16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei ( clear , partially filled , almost filled and filled ) . Spheroids stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of MCF10A spheroids categorized into 4 groups according to the filling of the lumen with cell nuclei (clear, partially filled, almost filled and filled). Spheroids stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red).


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Macrophage - conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control . Error bars represent mean ± SEM from 11 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces filling of the spheroid lumen with cell nuclei compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; 50 spheroids each). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Representative images of Ki67 positive nuclei of MCF10A cells . Spheroids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and Ki67 ( green ) .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Representative images of Ki67 positive nuclei of MCF10A cells. Spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and Ki67 (green).


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage - conditioned media . Error bars represent mean ± SEM from 3 independent experiments ( 20 - 30 non - empty spheroids per condition ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Quantitative analysis of Ki67 positive nuclei per spheroid indicates enhanced proliferation of MCF10A cells upon stimulation with M1 macrophage-conditioned media. Error bars represent mean ± SEM from 3 independent experiments (20-30 non-empty spheroids per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . NSC23766 ( Rac1 inhibitor ; 50 μM ) reduces the filling of the spheroid lumen with cell nuclei upon macrophage - conditioned medium stimulation compared to control . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Error bars represent mean ± SEM from 3 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled MCF10A spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. NSC23766 (Rac1 inhibitor; 50 μM) reduces the filling of the spheroid lumen with cell nuclei upon macrophage-conditioned medium stimulation compared to control. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled MCF10A spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Invasive protrusions of spheroids into matrigel / collagen mix marked with a white arrow .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Invasive protrusions of spheroids into matrigel/collagen mix marked with a white arrow.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . 3D structures of spheroids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and laminin V ( green ) . Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions . Invasive protrusions are marked with a white arrow .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. 3D structures of spheroids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and laminin V (green). Laminin V staining indicates the loss of basal membrane continuity at invasive protrusions. Invasive protrusions are marked with a white arrow.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . Macrophage - conditioned medium induces invasive protrusions in MCF10A spheroids compared to control . Error bars represent mean ± SEM from 11 independent experiments ( n = 2 per condition ; at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces invasive protrusions in MCF10A spheroids compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from ND mice were cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24 h . Representative images of organoids in matrigel / collagen mix .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h. Representative images of organoids in matrigel/collagen mix.


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from ND mice were cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24 h . Macrophage - conditioned medium induces invasive protrusions in organoids as compared to control . Error bars represent mean ± SEM from 3 independent experiments , with each mouse labelled with a different symbol shape ( n = 2 per condition ; 15 - 25 organoids each ) . Macrophage donors are indicated as D14 , D15 , D17 , D18 . M1A - M1 activated , M2A - M2 activated macrophages .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h. Macrophage-conditioned medium induces invasive protrusions in organoids as compared to control. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from ND mice were cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24 h . 3D structure of organoids stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and α - SMA ( green ) . Bi - layered structure of internal luminal cells and external basal myoepithelial cells is established for non - invasive organoids .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from ND mice were cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h. 3D structure of organoids stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). Bi-layered structure of internal luminal cells and external basal myoepithelial cells is established for non-invasive organoids.


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids isolated from ND or HFD mice were cultured in collagen for 2 days . Representative images of organoids cultured in collagen for 2 days . The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND . Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse ( labelled with a different symbol shape ) .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids isolated from ND or HFD mice were cultured in collagen for 2 days. Representative images of organoids cultured in collagen for 2 days. The number of invasive protrusions per organoid is higher for organoids isolated from mice on HFD compared to mice on ND. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on high fat diet ( HFD ) ( n = 3 ) . Organoids isolated from HFD mice were cultured in collagen for 2 days . 3D structure of organoid stained for DNA ( Hoechst 33342 in blue ) , F - actin ( Phalloidin in red ) and α - SMA ( green ) . α - SMA , myoepithelial cell marker , appeared to be loss at invasive protrusions .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on high fat diet (HFD) (n=3). Organoids isolated from HFD mice were cultured in collagen for 2 days. 3D structure of organoid stained for DNA (Hoechst 33342 in blue), F-actin (Phalloidin in red) and α-SMA (green). α-SMA, myoepithelial cell marker, appeared to be loss at invasive protrusions.


Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium . Representative images of ductal structures cultured with conditioned medium . Ducts were stained for DNA ( DAPI in blue ) and F - actin ( Phalloidin in red ) . Macrophage - conditioned medium induces filling of the duct lumen with cell nuclei compared to control . Error bars represent mean ± SEM ( A total of 24 structures was counted per each condition : 2 patient ductal structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 ducts each ) . Filling of the lumen was determined as % of luminal space filled with cells . Macrophage donors are indicated as D14 , D15 , D17 , D18 . M1A - M1 activated , M2A - M2 activated macrophages .

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

Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium. Representative images of ductal structures cultured with conditioned medium. Ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Macrophage-conditioned medium induces filling of the duct lumen with cell nuclei compared to control. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as D14, D15, D17, D18. M1A - M1 activated, M2A - M2 activated macrophages.


IKKε expression was assessed semi - quantitatively using a 0 - 3 grade scale ( " 0 " : no staining ; " 1 " : weak ; " 2 " : moderate ; " 3 " : strong ) on human breast cancer sections using immunohistochemistry . Representative images according to the IKKε staining grade are shown . Scale bar : 100 μm . Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade .

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

IKKε expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the IKKε staining grade are shown. Scale bar: 100 μm. Distribution table of IKKε expression in the cohort of 66 human breast carcinomas according to the staining grade.


Correlation between IKBKE mRNA levels ( log2 ratio ) and immune signature ( Yoshihara et al , 2013 ) in the METABRIC transcriptomic dataset from 1981 breast cancer patients ( Curtis et al , 2012 ) . Pearson ' s correlation rho coefficient and significance of difference from slope = 0 ( p ) are shown . IKBKE gene copy number and mRNA levels ( log2 ratio ) from the METABRIC transcriptomic dataset ( see D ) are plotted . Estimated + 1 copy number values are shown by the dotted vertical line . No significant correlation by Pearson ' s correlation rho coefficient and significance of difference from slope = 0 ( p ) was detected .

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

Correlation between IKBKE mRNA levels (log2 ratio) and immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patients (Curtis et al, 2012). Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) are shown. IKBKE gene copy number and mRNA levels (log2 ratio) from the METABRIC transcriptomic dataset (see D) are plotted. Estimated +1 copy number values are shown by the dotted vertical line. No significant correlation by Pearson's correlation rho coefficient and significance of difference from slope = 0 (p) was detected.


Amlexanox inhibits colony formation of MCF10A cells grown in soft agar with macrophage - conditioned medium for 5 weeks compared to controls . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 10 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

Amlexanox inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Representative images showing the filling of the spheroid lumen with cell nuclei . The spheroids were stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 50 μm . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Error bars represent mean ± SEM from 7 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) . Error bars represent mean ± SEM from 7 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) for statistical analysis . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Error bars represent mean ± SEM from 7 independent experiments (n=2 per condition; 50 spheroids each). Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids) for statistical analysis. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces the number of spheroids with invasive protrusions . Error bars represent mean ± SEM from 8 independent experiments ( n = 2 per condition , at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of spheroids with invasive protrusions. Error bars represent mean ± SEM from 8 independent experiments (n=2 per condition, at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) Organoids isolated from mice on ND were cultured in matrigel / collagen mix for 2 days and then stimulated with macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces the number of invasive organoids . Error bars represent mean ± SEM from 3 independent experiments , with each mouse labelled with a different symbol shape ( n = 2 per condition ; 15 - 25 organoids each ) . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) Organoids isolated from mice on ND were cultured in matrigel/collagen mix for 2 days and then stimulated with macrophage-conditioned or control medium in presence of amlexanox. Amlexanox reduces the number of invasive organoids. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


Mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically . Amlexanox reduces the number of invasive protrusions in organoids isolated from mice on G ) ND or H ) HFD . Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse ( labelled with a different symbol shape ) .

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

Mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. Amlexanox reduces the number of invasive protrusions in organoids isolated from mice on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).


Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in presence of amlexanox . Representative images showing the filling of the ductal lumen with cell nuclei . The ducts were stained for DNA ( DAPI in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 20 μm . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in presence of amlexanox. Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in presence of amlexanox . Amlexanox inhibits filling of the duct lumen with cell nuclei . Error bars represent mean ± SEM ( A total of 24 structures was counted per each condition : 2 patient ductal structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 ducts each ) . Filling of the lumen was determined as % of luminal space filled with cells . Macrophage donors are indicated as M1A - M1 activated , M2 activated macrophages .

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

Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in presence of amlexanox. Amlexanox inhibits filling of the duct lumen with cell nuclei. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as M1A - M1 activated, M2 activated macrophages.


IKBKE gene ( encoding for IKKε ) was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . IKKε CRISPR - Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage - conditioned medium for 5 weeks compared to CRISPR - Cas9 control ( nontargeting crRNA ) cells . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. IKKε CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


IKBKE gene ( encoding for IKKε ) was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . IKKε CRISPR - Cas9 knockout MCF10A cells are less invasive compared CRISPR Control ( nontargeting crRNA ) cells . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

IKBKE gene (encoding for IKKε) was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. IKKε CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


PSAT1 expression was assessed semi - quantitatively using a 0 - 3 grade scale ( " 0 " : no staining ; " 1 " : weak ; " 2 " : moderate ; " 3 " : strong ) on human breast cancer sections using immunohistochemistry . Representative images according to the PSAT1 staining grade are shown . Scale bar : 50 μm .

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

PSAT1 expression was assessed semi-quantitatively using a 0-3 grade scale ("0": no staining; "1": weak; "2": moderate; "3": strong) on human breast cancer sections using immunohistochemistry. Representative images according to the PSAT1 staining grade are shown. Scale bar: 50 μm.


Correlation of mRNA levels of the SBP enzyme genes PSAT1 ( E ) , PHGDH ( F ) , PSPH ( G ) with the immune signature ( Yoshihara et al , 2013 ) in the METABRIC transcriptomic dataset from 1981 breast cancer patient ( Curtis et al , 2012 ) .

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

Correlation of mRNA levels of the SBP enzyme genes PSAT1 (E), PHGDH (F), PSPH (G) with the immune signature (Yoshihara et al, 2013) in the METABRIC transcriptomic dataset from 1981 breast cancer patient (Curtis et al, 2012).


NCT502 ( 10 μM ) inhibits colony formation of MCF10A cells grown in soft agar with macrophage - conditioned medium for 5 weeks compared to controls . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 5 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

NCT502 (10 μM) inhibits colony formation of MCF10A cells grown in soft agar with macrophage-conditioned medium for 5 weeks compared to controls. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 5 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . Representative images showing the filling of the spheroid lumen with cell nuclei . The spheroids were stained for DNA ( Hoechst 33342 in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 50 μm . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). Representative images showing the filling of the spheroid lumen with cell nuclei. The spheroids were stained for DNA (Hoechst 33342 in blue) and F-actin (Phalloidin in red). Scale bar: 50 μm. Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . NCT502 reduces the filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei categorized into 4 groups ( clear , partially filled , almost filled and filled ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei categorized into 4 groups (clear, partially filled, almost filled and filled). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . NCT502 reduces the filling of the spheroid lumen with cell nuclei . Filling of the spheroid lumen with cell nuclei . Partially filled , almost filled and filled spheroids were combined together ( non - empty spheroids ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the filling of the spheroid lumen with cell nuclei. Filling of the spheroid lumen with cell nuclei. Partially filled, almost filled and filled spheroids were combined together (non-empty spheroids). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of NCT502 ( 10 μM ) . NCT502 reduces the number of spheroids with invasive protrusions ( n = 2 per condition ) . Error bars represent mean ± SEM from 3 independent experiments ( n = 2 per condition ; at least 15 spheroids each from at least 2 fields of view ) . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium in presence of NCT502 (10 μM). NCT502 reduces the number of spheroids with invasive protrusions (n=2 per condition). Error bars represent mean ± SEM from 3 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D25. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


Mouse mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice that were either on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were isolated from mice on ND and cultured in matrigel / collagen mix for 2 days followed by macrophage - conditioned or control medium stimulation for 24h in presence of NCT502 ( 10 μM ) . NCT502 reduces the number of invasive organoids . Error bars represent mean ± SEM from 3 independent experiments , with each mouse labelled with a different symbol shape ( n = 2 per condition ; 15 - 25 organoids each ) . Macrophage donors are indicated as M1A - M1 activated , , M2A - M2 activated macrophages .

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

Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were isolated from mice on ND and cultured in matrigel/collagen mix for 2 days followed by macrophage-conditioned or control medium stimulation for 24h in presence of NCT502 (10 μM). NCT502 reduces the number of invasive organoids. Error bars represent mean ± SEM from 3 independent experiments, with each mouse labelled with a different symbol shape (n=2 per condition; 15-25 organoids each). Macrophage donors are indicated as M1A - M1 activated, , M2A - M2 activated macrophages.


Mouse mammary organoids were isolated from 19 - 20 weeks old C57Bl / 6 mice that were either on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically . NCT502 ( 10 μM ) reduces the number of invasive protrusions in organoids isolated from mice that were either on G ) ND or H ) HFD . Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse ( labelled with a different symbol shape ) .

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

Mouse mammary organoids were isolated from 19-20 weeks old C57Bl/6 mice that were either on normal (ND) or high fat diet (HFD) (n=3). Organoids were cultured in collagen for 2 days after which the number of invasive protrusions per each organoid was determined microscopically. NCT502 (10 μM) reduces the number of invasive protrusions in organoids isolated from mice that were either on G) ND or H) HFD. Error bars represent mean ± SD from 3 independent experiments where 30 organoids were counted per each mouse (labelled with a different symbol shape).


Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in the presence of NCT502 ( 10μM ) . Representative images showing the filling of the ductal lumen with cell nuclei . The ducts were stained for DNA ( DAPI in blue ) and F - actin ( Phalloidin in red ) . Scale bar : 20 μm . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in the presence of NCT502 (10μM). Representative images showing the filling of the ductal lumen with cell nuclei. The ducts were stained for DNA (DAPI in blue) and F-actin (Phalloidin in red). Scale bar: 20 μm. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment . Reformed ducts were then cultured for 7 days in macrophage - conditioned or control medium in the presence of NCT502 ( 10μM ) . NCT502 inhibits filling of the duct lumen with cell nuclei . Error bars represent mean ± SEM ( A total of 24 structures was counted per each condition : 2 patient ductal structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 ducts each ) . Filling of the lumen was determined as % of luminal space filled with cells . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

Human myoepithelial and luminal cells isolated from breast specimens were cultured in collagen gels for 14 days to reform ductal structures with luminal compartment. Reformed ducts were then cultured for 7 days in macrophage-conditioned or control medium in the presence of NCT502 (10μM). NCT502 inhibits filling of the duct lumen with cell nuclei. Error bars represent mean ± SEM (A total of 24 structures was counted per each condition: 2 patient ductal structures (n=2), each labelled with a different symbol shape, were cultured with macrophage-conditioned media from either donor 17 or donor 18, 3 ducts each). Filling of the lumen was determined as % of luminal space filled with cells. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


PHGDH gene was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . PHGDH CRISPR - Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage - conditioned medium for 5 weeks compared to CRISPR - Cas9 control ( nontargeting crRNA ) cells . Colonies ≥ 50 μm were counted . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as , M1A - M1 activated , M2A - M2 activated macrophages .

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

PHGDH gene was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. PHGDH CRISPR-Cas9 knockout MCF10A cells form less colonies when grown in soft agar with macrophage-conditioned medium for 5 weeks compared to CRISPR-Cas9 control (nontargeting crRNA) cells. Colonies ≥ 50 μm were counted. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as , M1A - M1 activated, M2A - M2 activated macrophages.


PHGDH gene was deleted in MCF10A cells via CRISPR - Cas9 technology . 3 knockout clones were combined for each cell line . 16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium . PHGDH CRISPR - Cas9 knockout MCF10A cells are less invasive compared CRISPR Control ( nontargeting crRNA ) cells . Error bars represent mean ± SEM from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 ratio in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

PHGDH gene was deleted in MCF10A cells via CRISPR-Cas9 technology. 3 knockout clones were combined for each cell line. 16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. PHGDH CRISPR-Cas9 knockout MCF10A cells are less invasive compared CRISPR Control (nontargeting crRNA) cells. Error bars represent mean ± SEM from 3 independent experiments (n=3 per condition). Media from two macrophage donors were combined in 1:1 ratio in each experiment. Macrophage donors are indicated as M1A - M1 activated, M2A - M2 activated macrophages.


A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Wild - type mice on HFD gain more weight compared to ND mice . * p < 0 . 05 by one - tailed students t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Wild-type mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test.


A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Average adipocytes size ( major diameter ) in mammary fat pads is larger in HFD mice compared to ND mice based on the analysis of at least 100 adipocytes per mice on H & E stained sections . * p < 0 . 05 by two - tailed students t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Average adipocytes size (major diameter) in mammary fat pads is larger in HFD mice compared to ND mice based on the analysis of at least 100 adipocytes per mice on H&E stained sections. *p < 0.05 by two-tailed students t-test.


A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . The number of F4 / 80 + macrophages in mammary fat pads is increased in HFD mice compared to ND mice . The average of F4 / 80 + macrophages in at least 10 fields of view per mouse is shown . * p < 0 . 05 by two - tailed students t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). The number of F4/80+ macrophages in mammary fat pads is increased in HFD mice compared to ND mice. The average of F4/80+ macrophages in at least 10 fields of view per mouse is shown. *p < 0.05 by two-tailed students t-test.


A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Mice were administered amlexanox ( 17 mM ) or vehicle control daily by oral gavage ( Reilly et al , 2013 ) . A control group ( wild - type mice ) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV - PyMT gene . PyMT - MMTV + / - mice on HFD gain more weight compared to ND mice . * p < 0 . 05 by one - tailed students t - test . Amlexanox does not affect weight gain either in mice on F ) ND or G ) HFD .

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

A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene. PyMT-MMTV+/- mice on HFD gain more weight compared to ND mice. *p < 0.05 by one-tailed students t-test. Amlexanox does not affect weight gain either in mice on F) ND or G) HFD.


A - J ) Ten per group female C57Bl / 6 mice heterozygous for the PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 weeks old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Mice were administered amlexanox ( 17 mM ) or vehicle control daily by oral gavage ( Reilly et al , 2013 ) . A control group ( wild - type mice ) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV - PyMT gene . Mammary tumours are developing earlier in PyMT - MMTV + / - mice on HFD compared to ND mice . Amlexanox does not affect tumour development in PyMT - MMTV + / - mice on ND , but delays tumour formation in J ) HFD mice .

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

A-J)Ten per group female C57Bl/6 mice heterozygous for the PyMT-MMTV started high fat diet (HFD) when 6-7 weeks old (prior of mammary tumour development) together with control mice on normal diet (ND; n=10). Mice were administered amlexanox (17 mM) or vehicle control daily by oral gavage (Reilly et al, 2013). A control group (wild-type mice) was also included to confirm the efficacy of the diet and to control for possible unpredicted interactions of the MMTV-PyMT gene. Mammary tumours are developing earlier in PyMT-MMTV+/- mice on HFD compared to ND mice. Amlexanox does not affect tumour development in PyMT-MMTV+/- mice on ND, but delays tumour formation in J) HFD mice.


A ) Upper panels : Representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of AC16 cells kept at 37 ° C or exposed to different cold temperatures for 24h . Lower panels : DAPI signal segmented into chromatin and interchromatin regions according to relative intensity . Scale bar : 5 μm . Arrows mark chromatin concentrated at the nuclear periphery ( P ) and around nucleoli ( N ) . B ) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions . Median ( central line ) , interquartile range ( IQR , box edges ) , most extreme data point no more than 1 . 5x IQR from box edges ( whiskers ) . Number of nuclei : 37 ° C ( 31 ) , 28 ° C 24h ( 26 ) , 18 ° C 5h ( 19 ) , 18 ° C 24h ( 22 ) , 18 ° C 24h then 37 ° C 2h ( 25 ) , 8 ° C 24h ( 23 ) . C ) Left and central panels : representative single z - planes from 3D - SIM image stacks of the DAPI - stained nuclei of cells kept at 37 ° C or exposed to 18 ° C for 24h showing the spatial distribution of the immunofluorescence ( IF ) signal of hnRNPC ( green ) and nuclear pore complexes ( NPCs , red ) . Right panel : DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z - plane shown . Scale bar whole nuclei : 5 μm . Scale bar enlarged section : 1 μm . D ) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition . Number of distances : 37 ° C ( 112511 ) , 28 ° C 24h ( 94521 ) , 18 ° C 5h ( 72340 ) , 18 ° C 24h ( 74998 ) , 18 ° C 24h then 37 ° C 2h ( 92896 ) , 8 ° C 24h ( 80570 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N). B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23). C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm. D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).


A ) Number of genes showing significant ( adjusted p < 0 . 05 , Wald test ) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h .

O O O O O O O O O O O O O O O O O O O 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) Number of genes showing significant (adjusted p < 0.05, Wald test) up or downregulation in nuclear or cytoplasmic RNA levels upon transfer of AC16 cells from 37°C to different cold temperatures for 24h.


B ) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37 ° C to different cold temperatures for 24h . Points are color - coded from low to high density ( black < blue < red < yellow ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relationship between the log2 fold change in RNA level in the nucleus and that in the cytoplasm for all genes upon transfer of AC16 cells from 37°C to different cold temperatures for 24h. Points are color-coded from low to high density (black < blue < red < yellow).


C ) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37 ° C to 28 ° C or 18 ° C ( top ) , 28 ° C or 8 ° C ( middle ) and 18 ° C or 8 ° C ( bottom ) for 24h in both the nucleus and cytoplasm . Points are shaded from low to high density ( grey < black ) . Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relationship between the log2 fold change in RNA level for all genes upon transfer of AC16 cells from 37°C to 28°C or 18°C (top), 28°C or 8°C (middle) and 18°C or 8°C (bottom) for 24h in both the nucleus and cytoplasm. Points are shaded from low to high density (grey < black). Genes that show significant differential expression at a specific temperature or at both temperatures are highlighted with colored circles or triangles, respectively.


A ) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18 ° C for 24h time point . Range ( whiskers ) , interquartile range ( IQR , box edges ) , median + / - IQR / [ square root ( number of genes ) ] ( notch ) and median ( central 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18°C for 24h time point. Range (whiskers), interquartile range (IQR, box edges), median +/-IQR/[square root(number of genes)] (notch) and median (central line).


B ) Log2 fold change in cytoplasmic ( black line , left axis ) and nuclear ( red line , right axis ) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37 ° C to 18 ° C for 24h and then back to 37 ° C for 24h relative to cells kept at 37 ° C . Error bars show the standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Log2 fold change in cytoplasmic (black line, left axis) and nuclear (red line, right axis) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h relative to cells kept at 37°C. Error bars show the standard error.


C ) Nuclear and cytoplasmic full - length RNA - Seq data from AC16 cells kept at 37 ° C or exposed to 18 ° C for 24h for REV - ERBα and CRY2 . Data for each track have been normalized to show counts per million aligned counts ( CPM ) for each nucleotide . Number of 3 ' end RNA - seq sample replicates : 37 ° C ( C : 6 , N : 7 ) , 18 ° C 5h ( 4 ) , 10h ( 4 ) , 24h ( 6 ) , 18 ° C 24h then 37 ° C 2h ( 4 ) , 5h ( 2 ) , 10h ( 2 ) , 24h ( 2 ) . Number of cytoplasmic ( C ) and nuclear ( N ) samples are the same for each condition except 37 °

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°C


Western blot of Flag - tagged REV - ERBα levels ( short and long exposure ) in whole cell extracts from AC16 cells transferred from 37 ° C to 18 ° C for the time periods indicated and also returned after each of these time periods to 37 ° C for 2h . TP53 was used as a loading control as its transcript levels show minimal changes in response to 18 ° C exposure Ponceau stain is shown as an additional loading control .

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

Western blot of Flag-tagged REV-ERBα levels (short and long exposure) in whole cell extracts from AC16 cells transferred from 37°C to 18°C for the time periods indicated and also returned after each of these time periods to 37°C for 2h. TP53 was used as a loading control as its transcript levels show minimal changes in response to 18°C exposure Ponceau stain is shown as an additional loading control.


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV -

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

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for CRY2 ( B )

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

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for CRY2 (B)


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) AC16 exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for TP53 ( C )

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for TP53 (C)


Representative maximum intensity z - stack - projected images of DAPI - stained ( blue ) U2OS cells ( D ) , exposed to 18 ° C and returned to 37 ° C for the time periods indicated , probed by RNA - FISH for REV -

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

Representative maximum intensity z-stack-projected images of DAPI-stained (blue) U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα


Mean REV - ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( REV - ERBα ) : 37 ° C ( 65 ) , 18 ° C 5h ( 30 ) , 18 ° C 5h then 37 ° C 2h ( 21 ) , 18 ° C 24h ( 63 ) , 18 ° C 24h then 37 ° C 2h ( 64 )

Mean REV-ERBα per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (REV-ERBα): 37°C (65), 18°C 5h (30), 18°C 5h then 37°C 2h (21), 18°C 24h (63), 18°C 24h then 37°C 2h (64)


Mean CRY2 ( F ) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . ; AC16 cells ( CRY2 ) : 37 ° C ( 43 ) , 18 ° C 24h ( 42 ) , 18 ° C 24h then 37 ° C 2h ( 42 )

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

Mean CRY2 (F) per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. ; AC16 cells (CRY2): 37°C (43), 18°C 24h (42), 18°C 24h then 37°C 2h (42)


Mean TP53 ( G ) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . AC16 cells ( TP53 ) : 37 ° C ( 47 ) , 18 ° C 24h ( 45 ) , 18 ° C 24h then 37 ° C 2h ( 48 )

Mean TP53 (G) transcripts per AC16 cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. AC16 cells (TP53): 37°C (47), 18°C 24h (45), 18°C 24h then 37°C 2h (48)


Mean REV - ERBα per U2OS ( H ) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA - FISH data . U2OS cells ( REV - ERBα ) : 37 ° C ( 61 ) , 18 ° C 5h ( 61 ) , 18 ° C 5h then 37 ° C 2h ( 69 ) , 18 ° C 24h ( 53 ) , 18 ° C 24h then 37 ° C 2h ( 65 )

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

Mean REV-ERBα per U2OS (H) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. U2OS cells (REV-ERBα): 37°C (61), 18°C 5h (61), 18°C 5h then 37°C 2h (69), 18°C 24h (53), 18°C 24h then 37°C 2h (65)


A , B ) Mean baseline - detrended bioluminescence profiles from plate wells containing either PER2 : : LUC U2OS cells with ( WT , left panel ) or without REV - ERBα ( KO , right panel ) recorded at 37 ° C before and after transfer to 18 ° C for 24h ( A ) or 5h ( B ) ( grey region ) from biological repeat 1 . 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18 ° C exposure ( time zero ) .

A,B) Mean baseline-detrended bioluminescence profiles from plate wells containing either PER2::LUC U2OS cells with (WT, left panel) or without REV-ERBα (KO, right panel) recorded at 37°C before and after transfer to 18°C for 24h (A) or 5h (B) (grey region) from biological repeat 1. 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18°C exposure (time zero).


D ) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37 ° C or transferred to 18 ° C for 24h ( top panel ) or 5h ( bottom panel ) from biological repeats 1 and 2 . Points are colored according to their distinct phase prior to 18 ° C exposure . Colored lines ( bottom panel ) show the change in the mean for the points from each phase . Boxplots display median ( central line ) , interquartile range ( IQR , box edges ) and range ( whiskers ) . Number of wells : top panel WT cells : 37 ° C ( 120 ) , 18 ° C 24h ( 119 ) ; top panel KO cells : 37 ° C ( 120 ) , 18 ° C 24h ( 120 ) ; bottom panel WT cells : 37 ° C ( 58 ) , 18 ° C 5h ( 120 ) ; top panel KO cells : 37 ° C ( 60 ) , 18 ° C 5h ( 120 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37°C or transferred to 18°C for 24h (top panel) or 5h (bottom panel) from biological repeats 1 and 2. Points are colored according to their distinct phase prior to 18°C exposure. Colored lines (bottom panel) show the change in the mean for the points from each phase. Boxplots display median (central line), interquartile range (IQR, box edges) and range (whiskers). Number of wells: top panel WT cells: 37°C (120), 18°C 24h (119); top panel KO cells: 37°C (120), 18°C 24h (120); bottom panel WT cells: 37°C (58), 18°C 5h (120); top panel KO cells: 37°C (60), 18°C 5h (120).


E ) Bar graphs showing the mean amplitude change ( left axis ) following 18 ° C exposure for 5h for each group of plate well profiles ( grouped according to their distinct phase ( a - f ) prior to 18 ° C exposure , 10 wells per group ) for both WT and KO cells from biological repeats 1 and 2 . Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2 : LUC ( dashed line ) and predicted REV - ERBα expression level ( right axis ) ( grey shaded region ( light grey indicates REV - ERBα deletion ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graphs showing the mean amplitude change (left axis) following 18°C exposure for 5h for each group of plate well profiles (grouped according to their distinct phase (a-f) prior to 18°C exposure, 10 wells per group) for both WT and KO cells from biological repeats 1 and 2. Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2:LUC (dashed line) and predicted REV-ERBα expression level (right axis) (grey shaded region (light grey indicates REV-ERBα deletion)).


( A ) Upper panel : immunoblot analysis of COS7 cells treated for 12 h with MG132 ( 25 μM ) and vinblastine ( 25 μM ) . Lower panel : real ‐ time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure ( 25 μM , 12 h ) .

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

(A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h).


( B , C ) Immunofluorescence staining of indicated proteins in COS7 cells treated as in ( A ) . Scale bar , 20 μm .

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

(B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm.


( D ) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 ( 25 μM , 12 h ) . Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online ) . Diagram shows the percentage of aggresome ‐ positive cells .

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

(D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells.


( E ) Aggresome quantification as in supplementary Fig S1D online from cells analysed in ( H ) . Images are shown in supplementary Fig S1G online . Diagram shows the percentage of aggresome ‐ positive cells at 4 , 24 and 48 h after MG132 wash out .

(E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out.


( F ) Cells were treated as in ( H ) ; however , 4 h after MG132 wash out , BafA1 ( 5 μM ) was added for 20 h to inhibit aggresome clearance . Diagrams show the percentage of aggresome ‐ positive cells and the mean size of aggresomes ( supplementary Fig S1H online ) . Images are shown in supplementary Fig S1G online .

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

(F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes ( supplementary Fig S1H online). Images are shown in supplementary Fig S1G online.


( G ) Dot ‐ blot ( upper panel , quantification in supplementary Fig S1B online ) and western blot ( lower panel ) analyses of 0 . 5 % NP40 ‐ soluble and ‐ insoluble proteins from cells shown in Fig 1D ; supplementary Fig S1D online .

(G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online.


( H ) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 ( 25 μM , 18 h ) or DMSO as a control . After MG132 wash out , cells were allowed to recover for 4 , 24 and 48 h and 0 . 5 % NP40 ‐ soluble and ‐ insoluble proteins were analysed by immunoblotting .

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

(H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting.


( I ) COS7 cells were transfected for 24 h with GST or GST fused to DIC ( GST ‐ DIC ) and treated with MG132 ( 25 μM , 12 h ) . Indicated proteins in whole ‐ cell lysates and in precipitates obtained by GST pull ‐ down with glutathione beads were analysed by immunoblotting . BafA1 , bafilomycin A1 ; ctrl , control ; DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; Hsp70 , heat ‐ shock protein 70 ; nons , nonsense ; siRNA , short ‐ interfering RNA ; vinblast , vinblastine .

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(I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.


( A ) GST pull ‐ down analysis as in Fig 1I , but performed in cells transfected for 36 h with full ‐ length FLAG ‐ BAG3 ( I ) or the BAG3 deletion mutants FLAG - ( Δ421 - 498 ) - BAG3 ( II ) , FLAG - BAG ‐ domain ( III ) , and FLAG - PxxP - BAG ‐ domain ( IV ) . Asterisk , nonspecific band .

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(A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG-(Δ421-498)-BAG3 (II), FLAG-BAG‐domain (III), and FLAG-PxxP-BAG‐domain (IV). Asterisk, nonspecific band.


( B ) HEK cells were transfected for 18 h with SODG85R ‐ GFP , along with the indicated FLAG ‐ tagged BAG3 constructs ( shown in A ) and HA ‐ tagged Hsp70 ( HA ‐ Hsp70 ) . SODG85R ‐ GFP showed three distinct localization patterns : cytoplasmic , pre ‐ aggresomal and aggresomal ( see representative pictures on the right and supplementary Fig S2F online ) . The percentages of cells bearing pre ‐ aggresomal ( upper panel ) and aggresomal ( lower panel ) SODG85R ‐ GFP are shown . Scale bar , 10 μm .

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(B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm.


( C ) HEK cells transfected with SODG85R ‐ GFP , BAG3 and Hsp70 were additionally transfected with FLAG ‐ p50 , as indicated . Diagrams show the percentages of cells with pre ‐ aggresomal and aggresomal SODG85R ‐ GFP . Right panel : representative image showing pre ‐ aggresomal localization of FLAG ‐ p50 and SODG85R ‐ GFP . Scale bar , 5 μm .

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(C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm.


( D ) GST pull ‐ down analysis as in ( A ) , but in HEK cells transfected for 18 h with SODWT ‐ GFP or mutant SODG85R ‐ GFP , along with FLAG ‐ BAG3 .

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(D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3.


( E ) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA . At 6 h after transfection , lactacystin ( 10 μM ) or GST as vehicle control was added for GST and GST pull ‐ down analysis was performed as in ( D ) .

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(E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D).


( F ) Upper panel : sucrose ‐ density gradient analysis of extracts from HEK cells transfected with SODG85R ‐ GFP and GST ‐ DIC , along with FLAG ‐ BAG3 or vector control . Note the immunoreactive bands in high ‐ density fraction 14 on BAG3 overexpression ( arrow ) . Migration of a 20S marker protein is indicated . Lower panel : GST ‐ DIC pull ‐ down analyses of dynein complexes present in sucrose fraction 6 and 14 .

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(F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14.


( G ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells transfected with FLAG ‐ tagged BAG3 constructs ( I - IV ; indicated as in A ) .

(G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I-IV; indicated as in A).


( H ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells treated with PYR41 ( 10 μM ; 12 h ) , as indicated . DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short ‐ interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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

(H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( A ) Analysis of SODG85R ‐ GFP ( green ) localization with aggresome marker vimentin , the autophagic receptors p62 and NBR1 , the autophagosome marker LC3 and the lysosomal marker LAMP2 ( all in red ) in BAG3 / Hsp70 ‐ transfected HEK cells . Representative merged images of cells with cytoplasmic , pre ‐ aggresomal and aggresomal SOD distribution are shown . Arrows indicate LC3 ‐ positive autophagosomes . Scale bar , 5 μm .

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(A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm.


( B ) Transmission electron microscope analysis of SODG85R ‐ GFP aggresome in cells transfected as in ( A ) . Marked area in ( a ) is shown magnified in ( b ) . Arrows , autophagosomes . Arrowheads , autolysosomes . Scale bar , 1 μm .

(B) Transmission electron microscope analysis of SODG85R‐GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm.


( C ) HEK cells transfected for 8 h with SODG85R ‐ GFP along with FLAG ‐ BAG3 and HA ‐ Hsp70 , as indicated , were treated for 24 h with BafA1 ( 0 . 1 μM ) . Indicated proteins were analysed by immunoblotting . Diagram shows the ratio of SODG85R ‐ GFP levels in BafA1 ‐ treated cells to those in untreated cells .

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(C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells.


( D ) Same analysis as in ( C ) but with SODWT ‐ GFP .

(D) Same analysis as in (C) but with SODWT‐GFP.


( E ) Similar analysis as in ( C ) but in cells transfected 24 h before with nonsense or atg7 siRNA .

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(E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA.


( F ) HEK cells were transfected with SODG85R ‐ GFP and indicated FLAG ‐ tagged BAG3 constructs together with HA ‐ Hsp70 , and lysosomal degradation of SODG85R ‐ GFP was examined as in ( C ) . Corresponding immunoblots are shown in supplementary Fig S3F online . Ag , aggresome ; BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; N , nucleus ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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(F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( A ) Spinal cord sections of mice overexpressing SODWT and SODG85R ( disease end ‐ stage ) were immunostained for BAG3 and SOD1 . Arrows indicate SOD1 ‐ and BAG3 ‐ positive aggregates in the neuropil . Scale bar , 20 μm .

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(A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm.


( B ) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset . Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R . Scale bar , 10 μm . Magnific . , magnification ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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(B) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SODWT, wild‐type SOD.


( b ) Quantification of CTM - GFP and mCTM - GFP relative to WT - GFP in HEK cells 24 h after individual transfections with WT - GFP , mCTM - GFP ( n = 6 ) or CTM - GFP . Cells transfected with WT - GFP or CTM - GFP were treated without ( no treatment ; n = 13 ) or with serum deprivation ( + SD ; n = 5 ; to enhance CMA activity ) or with macroautophagy inhibitor 3 - methyladenine ( + 3 - MA ; 10 mM ; n = 5 ) , proteasome inhibitor ( + MG132 ; 5 μM ; n = 6 ) , lysosome inhibitor ( + NH4Cl ; 20 mM ; n = 6 ) or pepstatin A ( + Pep A ; 10 μM ; n = 5 ) . Top , representative immunoblots of cell lysates for GFP . Membranes reprobed for β - actin were used as a loading control . Bottom , means ± s . e . m . of relative protein levels , normalized to WT - GFP ( arbitrarily set as 1 ; white bar ) ; n represents the number of independent experiments from at least 4 separate cultures . One - way ANOVA with Fisher least significant difference test , F ( 12 , 73 ) = 20 . 939 . * * * P 0 . 001 versus WT - GFP ( white bar ) ; ΔΔΔP 0 . 001 relative to untreated CTM - GFP levels ( gray bar ) . Full - length blots are presented in Supplementary Figure 9 .

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(b) Quantification of CTM-GFP and mCTM-GFP relative to WT-GFP in HEK cells 24 h after individual transfections with WT-GFP, mCTM-GFP (n = 6) or CTM-GFP. Cells transfected with WT-GFP or CTM-GFP were treated without (no treatment; n = 13) or with serum deprivation (+ SD; n = 5; to enhance CMA activity) or with macroautophagy inhibitor 3-methyladenine (+ 3-MA; 10 mM; n = 5), proteasome inhibitor (+ MG132; 5 μM; n = 6), lysosome inhibitor (+ NH4Cl; 20 mM; n = 6) or pepstatin A (+ Pep A; 10 μM; n = 5). Top, representative immunoblots of cell lysates for GFP. Membranes reprobed for β-actin were used as a loading control. Bottom, means ± s.e.m. of relative protein levels, normalized to WT-GFP (arbitrarily set as 1; white bar); n represents the number of independent experiments from at least 4 separate cultures. One-way ANOVA with Fisher least significant difference test, F(12,73) = 20.939. ***P 0.001 versus WT-GFP (white bar); ΔΔΔP 0.001 relative to untreated CTM-GFP levels (gray bar). Full-length blots are presented in Supplementary Figure 9.


( c ) Representative confocal images of colocalization of GFP with the lysosome marker LAMP - 1 in COS - 7 cells transfected with either WT - GFP ( n = 17 ) or CTM - GFP ( n = 16 ) . Scale bar , 20 μm .

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(c) Representative confocal images of colocalization of GFP with the lysosome marker LAMP-1 in COS-7 cells transfected with either WT-GFP (n = 17) or CTM-GFP (n = 16). Scale bar, 20 μm.


b ) Reciprocal coimmunoprecipitation ( IP ) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1 . Flag - tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA - GluN2Bct - CTM or HA - GluN2Bct - CTMm at various ratios in HEK cells , and coimmunoprecipitation and / or immunoblotting were performed 24 h thereafter . Anti - HA was used to detect HA - GluN2Bct - CTM and HA - GluN2Bct - CTMm , anti - FLAG to detect wtDAPK1 and cDAPK1 .

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b) Reciprocal coimmunoprecipitation (IP) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1. Flag-tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA-GluN2Bct-CTM or HA-GluN2Bct-CTMm at various ratios in HEK cells, and coimmunoprecipitation and/or immunoblotting were performed 24 h thereafter. Anti-HA was used to detect HA-GluN2Bct-CTM and HA-GluN2Bct-CTMm, anti-FLAG to detect wtDAPK1 and cDAPK1.


( c ) HA - GluN2Bct - CTM specifically and dose - dependently decreased the level of cDAPK1 ( n = 4 independent experiments from 4 separate cell cultures and transfections ; P 0 . 001 ; F ( 5 , 18 ) = 18 . 27 ) but not

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(c) HA-GluN2Bct-CTM specifically and dose-dependently decreased the level of cDAPK1 (n = 4 independent experiments from 4 separate cell cultures and transfections; P 0.001; F(5,18) = 18.27) but not wtDAPK1


( d ; n = 3 independent experiments from 3 separate cell cultures and transfections ; P = 0 . 933 ; F ( 5 , 12 ) = 0 . 249 ) . HA - GluN2Bct - CTM - mediated cDAPK1 knockdown was significantly reduced by NH4Cl ( c ; 20 mM ; n = 4 ; ΔΔΔP 0 . 001 compared to HA - GluN2Bct - CTM : cDAPK1 8 : 1 group ) and by mutational inactivation of

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(d; n = 3 independent experiments from 3 separate cell cultures and transfections; P = 0.933; F(5,12) = 0.249). HA-GluN2Bct-CTM-mediated cDAPK1 knockdown was significantly reduced by NH4Cl (c; 20 mM; n = 4; ΔΔΔP 0.001 compared to HA-GluN2Bct-CTM:cDAPK1 8:1 group) and by mutational inactivation of CTM