( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

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

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 , 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 .

O O O O O O O O O O O O O O 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, 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 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 .

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

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 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

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 I-CONTROLLED_VAR I-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

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 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 .

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

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 morphology of cells expressing Atg18Sloop - GFP , imaged before and 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 cell 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 morphology of cells expressing Atg18DLoop - GFP , imaged as in d .

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


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

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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 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 ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 b, Atg18FGGG;


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 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 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 .

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

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

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


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

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 cells , assayed 15 min after a moderate hypertonic shock as in Fig . 1 .

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


d . Number of vacuoles per cell 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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

O O O O 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 O

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 the 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 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 .

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

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 the presence of increasing concentrations of liposomes containing 5 % PI ( 3 , 5 ) P2 .

O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

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 + / + 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 ) .

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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 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 .

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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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

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

O O 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

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


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 ) .

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

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 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 .

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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 ) 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 .

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 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

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 + / + 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 ) .

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

(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 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 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O 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

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 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 ) .

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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 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 ) .

O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 )

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

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 + / + 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 ) .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 + / + 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 .

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 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 .

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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 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 .

O O O O 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 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

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 + / + 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 )

O O 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

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 ) 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

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

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 images of new firing origins , and elongating forks showing stable replication and 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 + / + 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 .

O O O O 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

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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 + / + 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 ) .

O O O O O O O O O O O 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 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

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 + / + 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 and after ATO incubation at 1 : 5 molar ratio 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 the transfected DNMTs ( H ) and DNMT3A - C520A / C524A mutation ( I ) in 293T cells 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 protein levels in MEFs ( upper panel ) and CCRF - CEM cells ( lower panel ) treated with ATO in the 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 ) 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 .

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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 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 .

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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 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 .

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

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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 ) and CCRF - CEM cells ( lower panel ) treated with ATO or AsPh3 in the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 - 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 .

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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 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 .

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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 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 ) .

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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 ) , 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 ) 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 ) .

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

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 ) 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 ) .

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

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).


Hippocampal cultures were transfected with FLAG - β2AR and α11 . 2 - HA at 6 days in vitro ( DIV ) , treated with vehicle ( water ) or 1 M isoproterenol ( ISO ) for 5 min at 18 DIV , fixed and surface labeled for HA and FLAG . ( A , B ) Representative immunofluorescence microscopy images obtained by wide - field microscopy at lower and higher resolutions ( scale bar in A is 5 m ) . ( C ) Quantification of distance between centers of HA and FLAG puncta ( * * p < 0 . 001 , Mann - Whitney Rank Sum Test ) .

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

Hippocampal cultures were transfected with FLAG-β2AR and α11.2-HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 M isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.(A,B) Representative immunofluorescence microscopy images obtained by wide-field microscopy at lower and higher resolutions (scale bar in A is 5 m).(C) Quantification of distance between centers of HA and FLAG puncta (**p<0.001, Mann-Whitney Rank Sum Test).


Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( A ) β2AR was IPed before IB for α11 . 2 ( top part of IB ) , GluA1 ( middle part of IB ) , and β2AR ( bottom part of IB ) , as indicated . ( B , C ) For quantification , α11 . 2 and GluA1 IB signals were normalized to β2AR ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of the β2AR from α11 . 2 ( A , lanes 2 - 6 ) lasted at least 3 min but not 20 min ( compare lanes 6 and 7 ) .

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 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 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

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).


Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( D ) α11 . 2 and GluA1 were concurrently IPed from same samples as in ( A ) by simultaneous addition of anti - α11 . 2 and - GluA1 antibodies before probing and stripping / re - probing upper part of IB for pS1928 , pS1700 , and total α11 . 2 ( top three panels ) and middle part for pS845 , pS831 , and total GluA1 ( bottom three panels ) . ( E - H ) For quantification , pS1928 and pS1700 IB signals were normalized to total α11 . 2 and pS845 and pS831 signals to total GluA1 ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of the β2AR from α11 . 2 ( see A ) rendered α11 . 2 ( but not GluA1 ) refractory to re - phosphorylation of S1928 and S1700 upon a second ISO application of α11 . 2 ( D , compare lanes 5 and 6 ) .

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

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).


Forebrain slices from S1928A KI mice were treated with vehicle ( lanes 1 , 5 ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) and a second application of ISO for 5 min ( lane 6 ) , before solubilization and ultracentrifugation . ( A ) α11 . 2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti - α11 . 2 and - GluA1 antibodies before probing and stripping / re - probing upper part of IB for pS1928 , pS1700 , and total α11 . 2 and middle part for pS845 , pS831 , and total GluA1 . In S1928A KI mice , S1700 re - phosphorylation after a 3 min wash out of ISO was not blunted ( lane 6 ) in contrast to WT mice but rather augmented ( compare to lane 5 ) . ( B - E ) For quantification , pS1928 ( virtually absent ) and pS1700 IB signals were normalized to total α11 . 2 and pS845 and pS831 signals to total GluA1 ( * * 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-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 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

Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1,5) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re-phosphorylation after a 3 min wash out of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).(B-E) For quantification, pS1928 (virtually absent) and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ***p<0.001, ANOVA).


( A ) Representative single channel recordings from hippocampal neurons at 7 - 14 DIV with 500 nM BayK - 8644 , 1 µM ω - conotoxin GVIA , and 1 µM MVIIC in patch pipette upon depolarization from - 80 to - 30 , - 20 , - 10 , and 0 mV . The right panel depicts the single - channel current - voltage relationship . Mean amplitude of unitary currents for different membrane potential studied are - 2 . 07 pA ( - 30 mV ) , - 1 . 75 ( - 20 mV ) , - 1 . 53 ( - 10 mV ) and - 1 . 25 ( 0 mV ; n = 5 patches per test potential ) . Solid line represents best - fit of data using a linear equation ( R2 = 0 . 92 ) revealing a slope conductance for these channels of 27 ± 2 pS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative single channel recordings from hippocampal neurons at 7-14 DIV with 500 nM BayK-8644, 1 µM ω-conotoxin GVIA, and 1 µM MVIIC in patch pipette upon depolarization from -80 to -30, -20, -10, and 0 mV. The right panel depicts the single-channel current-voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are -2.07 pA (-30 mV), -1.75 (-20 mV), -1.53 (-10 mV) and -1.25 (0 mV; n=5 patches per test potential). Solid line represents best-fit of data using a linear equation (R2=0.92) revealing a slope conductance for these channels of 27 ± 2 pS.


( B - D ) Representative single channel traces and summary plot upon depolarization from - 80 to 0 mV under control conditions and in the presence of ISO in the patch pipette . Cultures were pre - incubated for 15 min with 10 M 11R - PKI if indicated . The patch pipette contained either vehicle for control , nifedipine ( nif ; 1M ) , ISO ( 1M ) , or ISO plus nifedipine , which blocked all currents . The ISO - induced increase in NPo was prevented by 11R - PKI .

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

(B-D) Representative single channel traces and summary plot upon depolarization from -80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre-incubated for 15 min with 10 M 11R-PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1M), ISO (1M), or ISO plus nifedipine, which blocked all currents. The ISO-induced increase in NPo was prevented by 11R-PKI.


( E - H ) Representative single channel currents upon depolarization from - 80 to 0 mV and summary plot after pretreatment of whole cultures with ISO . Cultures were pre - incubated with vehicle ( H2O , mock wash , E ) or 1 M ISO for 5 min ( F , G ) and washed for 3 ( E , F ) or 10 min ( G ) before forming the cell - attached patch with ISO present in the patch pipette . The upregulation of NPo to ~ 0 . 4 ( cf . C , D ) occurred only if neurons were pretreated with vehicle instead of ISO ( E , H ; mock wash ) or if ISO wash out duration was 10 min ( G , H ) but not if wash out was only 3 min ( F , H ) .

O O 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 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 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-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 O O O O O O O O O O O O O O O

(E-H) Representative single channel currents upon depolarization from -80 to 0 mV and summary plot after pretreatment of whole cultures with ISO. Cultures were pre-incubated with vehicle (H2O, mock wash, E) or 1 M ISO for 5 min (F,G) and washed for 3 (E,F) or 10 min (G) before forming the cell-attached patch with ISO present in the patch pipette. The upregulation of NPo to ~0.4 (cf. C,D) occurred only if neurons were pretreated with vehicle instead of ISO (E,H; mock wash) or if ISO wash out duration was 10 min (G,H) but not if wash out was only 3 min (F,H).


( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it membrane permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , and IP of β2AR ( A - C ) or simultaneously α11 . 2 and GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( A ) Myr - Pep 2 displaced α11 . 2 ( lane 5 vs . 3 , top of blot ) but not GluA1 ( middle , same blot ) from β2AR ( bottom , same blot ) ; the inverse was true for Myr - DSPL ( lane 1 vs . 3 ) . ( B , C ) For quantification , α11 . 2 and GluA1 immunosignals were normalized to β2AR signals ( * * p < 0 . 01 , * * * 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 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 O O 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-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA).


( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it membrane permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , and IP of β2AR ( A - C ) or simultaneously α11 . 2 and GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( D ) Myr - Pep2 blunted ISO - induced phosphorylation of α11 . 2 S1700 ( lane 6 vs . 4 , top of blot ) but not GluA1 S845 ( middle , same blot ) ; the inverse was true for Myr - DSPL ( lane 2 vs . 4 ) . ( E , F ) For quantification , pS1700 and pS845 immunosignals were normalized to α11 . 2 and GluA1 signals , respectively ( * * * 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 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 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

(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA).


( G ) Representative cell - attached recordings from hippocampal neurons as in Figure 7 . In interleafed experiments cultures were pre - incubated for 30 min with 10 M Myr - Pep2 or scrambled Myr - Pep2 ( Myr - Pep2scr ) . The patch pipette contained either vehicle ( H2O ; control ) or 1 µM ISO . The ISO induced increase in NPo was prevented by Myr - Pep2 but not Myr - Pep2scr . Arrows indicate the 0 - current level ( i . e . closed channel ) . ( H ) Summary plot for ( G ) . For statistical analysis , the NPo value was determined for each recording and pooled under each condition for comparison ( n = 10 - 12 patches ; * p < 0 . 05 , One Way ANOVA with Tukey post - hoc test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).


Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz / 3 min ( A - C ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO . Inserts show sample traces immediately before ( left ) and ~ 30 min after ( right ) tetani . ( A - C ) Litter - matched WT but not conditional Cav1 . 2 KO mice showed PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) . ( D - F ) WT as well as Cav1 . 2 KO mice showed NMDAR - dependent 100 Hz LTP .

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

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(A-C) Litter-matched WT but not conditional Cav1.2 KO mice showed PTT-LTP (**p < 0.001, one-way ANOVA).(D-F) WT as well as Cav1.2 KO mice showed NMDAR-dependent 100 Hz LTP.


Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz / 3 min ( A - C , G - I ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr - Pep2 or Myr - Pep2scr . Inserts show sample traces immediately before ( left ) and ~ 30 min after ( right ) tetani . ( G - I ) Myr - Pep2 but not Myr - Pep2scr blocked PTT - LTP ( * * 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 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

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C, G-I) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr-Pep2 or Myr-Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(G-I) Myr-Pep2 but not Myr-Pep2scr blocked PTT-LTP (**p < 0.001, one-way ANOVA).


Left : Immunofluorescence images of cGAS in the nucleus ( DAPI ) and cytosol ( phalloidin ) in BMDMos cultured at low / high density Scale bar : 50 μm . Right : Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells .

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

Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured at low/high density Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Left : Immunofluorescence images of cGAS in the nucleus ( DAPI ) and cytosol ( phalloidin ) in BMDMos cultured with / without serum Scale bar : 50 μm . Right : Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells .

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

Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without serum Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.