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

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

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


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

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

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


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

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

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

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


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

O O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

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


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

O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O

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


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

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

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


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

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O

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


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

O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-CELL_TYPE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

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


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

O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O B-CELL_TYPE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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


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

O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

B-TISSUE O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD 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 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 surface labeled for HA FLAG . ( A , B ) Representative immunofluorescence microscopy obtained by wide - field microscopy at lower higher resolutions ( scale bar in A is 5 m ) . ( C ) of between centers of HA FLAG ( * * p < 0 . 001 , Mann - Whitney Rank Sum ) .

B-TISSUE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD 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 ) a second application of ISO for 5 min ( lane 6 ) , before solubilization ultracentrifugation . ( A ) β2AR was IPed before IB for α11 . 2 ( top part of IB ) , GluA1 ( middle part of IB ) , β2AR ( bottom part of IB ) , as indicated . ( B , C ) For , α11 . 2 GluA1 IB signals were normalized to β2AR ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of β2AR from α11 . 2 ( A , lanes 2 - 6 ) lasted at least 3 min but not 20 min ( compare lanes 6 7 ) .

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

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 ) a second application of ISO for 5 min ( lane 6 ) , before solubilization ultracentrifugation . ( D ) α11 . 2 GluA1 were concurrently IPed from same samples as in ( A ) by simultaneous addition of anti - α11 . 2 - GluA1 antibodies before probing stripping / re - probing upper part of IB for pS1928 , pS1700 , total α11 . 2 ( top three panels ) middle part for pS845 , pS831 , total GluA1 ( bottom three panels ) . ( E - H ) For , pS1928 pS1700 IB signals were normalized to total α11 . 2 pS845 pS831 signals to total GluA1 ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of β2AR from α11 . 2 ( see A ) rendered α11 . 2 ( but not GluA1 ) refractory to re - phosphorylation of S1928 S1700 upon a second ISO application of α11 . 2 ( D , compare lanes 5 6 ) .

B-TISSUE O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD 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 ) a second application of ISO for 5 min ( lane 6 ) , before solubilization ultracentrifugation . ( A ) α11 . 2 GluA1 were concurrently IPed from same samples by simultaneous addition of anti - α11 . 2 - GluA1 antibodies before probing stripping / re - probing upper part of IB for pS1928 , pS1700 , total α11 . 2 middle part for pS845 , pS831 , 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 , pS1928 ( virtually absent ) pS1700 IB signals were normalized to total α11 . 2 pS845 pS831 signals to total GluA1 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) .

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

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 , 1 µM MVIIC in patch pipette upon depolarization from - 80 to - 30 , - 20 , - 10 , 0 mV . right panel depicts single - channel current - voltage relationship . Mean amplitude of unitary currents for different potential studied are - 2 . 07 pA ( - 30 mV ) , - 1 . 75 ( - 20 mV ) , - 1 . 53 ( - 10 mV ) - 1 . 25 ( 0 mV ; n = 5 patches per potential ) . Solid 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 B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 summary plot upon depolarization from - 80 to 0 mV under control conditions in presence of ISO in patch pipette . Cultures were pre - incubated for 15 min with 10 M 11R - PKI if indicated . patch pipette contained either vehicle for control , nifedipine ( nif ; 1M ) , ISO ( 1M ) , or ISO plus nifedipine , which blocked currents . 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 ) washed for 3 ( E , F ) or 10 min ( G ) before forming - attached patch with ISO present in patch pipette . 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 B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O 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 permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , IP of β2AR ( A - C ) or simultaneously α11 . 2 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 ) ; inverse was true for Myr - DSPL ( lane 1 vs . 3 ) . ( B , C ) For , α11 . 2 GluA1 immunosignals were normalized to β2AR signals ( * * p < 0 . 01 , * * * p < 0 . 001 , One Way ANOVA ) .

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

(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 permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , IP of β2AR ( A - C ) or simultaneously α11 . 2 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 ) ; inverse was true for Myr - DSPL ( lane 2 vs . 4 ) . ( E , F ) For , pS1700 pS845 immunosignals were normalized to α11 . 2 GluA1 signals , respectively ( * * * p < 0 . 001 , One Way ANOVA ) .

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

(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 - 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 ) . patch pipette contained either vehicle ( H2O ; control ) or 1 µM ISO . ISO induced increase in NPo was prevented by Myr - Pep2 but not Myr - Pep2scr . Arrows indicate 0 - current level ( i . e . closed channel ) . ( H ) Summary plot for ( G ) . For statistical , NPo value was determined for each recording pooled under each condition for comparison ( n = 10 - 12 patches ; * p < 0 . 05 , One Way ANOVA with Tukey post - hoc ) .

O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 after either a 5Hz / 3 min ( A - C ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani bars perfusion with 1 µM ISO . Inserts show sample traces immediately before ( left ) ~ 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 B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM 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 after either a 5Hz / 3 min ( A - C , G - I ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani bars perfusion with 1 µM ISO 10 µM of Myr - Pep2 or Myr - Pep2scr . Inserts show sample traces immediately before ( left ) ~ 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 B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of cGAS in nucleus ( DAPI ) cytosol ( phalloidin ) in BMDMos cultured at low / high density Scale bar : 50 μm . Right : Corresponding of cGAS from 6 different fields with n > 50 .

O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

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 of cGAS in nucleus ( DAPI ) cytosol ( phalloidin ) in BMDMos cultured with / without serum Scale bar : 50 μm . Right : Corresponding of cGAS from 6 different fields with n > 50 .

O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O B-GENEPROD 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.


Immunofluorescence of cGAS in nucleus ( DAPI ) cytosol ( phalloidin ) in BMDMos cultured with / without Aphidicolin ( Aphi ) ( C ) . Scale bar : 50 μm . Right : Corresponding of cGAS from 6 different fields with n > 50 .

B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

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


Immunoblot estimation of cGAS in / subcellular fraction of BMDMos cultured under indicated conditions . Lamin B α - Tubulin are markers respectively .

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

Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively.


Flow cytometric analysis of cell cycle of BMDMos


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . Scale bar : 10 μm ( A ) .

B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). Scale bar: 10 μm (A).


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . average MNs / . Graphs show Mean ± ( n = 3 independent experiments ) representing six different microscopic fields with over 200 .

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

Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). The average MNs/cell. Graphs show Mean ± SEM (n=3 independent experiments) representing six different microscopic fields with over 200 cells.


IFNB1 response in HEK293 stimulated with transfected plasmid . Mean ± of n = 3 independent experiments .

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IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n=3 independent experiments.


Micronuclei ( indicated by arrow head ) cGAS in WT cGAS - / - BMDMos exposed to γ - irradiation ( 10 Gy ) . Scale bar : 10 μm .

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Micronuclei (indicated by arrow head) and cGAS staining in WT and cGAS-/- BMDMos exposed to γ-irradiation (10 Gy). Scale bar: 10 μm.


Average MNs / in BMDMos . MN graphs show Mean ± ( n = 3 independent experiments ) representing eight different microscopic fields with over 200 .

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Average MNs/cell in BMDMos. MN graphs show Mean ± SEM (n=3 independent experiments) representing eight different microscopic fields with over 200 cells.


in WT cGAS - / - BMDMos that were first synchronized at G2 / M , then γ - irradiated ( 10 Gy ) followed by release at indicated time points . Mean ± SD , x biological triplicates ( n = 3 ) per treatment group are shown .

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Cell death in WT and cGAS-/- BMDMos that were first synchronized at G2/M, then γ-irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ±SD, x biological triplicates (n=3) per treatment group are shown.


BMDMos from cGAS - / - mice exhibit enhanced repair efficiency than those from WT Sting - / - mice . ( A ) Representative comet tails of WT , cGAS - / - Sting - / - BMDMos exposed to γ - irradiation ( IR : 10 Gy ) on ice then incubated at 37 ̊ C for indicated duration . ( B ) Corresponding of comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments .

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BMDMos from cGAS-/- mice exhibit enhanced DNA repair efficiency than those from WT and Sting-/- mice. (A) Representative comet tails of WT, cGAS-/- and Sting-/-BMDMos exposed to γ-irradiation (IR: 10 Gy) on ice then incubated at 37 ̊C for indicated duration. (B) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments.


cGAS promotes micronuclei generation in BMDMos independently of STING . Confocal microscopic visualization of micronuclei ( indicated by arrow head ) cGAS in WT , cGAS - / - Sting - / - BMDMos exposed to γ - irradiation ( 10Gy ) . Scale bar : 10 μm .

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cGAS promotes micronuclei generation in BMDMos independently of STING. Confocal microscopic visualization of micronuclei (indicated by arrow head) and cGAS staining in WT, cGAS-/- and Sting-/- BMDMos exposed to γ-irradiation (10Gy). Scale bar: 10 μm.


cGAS promotes micronuclei generation in BMDMos independently of STING . Average MNs / in corresponding representative . Bar graphs show mean values from eight different microscopic fields with over 200 .

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cGAS promotes micronuclei generation in BMDMos independently of STING. Average MNs/cell in corresponding representative images. Bar graphs show mean values from eight different microscopic fields with over 200 cells.


γ - irradiation - induced in WT , cGAS - / -

γ-irradiation-induced cell death in WT, cGAS-/- BMDMos


γ - irradiation - induced in Sting - / - cGAS - / - Sting - / - BMDMos ( F ) .

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γ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).


Kinetics of in vivo depletion of indicated bone marrow in WT mice ( n = 3 ) after γ - irradiation ( 9 Gy ) .

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Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy).


Bone marrow in WT ( n = 3 ) cGAS - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in WT (n=3) and cGAS-/- (n=3) mice 10 hours post γ-irradiation.


Bone marrow in Sting - / - ( n = 3 ) cGAS - / - Sting - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.


Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS . Immunoblot inserts in depict knockdown efficiency of cGAS Histone H1 . 2 .

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Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1.2.


Results showing effect of hcGAS , hcGAS △ cGAMP , hcGAS △ or hcGAS △ Oligo on HR in HEK293 . Corresponding immunoblot inserts depict cGAS expression .

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Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


Results showing effect of hcGAS , hcGAS △ cGAMP , hcGAS △ or hcGAS △ Oligo on NHEJ in HEK293 . Corresponding immunoblot inserts depict cGAS expression .

Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


cGAS is not recruited to DSB sites : Confocal microscopic of GFP - NLS - or GFP - hcGAS - expressing U2OS - DSB reporter incubated ( or not ) with Shield - 1 4 - OHT to induce expression translocation of mCherry - LacI - FokI ( red ) to specific DSB sites . Scale bar : 10μm . arrowheads indicate DSB sites .

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cGAS is not recruited to DSB sites: Confocal microscopic images of GFP-NLS- or GFP-hcGAS-expressing U2OS-DSB reporter cells incubated (or not) with Shield-1 and 4-OHT to induce the expression and translocation of mCherry-LacI-FokI (red) to specific DSB sites. Scale bar: 10μm. The arrowheads indicate DSB sites.


cGAS does not colocalize with γ - H2AX at DSB sites : GFP - NLS - or GFP - hcGAS - expressing HEK293 exposed ( or not ) to γ - irradiation ( IR : 10 Gy ) then stained for γ - H2AX . Scale bar : 10 μm .

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cGAS does not colocalize with γ-H2AX at DSB sites: GFP-NLS- or GFP-hcGAS- expressing HEK293 cells exposed (or not) to γ-irradiation (IR: 10 Gy) then stained for γ-H2AX. Scale bar: 10 μm.


cGAS is mainly - bound remains unaltered upon γ - irradiation . ( cyto ) fractions of γ - irradiated ( 10 Gy , 30 min ) BMDMos analyzed for cGAS indicated molecules .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic (cyto) and nuclear fractions of γ-irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules.


cGAS is mainly - bound remains unaltered upon γ - irradiation . , soluble fractions from BMDMos were immunoblotted for cGAS indicated .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic, soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins.


cGAS co - isolates with repair because of bound bridges . Nuclease digestion abrogates co - isolation of cGAS repair : Lysates of control ( - IR ) γ - irradiated ( + IR , 10 Gy , 30 min ) GFP - hcGAS expressing HEK293 were treated ( or not ) with Benzonase before cGAS immunoprecipitation for indicated . Agarose gel of in corresponding lysates in ( E ) .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Nuclease digestion abrogates the co-isolation of cGAS and DNA repair proteins: Lysates of control (-IR) and γ-irradiated (+IR, 10 Gy, 30 min) GFP-hcGAS expressing HEK293 cells were treated (or not) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins. Agarose gel analysis of DNA in corresponding cell lysates in (E).


cGAS co - isolates with repair because of bound bridges . Co - isolation of cGAS repair depends on its binding to : cGAS pulldowns along with lysate inputs of control γ - irradiated HEK293 expressing GFP - hcGAS or GFP - hcGASΔDNA probed for indicated .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Co-isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ-irradiated HEK293 cells expressing GFP-hcGAS or GFP-hcGASΔDNA probed for indicated proteins.


Confocal of γ - irradiated GFP - NLS - , GFP - hcGAS - expressing HEK293 stained for RAD51 ( red ) with or without γ - irradiation . Scale bar : 10 μm .

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Confocal images of γ-irradiated GFP-NLS-, GFP-hcGAS-expressing HEK293 cells stained for RAD51 (red) with or without γ-irradiation. Scale bar: 10 μm.


Pre - incubation of dsDNA with mcGAS - cat prevents D - loop formation by human RAD51 , but does not affect RAD1 activity once RAD51 filaments are bound to dsDNA . of D - loop formed in each reaction ( left ) was graphed as average of triplicates ± SD .

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Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD.


Pre - incubation of template dsDNA with hcGAScat blocks subsequent D - loop formation . of D - loop formation ( below ) was graphed as average of triplicates ± SD .

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Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.


Negative - stain electron of cGAS - dsDNA complexes following incubation of dsDNA with indicated cGAS variants . Scale bar : 100 nm .

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Negative-stain electron micrographs of cGAS-dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation when pre - incubated with dsDNA . of D - loop formed in each reaction ( left ) graphed as average of triplicates ± SD .

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Effect of indicated hcGAS variants on D-loop formation when pre-incubated with dsDNA. Percentage of D-loop formed in each reaction (left) graphed as the average of triplicates± SD.


DR - GFP showing that hcGASΔDNA - Y215E is impaired in HR inhibition .

DR-GFP assay showing that hcGASΔDNA-Y215E is impaired in HR inhibition.


hcGASΔDNA - Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24 .

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hcGASΔDNA-Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24.


hcGASΔDNA - Y215E hcGASΔcGAMP are defective in synthase activity .

hcGASΔDNA-Y215E and hcGASΔcGAMP are defective in synthase activity.


Negative - stain electron showing that hcGAScat - ΔDNA - Y215E is defective in inducing cGAS - dsDNA complexes . Scale bar : 100 nm .

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Negative-stain electron micrographs showing that hcGAScat-ΔDNA-Y215E is defective in inducing cGAS-dsDNA complexes. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation .

Effect of indicated hcGAS variants on D-loop formation.


( E ) Representative photos of iWAT , eWAT BAT from Ftoflox / flox FtoAKO mice fed a HFD . Scale bar , 1 cm . ( F ) Representative H & E of iWAT , eWAT BAT from Ftoflox / flox FtoAKO mice fed a HFD . Scale bar , 200 μm . (

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(E) Representative photos of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 1 cm. (F) Representative H&E staining of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 200 μm. (


( I - K ) Serum of glucose ( I ) , triglyceride ( J ) free fatty acid ( FFA ) ( K ) in Ftoflox / flox FtoAKO mice fed a HFD followed by fasting . Data information : data were presented as mean ± SD ( n = 6 ) . Statistical analyses were performed using Student t ‐ . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(I-K) Serum levels of glucose (I), triglyceride (J) and free fatty acid (FFA) (K) in Ftoflox/flox and FtoAKO mice fed a HFD followed by fasting. Data information: The data were presented as the mean ± SD (n = 6). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( A ) O2 consumption of 16 - week - old Ftoflox / flox FtoAKO mice fed a HFD . gray areas in graphs indicate day night , respectively . ( B ) CO2 generation of 16 - week - old Ftoflox / flox FtoAKO mice fed a HFD . gray areas in graphs indicate day night , respectively . ( Data information : data were presented as mean ± SD of n = 6 mice per group or triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(A) O2 consumption of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. (B) CO2 generation of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. ( Data information: The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) qPCR of genes involved in thermogenesis , mitochondrial biogenesis adipogenesis in iWAT of Ftoflox / flox FtoAKO mice .

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(F) qPCR analysis of genes involved in thermogenesis, mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox/flox and FtoAKO mice.


( G ) Western blot of of UCP1 , PPARGC1A PPARG in iWAT of Ftoflox / flox FtoAKO mice .

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(G) Western blot analysis of the protein levels of UCP1, PPARGC1A and PPARG in iWAT of Ftoflox/flox and FtoAKO mice.


( I ) Immunofluorescence detection of UCP1 in iWAT from Ftoflox / flox FtoAKO mice following 6 days cold exposure ( 4 ° C ) . Scale bar , 20 μm .

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(I) Immunofluorescence detection of UCP1 in iWAT from Ftoflox/flox and FtoAKO mice following 6 days cold exposure (4°C). Scale bar, 20 μm.


( M ) Western blot of UCP1 , PPARGC1A PPARG in differentiated beige adipocytes described in ( J ) .

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(M) Western blot analysis of UCP1, PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in (J).


( N ) Immunofluorescent of UCP1 in differentiated beige adipocytes described in ( J ) . Scale bar , 20 μm .

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(N) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (J). Scale bar, 20 μm.


( G ) Western blot of of FTO , HIF1A , UCP1 , PPARGC1A PPARG in differentiated beige adipocytes described in ( F ) .

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(G) Western blot analysis of the protein levels of FTO, HIF1A, UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (F).


( C ) O2 consumption rate ( OCR ) in differentiated beige adipocytes from 3T3 - L1 overexpressing Vec or HIF1A plasmid . average basal maximal respiration rates are shown in right panel . Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(C) The O2 consumption rate (OCR) in differentiated beige adipocytes from 3T3-L1 cells overexpressing Vec or HIF1A plasmid. The average basal and maximal respiration rates are shown in right panel. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( G ) qPCR of genes involved in thermogenesis in differentiated beige adipocytes described in ( C ) . ( H ) qPCR of genes involved in thermogenesis in differentiated beige adipocytes described in ( D ) . ( Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C). (H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D). ( Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( I ) Immunofluorescent of UCP1 in differentiated beige adipocytes described in ( D ) . Scale bar , 20 μm .

(I) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (D). Scale bar, 20 μm.


( C ) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl siFto 3T3 - L1 . Blue boxes represent exons , blue lines represent introns . m6A peaks were in dashed red box .

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(C) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A peaks were in the dashed red box.


( D ) Methylated immunoprecipitation ( MeRIP ) - qPCR of m6A of Hif1a mRNA in siCtrl siFto 3T3 - L1 . Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(D) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


( H ) Western blot of of HIF1A , UCP1 , PPARGC1A , PPARG GFP in 3T3 - L1 infected with lentiviruses pCDHpuro expressing Vec , WT - or MUT - HIF1A .

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(H) Western blot analysis of the protein levels of HIF1A, UCP1, PPARGC1A, PPARG and GFP in 3T3-L1 cells infected with lentiviruses pCDHpuro expressing Vec, WT- or MUT-HIF1A.


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(A) Western blot analysis of the protein levels of YTHDF1, YTHDF2 and HIF1A in siCtrl, siYthdf1 and siYthdf2 3T3-L1 cells.


( G ) Polysome profiling of 3T3 - L1 overexpressing Vec or YTHDC2 plasmid . ( H ) qPCR of mRNA of Hif1a in 40 - 80S or polysome fraction in 3T3 - L1 overexpressing Vec or YTHDC2 plasmid . ( I ) Polysome profiling of siCtrl siYthdc2 3T3 - L1 . ( J ) qPCR of mRNA of Hif1a in 40 - 80S or polysome fraction in siCtrl siYthdc2 3T3 - L1 . Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(G) Polysome profiling of 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (H) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (I) Polysome profiling of siCtrl and siYthdc2 3T3-L1 cells. (J) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in siCtrl and siYthdc2 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) in response to different of KCl stress . Points reflect measured values , lines correspond to best model fit .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) in response to different levels of KCl stress. Points reflect the measured values, and lines correspond to best model fit.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) at 0 . 4M KCl .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) at 0.4M KCl.


Representation of experiments shown in ( B , C ) in parameter space . Connected shows progression in response to increasing KCl inferred tonparameter ( Y axis ) vs . KCl ( X axis ) of KCl gradient experiment inferred total open time - toff − ton ( Y axis ) vs . inferred production rate ( X axis ) of KCl gradient

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Representation of the experiments shown in (B,C) in parameter space. Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter (Y axis) vs. KCl concentration (X axis) of the KCl gradient experiment The inferred total open time - toff− ton(Y axis) vs. the inferred production rate (X axis) of the KCl gradient experiment


Summary of Hsp12 - GFP dynamics single mutant screen . 68 mutants were divided into three groups : increased / decreased / did not change total GFP produced . Each group was independently hierarchically clustered . values shown are Z - Scores of each parameter : ton - response onset time ; production rate ; total open time - of transcriptional window ( toff − ton ) ; Basal GFP - GFP measured before exposure to stress ; Delta GFP - amount of GFP produced throughout experiment ( Max GFP - Basal GFP ) .

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Summary of Hsp12-GFP dynamics single mutant screen. 68 mutants were divided into three groups: increased/decreased/did not change the total GFP produced. Each group was independently hierarchically clustered. The values shown are the Z-Scores of each parameter: ton- response onset time; production rate; total open time - length of transcriptional window (toff − ton); Basal GFP - GFP levels measured before exposure to stress; Delta GFP - amount of GFP produced throughout the experiment (Max GFP - Basal GFP).


mRNA of HSP12 ( Y axis ) as a function of time ( X axis ) in six representative strains .

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mRNA levels of HSP12 (Y axis) as a function of time (X axis) in six representative strains.


Comparison of to . X axis is sum over HSP12 mRNA ( see D ) . Y axis is maximum over Hsp12 - GFP values . red indicates best linear fit .

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Comparison of RNA levels to protein levels. X axis is the sum over the HSP12 mRNA counts (see D). Y axis is the maximum over the Hsp12-GFP values. The red line indicates the best linear fit.


Comparing total open time parameter inferred from mRNA data ( X axis ) to one inferred from data ( Y axis ) .

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Comparing the total open time parameter inferred from mRNA data (X axis) to the one inferred from protein data (Y axis).


Time - lapse microscopy of Hsp12 - GFP in response to stress ( 0 . 4M KCl ) . Examples of raw ( top ) traces of ( bottom ) . Traces are colored according to their basal GFP level .

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Time-lapse microscopy of Hsp12-GFP in response to stress (0.4M KCl). Examples of raw images (top) and traces of individual cells (bottom). Traces are colored according to their basal GFP level.


Scatter plot showing production rate estimated from single traces vs . production rate estimated from population average . Each point represents a different yeast .

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Scatter plot showing the production rate estimated from single traces vs. the production rate estimated from the population average. Each point represents a different yeast strain.


Estimated parameters ( median over repeats ) of ~ 1600 double mutant strains . color represents total Hsp12 - GFP produced . same amount of GFP can be produced with multiple parameter combinations . ellipses illustrate iso - expression areas in graph . Three examples of strains ( marked in B ) with nearly same total expression but dramatically different dynamics .

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Estimated parameters (median over repeats) of all ~1600 double mutant strains. The color represents the total Hsp12-GFP produced. The same amount of GFP can be produced with multiple parameter combinations. The ellipses illustrate iso-expression areas in the graph. Three examples of individual strains (marked in B) with nearly the same total expression but dramatically different dynamics.


Illustration of genetic interaction trends of mutants . For each single mutant we plot median of double mutants that contain it ( purple dots ) . gray dots are double mutant strains ( B ) .

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Illustration of the genetic interaction trends of individual mutants. For each single mutant we plot the median of all the double mutants that contain it (purple dots). The gray dots are all individual double mutant strains (B).