; ***p< 0.001; **** p< 0.0001).


B ) Distribution of replication rate after treatment with / without ATM inhibitor . Statistical of distributions was carried out using an unpaired Mann Whitney U . n = 4 . A minimum of 270 forks was measured for each condition ( * * * * p < 0 . 0001 ) .

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

B) Distribution of replication rate after treatment with/without ATM inhibitor. Statistical analysis of distributions was carried out using an unpaired Mann Whitney U test. n=4. A minimum of 270 forks was measured for each condition ( **** p< 0.0001).


C ) Distribution of average IdU / CldU in Mybl2 + / + Mybl2Δ / Δ ESCs with or without ATM inhibitor treatment . of highly unstable forks ( above 2 ) are indicated . Dotted at 1 indicates positive . Y - axis was cut to change scale display large values . Statistical was carried out using Mann Whitney U . At least 260 ratios were calculated per treatment group from 4 separate experiments ( * * * * p < 0 . 0001 ) .

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

C) Distribution of the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U. At least 260 ratios were calculated per treatment group from 4 separate experiments ( **** p< 0.0001).


D ) Frequency of new fired origins for Mybl2 + / + Mybl2Δ / Δ ESCs with or without ATM inhibitor treatment . Error bars represent . Statistical was carried out using unpaired t - . At least 500 replication structures were counted per treatment group from 4 separate repeats .

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

D) Frequency of new fired origins for Mybl2+/+ and Mybl2Δ/Δ ESCs with or without ATM inhibitor treatment. Error bars represent SEM. Statistical analysis was carried out using unpaired t-test. At least 500 replication structures were counted per treatment group from 4 separate repeats.


E ) Frequency of anaphases positive for UFB positive ( on ERCC / PICH positive immunostaining ) for Mybl2 + / + , Mybl2Δ / Δ Mybl2 + / + treated for 2 hours with ATM inhibitor ( KU60019 ) . Data represents at least 100 anaphases from each group from 2 experimental repeats . Statistical was performed using an unpaired two - t - .

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

E) Frequency of anaphases positive for UFB positive cells (based on ERCC/PICH positive immunostaining) for Mybl2+/+, the Mybl2Δ/Δ and the Mybl2+/+ treated for 2 hours with ATM inhibitor (KU60019). Data represents at least 100 anaphases from each group from 2 experimental repeats. Statistical analysis was performed using an unpaired two-tailed t-test.


F ) Immunoblot showing of phosphorylated ATM ( Ser1987 ) ATM in Mybl2 + / + Mybl2Δ / Δ ESCs with or without CPT treatment ( 10µM CPT for 2 hours ) . Bar graph ( lower panel ) represents average band density of P - ATM in Mybl2Δ / Δ ESCs treated with CPT relative to ATM relative to wild type CPT treated from three independent repeats . Error bars represent SD . Statistical was carried out using a two - unpaired t - .

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

F) Immunoblot showing the levels of phosphorylated ATM (Ser1987) and ATM in the Mybl2+/+ and Mybl2Δ/Δ ESCs with or without CPT treatment (10µM CPT for 2 hours). Bar graph (lower panel) represents average band density of P-ATM in Mybl2Δ/Δ ESCs treated with CPT relative to ATM and relative to wild type CPT treated cells from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test.


G ) Distribution plot of replication speed in Mybl2 + / + ATM - / - ESC treated or not for 90 minutes with 10µM KU60019 ( ATMi ) , before sequential addition of IdU CldU for 20 minutes each . Statistical was carried out using Mann Whitney U . At least 200 replication forks were counted from 3 separate experiments for wild type ATM - / - two separate experiments for ATM - / - treated with ATM inhibitor . ( * * * * p < 0 . 0001 )

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

G) Distribution plot of replication speed in Mybl2+/+and ATM-/- ESC treated or not for 90 minutes with 10µM KU60019 (ATMi), before sequential addition of IdU and CldU for 20 minutes each. Statistical analysis was carried out using the Mann Whitney U test. At least 200 replication forks were counted from 3 separate experiments for wild type and ATM-/- and two separate experiments for ATM-/- treated with ATM inhibitor . ( **** p< 0.0001)


H ) of number of replication factories per for different genotypes treatments . A minimum of 65 early S - phase nuclei were counted per condition from two independent repeats . Statistical was performed using a two - unpaired unequal variance t - . ( * * * * p < 0 . 0001 )

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

H) Quantification of the number of replication factories per cell for the different genotypes and treatments. A minimum of 65 early S-phase nuclei were counted per condition from two independent repeats. Statistical analysis was performed using a two-tailed unpaired unequal variance t-test.( **** p< 0.0001)


I ) Distribution plot of replication speed in human iPSC MEFs ( immortal ) . Statistical was carried out using Mann Whitney U . At least 400 replication forks for iPSC , 200 for MEFS 250 for immortal MEFs were counted from 3 independent experiments . ( * * * * p < 0 . 0001 )

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

I) Distribution plot of replication speed in human iPSC and MEFs (primary and immortal). Statistical analysis was carried out using the Mann Whitney U test. At least 400 replication forks for iPSC,200 for primary MEFS and 250 for immortal MEFs were counted from 3 independent experiments. ( **** p< 0.0001)


B C ) Distribution curve of replication fork rates for Mybl2 + / + Mybl2Δ / Δ ESCs treated with mirin . Statistical was performed using Mann Whitney U . n = 3 experimental replicates . value of mean fork is indicated above arrow ( * * * * p < 0 . 0001 )

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

B and C) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs treated with mirin. Statistical analysis was performed using the Mann Whitney U test. n=3 experimental replicates. The value of the mean fork length is indicated above arrow ( **** p< 0.0001)


D ) Representative of new firing origins , elongating forks showing stable replication fork instability .

D) Representative images of new firing origins, and elongating forks showing stable replication and fork instability.


E ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + Mybl2Δ / Δ ESCs treated or not with Mirin . At least 450 replication structures were counted per treatment from 3 independent repeats . Error bars represent . Statistical using two - unpaired t - .

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

E) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with Mirin. At least 450 replication structures were counted per treatment from 3 independent repeats. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test.


F ) Distribution average IdU / CldU in Mybl2 + / + Mybl2Δ / Δ ESCs with or without MRE11 inhibitor treatment . of both incorporated labels for each elongating fork was measured in µm positive was calculated . of highly unstable forks ( above 2 ) are indicated . Dotted at 1 indicates positive . Y - axis was cut to change scale display large values . Statistical was carried out using Mann Whitney U . At least 260 ratios were calculated per treatment group from 3 separate experiments . ( * * * * p < 0 . 0001 )

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

F) Distribution and the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without MRE11 inhibitor treatment. The length of both incorporated labels for each elongating fork was measured in µm and the positive ratio was calculated. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 260 ratios were calculated per treatment group from 3 separate experiments.( **** p< 0.0001)


C ) Frequency of new firing origins ( relative to total structures counted ) from Mybl2 + / + Mybl2Δ / Δ ESCs treated or not with indicated inhibitors : CDC7 inhibitor ( PHA - 767491 ) / or ATM inhibitor ( Ku60019 ) . At least 300 replication structures were counted per treatment . Error bars represent . Statistical using two - unpaired t - ( * p < 0 . 05 ) .

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

C ) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with the indicated inhibitors: CDC7 inhibitor (PHA-767491) and/or ATM inhibitor (Ku60019). At least 300 replication structures were counted per treatment. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test (*p < 0.05).


D ) Replication rate ( kb / min ) of Mybl2 + / + Mybl2Δ / Δ ESCs treated with indicated inhibitors . Statistical was carried out using an unpaired Mann Whitney U ( * * * * p < 0 . 0001 ) . Minimum three independent experiments . At least 270 forks were scored for non - ATM inhibitor treated groups , a minimum of 130 for ATM inhibitor treated groups .

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 O O 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 B-CONTROLLED_VAR O O O O

D ) Replication rate (kb/min) of Mybl2+/+ and Mybl2Δ/Δ ESCs treated with the indicated inhibitors. Statistical analysis was carried out using an unpaired Mann Whitney U test (**** p< 0.0001). Minimum three independent experiments. At least 270 forks were scored for non-ATM inhibitor treated groups, and a minimum of 130 for ATM inhibitor treated groups.


E ) Fork stability ( average IdU / CldU ) of Mybl2 + / + ESC Mybl2Δ / Δ ESCs treated with indicated inhibitors alone or in combination . At least 240 ratios were calculated for non - ATM inhibitor treated groups , a minimm of 130 for ATM inhibitor treated groups . Y - axis was cut to change scale display large values . Statistical was carried out using Mann Whitney U ( * * * * p < 0 . 0001 ) . Data for Mybl2 + / + ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor , was collected from two independent experiments . For of conditions , a minimum of three independent repeats was performed .

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

E ) Fork stability (average IdU/CldU ratio) of Mybl2+/+ ESC and Mybl2Δ/Δ ESCs treated with the indicated inhibitors alone or in combination. At least 240 ratios were calculated for non-ATM inhibitor treated groups, and a minimm of 130 for ATM inhibitor treated groups. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using Mann Whitney U test (**** p< 0.0001). Data for Mybl2+/+ ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor, was collected from two independent experiments. For the rest of the conditions, a minimum of three independent repeats was performed.


F ) Immunoblot showing of CDC7 actin in Mybl2 + / + Mybl2Δ / Δ ESCs . Bar graph ( lower panel ) represents average band density of CDC7 in Mybl2Δ / Δ ESCs relative to actin relative to Mybl2 + / + ESCs from three independent repeats . Error bars represent SD . Statistical was carried out using a two - unpaired t - ( ns = no significant ) .

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F) Immunoblot showing the levels of CDC7 and actin in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Bar graph (lower panel) represents average band density of CDC7 in Mybl2Δ/Δ ESCs relative to actin and relative to Mybl2+/+ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (ns=no significant).


A LC - ESI - MS / MS spectrum of Dnmt3a pulled down by Biotin - As in NIH3T3 . NIH3T3 were treated with 10 μg / mL Biotin - As for 2 hours , followed by pull - down determination using LC - ESI - MS / MS .

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A LC-ESI-MS/MS spectrum of Dnmt3a pulled down by Biotin-As in NIH3T3 cells. NIH3T3 cells were treated with 10 μg/mL Biotin-As for 2 hours, followed by pull-down assay and determination using LC-ESI-MS/MS.


B - D Validation of binding between DNMT3A Biotin - As at 5 μg / mL in NIH3T3 ( B ) , MEFs ( C ) CCRF - CEM ( D ) in Biotin - As pull - down .

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B-D Validation of the binding between DNMT3A and Biotin-As at 5 μg/mL in NIH3T3 (B), MEFs (C) and CCRF-CEM (D) cells in the Biotin-As pull-down assay.


F Measurement of DNMT3A - ADD thermodynamic stability upon ATO treatment . Purified DNMT3A - ADD was mixed with ATO at indicated ratios in 20 mM HEPES ( pH 7 . 5 ) for 1 hour . Melting curves were measured by DSF ( left panel ) , calculated ΔTm values are shown ( right panel ) .

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F Measurement of DNMT3A-ADD thermodynamic stability upon ATO treatment. Purified DNMT3A-ADD was mixed with ATO at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Melting curves were measured by DSF (left panel), and the calculated ΔTm values are shown (right panel).


G LC - MS spectra of purified DNMT3A - ADD before after ATO incubation at 1 : 5 molar for 16 hours .

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G LC-MS spectra of purified DNMT3A-ADD before and after ATO incubation at 1:5 molar ratio for 16 hours.


H , I Biotin - As pull - down assays for transfected DNMTs ( H ) DNMT3A - C520A / C524A mutation ( I ) in 293T treated with 5 μg / mL Biotin - As .

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H, I Biotin-As pull-down assays for the transfected DNMTs (H) and DNMT3A-C520A/C524A mutation (I) in 293T cells treated with 5 μg/mL Biotin-As.


A Immunoblotting of DNMT3A in MEFs ( upper panel ) CCRF - CEM ( lower panel ) treated with ATO in indicated conditions .

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A Immunoblotting of DNMT3A protein levels in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO in the indicated conditions.


B MEFs ( upper panel ) CCRF - CEM ( lower panel ) were pretreated with 20 μM DMSO , 1 μM MG132 , 0 . 02 μM Concanamycin A ( Conc A ) , 20 μM Calpeptin ( Calp ) , or 20 μM Z - VAD - FMK ( Z - VAD ) for 2 hours , followed by 16 - hour ATO co - treatment at 1 μg / mL for MEFs or 0 . 3 μg / mL for CCRF - CEM . Immunoblotting of DNMT3A in whole lysate is shown .

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B MEFs (upper panel) and CCRF-CEM cells (lower panel) were pretreated with 20 μM DMSO, 1 μM MG132, 0.02 μM Concanamycin A (Conc A), 20 μM Calpeptin (Calp), or 20 μM Z-VAD-FMK (Z-VAD) for 2 hours, followed by 16-hour ATO co-treatment at 1 μg/mL for MEFs or 0.3 μg/mL for CCRF-CEM cells. Immunoblotting of DNMT3A in whole cell lysate is shown.


C HCT116 were pretreated with 3 μM MG132 for 2 hours , followed by ATO co - treatment for 16 hours at indicated concentrations . Immunoblotting of DNMT3A in whole lysate is shown .

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C HCT116 cells were pretreated with 3 μM MG132 for 2 hours, followed by ATO co-treatment for 16 hours at the indicated concentrations. Immunoblotting of DNMT3A in whole cell lysate is shown.


D In vivo ubiquitination . 293T were transiently transfected with HA - ubiquitin Myc - DNMT3A . After 24 hours , were treated with or without MG132 for 2 hours at indicated concentrations , followed by ATO co - treatment for 6 hours at indicated concentrations . Myc - DNMT3A was immunoprecipitated ( IP ) with anti - Myc antibody , followed by immunoblotting ( IB ) as indicated .

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D In vivo ubiquitination assay. 293T cells were transiently transfected with HA-ubiquitin and Myc-DNMT3A. After 24 hours, cells were treated with or without MG132 for 2 hours at the indicated concentrations, followed by ATO co-treatment for 6 hours at the indicated concentrations. Myc-DNMT3A was immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) as indicated.


E Immunoblotting of transfected wild - type DNMT3A , C520A / C524A C541A mutants in 293T upon 1 μg / mL ATO treatment for indicated durations .

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E Immunoblotting of the transfected wild-type DNMT3A, C520A/C524A and C541A mutants in 293T cells upon 1 μg/mL ATO treatment for the indicated durations.


F Measurement of DNMT3A - ADD thermodynamic stability upon ATO or AsPh3 treatment . Purified DNMT3A - ADD was mixed with ATO or AsPh3 at indicated ratios in 20 mM HEPES ( pH 7 . 5 ) for 1 hour . Chemical structure of AsPh3 is shown . Melting curves were measured by DSF ( upper panel ) , calculated ΔTm values are shown ( lower panel ) .

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F Measurement of DNMT3A-ADD thermodynamic stability upon ATO or AsPh3 treatment. Purified DNMT3A-ADD was mixed with ATO or AsPh3 at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Chemical structure of AsPh3 is shown. Melting curves were measured by DSF (upper panel), and the calculated ΔTm values are shown (lower panel).


G Immunoblotting of DNMT3A in MEFs ( upper panel ) CCRF - CEM ( lower panel ) treated with ATO or AsPh3 in indicated conditions .

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G Immunoblotting of DNMT3A in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO or AsPh3 in the indicated conditions.


H , I Immunoblotting of endogenous DNMT3A , DNMT3B DNMT1 in CCRF - CEM ( H ) THP - 1 ( I ) treated with DAC or ATO in indicated conditions . DAC : + , 0 . 2 μM . ATO : + , 0 . 06 μg / mL ; + + , 0 . 2 μg / mL ; + + + , 0 . 6 μg / mL . Red asterisk indicates specific band .

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H, I Immunoblotting of endogenous DNMT3A, DNMT3B and DNMT1 protein levels in CCRF-CEM (H) and THP-1 (I) cells treated with DAC or ATO in the indicated conditions. DAC: +, 0.2 μM. ATO: +, 0.06 μg/mL; ++, 0.2 μg/mL; +++, 0.6 μg/mL. Red asterisk indicates the specific band.


A Dot blot of global methylation in MEFs upon ATO or DAC treatment at indicated concentrations for 7 days . relative 5meC were quantified by ImageJ . full blotting image of Fig 4A is shown in Fig EV4D , treatment is same .

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A Dot blot analysis of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ. The full blotting image of Fig 4A is shown in Fig EV4D, the treatment is the same.


B COBRA - IAP of global methylation in MEFs upon ATO or DAC treatment at indicated concentrations for 7 days . signal density of unmethylated methylated were quantified by ImageJ .

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B COBRA-IAP of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The signal density of unmethylated and methylated DNA were quantified by ImageJ.


C 5meC enrichment of IAP , Xist , H19 loci in MEFs upon ATO treatment at 0 . 3 μg / mL for 4 or 7 days as measured by MeDIP - qPCR .

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C 5meC enrichment analysis of the IAP, Xist, H19 loci in MEFs upon ATO treatment at 0.3 μg/mL for 4 or 7 days as measured by MeDIP-qPCR.


D Dot blot of global methylation in CCRF - CEM upon ATO or DAC treatment at indicated concentrations for 7 days . relative 5meC were quantified by ImageJ .

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

D Dot blot analysis of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ.


E COBRA - - 1 of global methylation in CCRF - CEM upon ATO or DAC treatment in indicated conditions . signal density of unmethylated methylated were quantified by ImageJ .

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E COBRA-LINE-1 of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment in the indicated conditions. The signal density of unmethylated and methylated DNA were quantified by ImageJ.


F 5meC enrichment of XIST , H19 loci in CCRF - CEM upon ATO treatment at indicated concentrations for 63 days as measured by MeDIP - qPCR .

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F 5meC enrichment analysis of the XIST, H19 loci in CCRF-CEM cells upon ATO treatment at the indicated concentrations for 63 days as measured by MeDIP-qPCR.


( B ) Pull - Down of GST - tagged α11 . 2 segments ( top immunoblot ; IB ) by immobilized MBP - β2AR C - terminus ( residues 326 - 413 of human β2AR ) . GST fusion were detected by an anti - GST antibody MBP fusion by an anti - MBP antibody . Middle IB shows that comparable amounts of various GST fusion had been added to resin samples bottom IB illustrates equal loading of amylose resin samples with MBP - β2AR . ( C ) of ( B ) ( * * * p < 0 . 001 , ANOVA ) .

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(B) Pull-Down of GST-tagged α11.2 segments (top immunoblot; IB) by immobilized MBP-β2AR C-terminus (residues 326-413 of human β2AR). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody. Middle IB shows that comparable amounts of the various GST fusion proteins had been added to the resin samples and bottom IB illustrates equal loading of all amylose resin samples with MBP-β2AR.(C) Quantification of (B) (***p<0.001, ANOVA).


( D ) Pull - Down of GST - CTC but not GST - CTD or GST - CTE ( middle IB ) by immobilized MBP - β2AR C - terminus ( left ) but not MBP alone ( right ) , of which were present at comparable amounts ( bottom top IBs , respectively ) . GST fusion were detected by an anti - GST antibody MBP fusion by an anti - MBP antibody . ( E ) of ( D ) ( * * * p < 0 . 001 , ANOVA ) .

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(D) Pull-Down of GST-CTC but not GST-CTD or GST-CTE (middle IB) by immobilized MBP-β2AR C-terminus (left) but not MBP alone (right), all of which were present at comparable amounts (bottom and top IBs, respectively). GST fusion proteins were detected by an anti-GST antibody and MBP fusion proteins by an anti-MBP antibody.(E) Quantification of (D) (***p<0.001, ANOVA).


( F ) IP of β2AR in presence of 10 μM GST ( Control ; Ctr ) or GST - tagged C - terminal fragments as indicated . CT4 CTC specifically displaced α11 . 2 ( top of IB ) but not GluA1 ( middle of same IB ) from β2AR ( bottom of same IB ) . Use of non - specific IgG ( left lane in right panels ) indicates specificity of IP . ( G , H ) . For of coIPs , α11 . 2 GluA1 immunosignals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) . ( I ) Representative IB showing amounts of GST fusion that were added in ( F ) , as detected by anti - GST antibodies .

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(F) IP of β2AR in the presence of 10 μM GST (Control; Ctr) or GST-tagged C-terminal fragments as indicated. CT4 and CTC specifically displaced α11.2 (top of IB) but not GluA1 (middle of same IB) from β2AR (bottom of same IB). Use of non-specific IgG (left lane in right panels) indicates specificity of IP.(G, H). For quantification of coIPs, α11.2 and GluA1 immunosignals were normalized to β2AR signals (***p<0.001, ANOVA).(I) Representative IB showing amounts of the GST fusion proteins that were added in (F), as detected by anti-GST antibodies.


( A ) Titration of polarization of FITC - peptides ( 100 nM ) spanning α11 . 2 aa 1906 - 1959 with purified MBP - β2AR C - terminus . Kd value was obtained by fitting a non - linear direct binding curve to Pep2 .

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(A) Titration of fluorescence polarization of FITC-peptides (100 nM) spanning α11.2 aa 1906-1959 with purified MBP-β2AR C-terminus. Kd value was obtained by fitting a non-linear direct binding curve to Pep2.


( B ) IP of β2AR in absence ( Control ) or presence of 10 μM peptides , as indicated . Pep2 specifically displaced α11 . 2 ( top of IB ) but not GluA1 ( middle , same IB ) from β2AR ( bottom , same IB ) . Use of non - specific IgG ( left lane ) indicates specificity of coIPs . ( C , D ) . For , α11 . 2 GluA1IB signals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) .

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(B) IP of β2AR in the absence (Control) or presence of 10 μM peptides, as indicated. Pep2 specifically displaced α11.2 (top of IB) but not GluA1 (middle, same IB) from β2AR (bottom, same IB). Use of non-specific IgG (left lane) indicates specificity of coIPs.(C, D). For quantification, α11.2 and GluA1IB signals were normalized to β2AR signals (***p<0.001, ANOVA).


( E ) IP of β2AR in absence ( Control ) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated ( phPep2 ) , which did not displaced α11 . 2 . ( F ) α11 . 2 GluA1 IB signals were normalized to β2AR signals ( * * * p < 0 . 001 , ANOVA ) .

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(E) IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displaced α11.2.(F) α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).


Forebrain slices from WT ( A - F ) S1928A KI mice ( G - L ) were treated with vehicle ( water ) or 10 M isoproterenol ( ISO ) for 0 . 5 - 10 min before solubilization , ultracentrifugation , IP of α11 . 2 ( A - C , G - I ) or β2AR ( D - F , J - L ) , sequential IB for pS1928 , pS1700 , α11 . 2 , for GluA1 , or for β2AR , of corresponding regions of blots , as indicated . α11 . 2 IPs in A - C G - I were from same samples ( which were split in half for parallel IP ) as β2AR IPs in D - F J - L , respectively ( for of coIP of β2AR with α11 . 2 see Figure EV1F , G ) . ( A - F ) In WT , time - dependent increase in S1928 S1700 phosphorylation ( A - C ) paralleled decrease in coIP of β2AR with α11 . 2 ( A bottom , Figure EV1F ) of α11 . 2 with β2AR ( D - F ) . ( G - L ) In S1928A KI mice , ISO induced S1700 phosphorylation ( G , I ) but did not disrupt α11 . 2 - β2AR interaction ( G bottom , J - L , Figure EV1G ) . ( B , C , H , I ) For of α11 . 2 phosphorylation , pS1928 pS1700 signals were normalized to α11 . 2 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) .

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

Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).


Forebrain slices from WT ( A - F ) S1928A KI mice ( G - L ) were treated with vehicle ( water ) or 10 M isoproterenol ( ISO ) for 0 . 5 - 10 min before solubilization , ultracentrifugation , IP of α11 . 2 ( A - C , G - I ) or β2AR ( D - F , J - L ) , sequential IB for pS1928 , pS1700 , α11 . 2 , for GluA1 , or for β2AR , of corresponding regions of blots , as indicated . α11 . 2 IPs in A - C G - I were from same samples ( which were split in half for parallel IP ) as β2AR IPs in D - F J - L , respectively ( for of coIP of β2AR with α11 . 2 see Figure EV1F , G ) . ( A - F ) In WT , time - dependent increase in S1928 S1700 phosphorylation ( A - C ) paralleled decrease in coIP of β2AR with α11 . 2 ( A bottom , Figure EV1F ) of α11 . 2 with β2AR ( D - F ) . ( G - L ) In S1928A KI mice , ISO induced S1700 phosphorylation ( G , I ) but did not disrupt α11 . 2 - β2AR interaction ( G bottom , J - L , Figure EV1G ) . ( B , C , H , I ) For of α11 . 2 phosphorylation , pS1928 pS1700 signals were normalized to α11 . 2 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) . ( E , F , K , L ) For of coIP , α11 . 2 GluA1 signals were normalized to β2AR ( * * * p < 0 . 001 , One Way ANOVA ) .

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

Forebrain slices from WT (A-F) and S1928A KI mice (G-L) were treated with vehicle (water) or 10 M isoproterenol (ISO) for 0.5 - 10 min before solubilization, ultracentrifugation, IP of α11.2 (A-C, G-I) or β2AR (D-F, J-L), and sequential IB for pS1928, pS1700, and α11.2, for GluA1, or for β2AR, of corresponding regions of the blots, as indicated. All the α11.2 IPs in A-C and G-I were from the same samples (which were split in half for parallel IP) as the β2AR IPs in D-F and J-L, respectively (for quantification of coIP of β2AR with α11.2 see Figure EV1F,G).(A-F) In WT, the time-dependent increase in S1928 and S1700 phosphorylation (A-C) paralleled the decrease in coIP of β2AR with α11.2 (A bottom, Figure EV1F) and of α11.2 with β2AR (D-F).(G-L) In S1928A KI mice, ISO induced S1700 phosphorylation (G,I) but did not disrupt the α11.2 - β2AR interaction (G bottom, J-L, Figure EV1G).(B,C,H,I) For quantification of α11.2 phosphorylation, pS1928 and pS1700 signals were normalized to α11.2 (**p<0.01, ***p<0.001, ANOVA).(E,F,K,L) For quantification of coIP, α11.2 and GluA1 signals were normalized to β2AR (***p<0.001, One Way ANOVA).