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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) Representative photographs of Bax / Bak − / − MEFs treated with etoposide . Bax / Bak − / − MEFs were treated with 20 μM etoposide in the presence of PI ( 2 μM ) for the indicated times , and were analysed under a phase - contrast and fluorescence ( PI ) microscope .

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

(a) Representative photographs of Bax/Bak−/− MEFs treated with etoposide. Bax/Bak−/− MEFs were treated with 20 μM etoposide in the presence of PI (2 μM) for the indicated times, and were analysed under a phase-contrast and fluorescence (PI) microscope.


( b , c ) Reduced viability of Bax / Bak − / − MEFs after exposure to etoposide . WT ( circles ) and Bax / Bak − / − ( squares ) MEFs were treated with 20 μM etoposide in the presence ( closed symbols ) or absence ( open symbols ) of 100 μM zVAD - fmk . Then cell viability was measured by the PI staining ( b ) and CTB assay ( c ) , and expressed as a percentage of the initial value without etoposide . Data are shown as mean ± s . d . ( n = 4 ) .

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

(b, c) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide. WT (circles) and Bax/Bak−/− (squares) MEFs were treated with 20 μM etoposide in the presence (closed symbols) or absence (open symbols) of 100 μM zVAD-fmk. Then cell viability was measured by the PI staining (b) and CTB assay (c), and expressed as a percentage of the initial value without etoposide. Data are shown as mean ± s.d. (n = 4).


( d ) Reduced viability of etoposide - treated Bax / Bak − / − MEFs as assessed by proliferation assay . Bax / Bak − / − MEFs were either not treated ( 0 h ) or were treated with 20 μM etoposide for 12 and 24 h , then all cells were recovered and 5 , 000 cells were reseeded . Viable cell numbers were measured on the indicated days by the CTB assay . Results were normalized by adjusting the day 0 value . n = 4 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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) Reduced viability of etoposide-treated Bax/Bak−/− MEFs as assessed by proliferation assay. Bax/Bak−/− MEFs were either not treated (0 h) or were treated with 20 μM etoposide for 12 and 24 h, then all cells were recovered and 5,000 cells were reseeded. Viable cell numbers were measured on the indicated days by the CTB assay. Results were normalized by adjusting the day 0 value. n = 4.


e ) Reduced viability of Bax / Bak − / − MEFs after exposure to etoposide and staurosporine , as assessed by clonogenicity assay . MEFs were treated with etoposide ( 20 μM ) or staurosporine ( 1 μM ) at the indicated times , collected , and 2 , 000 cells were seeded in the normal medium . After 1 week , colonies were counted . n =

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 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

e) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine, as assessed by clonogenicity assay. MEFs were treated with etoposide (20 μM) or staurosporine (1 μM) at the indicated times, collected, and 2,000 cells were seeded in the normal medium. After 1 week, colonies were counted. n = 4


f - i ) Induction of autophagy in Bax / Bak − / − MEFs by etoposide . ( f ) Electron micrograph ( × 14 , 800 ) of Bax / Bak − / − MEF treated with etoposide ( 20 μM ) for 18 h . Representative features of Bax / Bak − / − MEFs that have received no treatment ( NT ; that is , are healthy ) are also shown ( NT : × 14 , 000 ) .

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-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

f-i) Induction of autophagy in Bax/Bak−/− MEFs by etoposide. (f) Electron micrograph (×14,800) of Bax/Bak−/− MEF treated with etoposide (20 μM) for 18 h. Representative features of Bax/Bak−/− MEFs that have received no treatment (NT; that is, are healthy) are also shown (NT: ×14,000).


( g ) In magnified photographs , a large number of autolysosomes / autophagosomes ( left : × 26 , 000 ) were observed , but the mitochondria were unaffected ( right : × 17 , 000 ) .

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

(g) In magnified photographs, a large number of autolysosomes/autophagosomes (left: ×26,000) were observed, but the mitochondria were unaffected (right: ×17,000).


( h , i ) Punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs treated with etoposide . GFP - LC3 - transfected cells were incubated with and without etoposide ( 20 μM ) for 24 h ( h ) or the indicated times ( i ) , and then examined by fluorescent microscopy . ( h ) Representative photographs of healthy ( NT ) and etoposide - treated Bax / Bak − / − MEFs are shown . ( i ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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

(h, i) Punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs treated with etoposide. GFP-LC3-transfected cells were incubated with and without etoposide (20 μM) for 24 h (h) or the indicated times (i), and then examined by fluorescent microscopy. (h) Representative photographs of healthy (NT) and etoposide-treated Bax/Bak−/− MEFs are shown. (i) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-positive cells. Data are shown as mean ± s.d. (n = 4).


( a , b ) Bax / Bak − / − MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3 - MA for 24 h , and then were examined by phase - contrast microscopy ( a ) and electron microscopy ( × 8 , 400 ) ( b ) . Autolysosomes / autophagosomes were hardly observed . (

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O 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, b) Bax/Bak−/− MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3-MA for 24 h, and then were examined by phase-contrast microscopy (a) and electron microscopy (×8,400) (b). Autolysosomes/autophagosomes were hardly observed. (


c , d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by 3 - MA . Bax / Bak − / − MEFs that were transfected with GFP - LC3 were incubated without ( NT ) or with 20 μM etoposide in the presence or absence of 10 mM 3 - MA for 24 h , and then were examined by confocal fluorescent microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in the presence of 3 - MA . ( d ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).


( c , d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by 3 - MA . Bax / Bak − / − MEFs that were transfected with GFP - LC3 were incubated without ( NT ) or with 20 μM etoposide in the presence or absence of 10 mM 3 - MA for 24 h , and then were examined by confocal fluorescent microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in the presence of 3 - MA . ( d ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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

(c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).


( e - g ) Inhibition of etoposide - and staurosporine - induced cell death by 3 - MA . Bax / Bak − / − MEFs were treated with 20 μM etoposide ( e - g ) or 1 μM staurosporine ( g ) in the absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA for the indicated time . Cell viability was measured by PI staining ( e ) , CTB assay ( f ) and clonogenicity assay ( g ) ( as in Fig . 1e ) . Upper panels in g : viable cells were visualized by staining with calcein - AM . n = 4 .

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

(e-g) Inhibition of etoposide- and staurosporine-induced cell death by 3-MA. Bax/Bak−/− MEFs were treated with 20 μM etoposide (e-g) or 1 μM staurosporine (g) in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA for the indicated time. Cell viability was measured by PI staining (e), CTB assay (f) and clonogenicity assay (g) (as in Fig. 1e). Upper panels in g: viable cells were visualized by staining with calcein-AM. n = 4.


( a , b ) Bax / Bak − / − MEFs were treated with the indicated siRNAs ( 10 μg ) for 24 h and then incubated with 20 μM etoposide for the indicated times . Expression of APG5 - APG12 complex ( a ) , Beclin 1 ( b ) and VDAC ( loading control ) was analysed by Western blot analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex (a), Beclin 1 (b) and VDAC (loading control) was analysed by Western blot analysis.


( c ) Inhibition of etoposide - induced autophagy by silencing of Beclin 1 . Bax / Bak − / − MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy ( × 8 , 500 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inhibition of etoposide-induced autophagy by silencing of Beclin 1. Bax/Bak−/− MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy (×8,500).


d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by silencing of Beclin 1 and APG5 . Bax / Bak − / − MEFs that were transfected with GFP - LC3 together with the indicated siRNAs , were incubated without ( NT ) or with 20 μM etoposide for 24 h , and then examined by confocal fluorescent microscopy . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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

d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were transfected with GFP-LC3 together with the indicated siRNAs, were incubated without (NT) or with 20 μM etoposide for 24 h, and then examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).


( e , f ) Inhibition of etoposide - and staurosporine - induced cell death by silencing of Beclin 1 and APG5 . Bax / Bak − / − MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide ( e , f ) or 1 μM staurosporine ( f ) for the indicated time . Cell viability was measured by CTB assay ( e ) and clonogenicity assay ( f ) . n = 4 .

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(e, f) Inhibition of etoposide- and staurosporine-induced cell death by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide (e, f) or 1 μM staurosporine (f) for the indicated time. Cell viability was measured by CTB assay (e) and clonogenicity assay (f). n = 4.


( a , b ) APG5 - dependent punctate GFP - LC3 fluorescence in Bcl - 2 - transfected MEFs after etoposide treatment . APG5 + / + and APG5 − / − MEFs that were transfected with both GFP - LC3 and the indicated plasmids ( Bcl - 2 or vector ) were incubated without or with 20 μM etoposide for 24 h , then examined by confocal fluorescent microscopy ( a ) . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells ( b ) .

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(a, b) APG5-dependent punctate GFP-LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment. APG5+/+ and APG5−/− MEFs that were transfected with both GFP-LC3 and the indicated plasmids (Bcl-2 or vector) were incubated without or with 20 μM etoposide for 24 h, then examined by confocal fluorescent microscopy (a). The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells (b).


( c ) Accumulation of Beclin 1 and APG5 - APG12 complex in Bcl - 2 - transfected MEFs after etoposide treatment . WT MEFs were transfected with the indicated plasmids , and were incubated without ( NT ) or with 20 μM etoposide for 24 h .

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(c) Accumulation of Beclin 1 and APG5-APG12 complex in Bcl-2-transfected MEFs after etoposide treatment. WT MEFs were transfected with the indicated plasmids, and were incubated without (NT) or with 20 μM etoposide for 24 h.


( d , e ) Occurrence of etoposide - induced 3 - MA - inhibitable non - apoptotic death in Bcl - xL - overexpressing APG5 + / + MEFs , but not APG5 − / − MEFs . APG5 + / + ( open columns ) and APG5 − / − ( closed columns ) MEFs were transfected with the indicated plasmids ( 2 μg ) . After 24 h , transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3 - MA for 24 h , and cell viability was measured by the CTB assay ( d ) and apoptotic nuclear morphology ( e ) . Data are shown as mean ± s . d . ( n = 4 ) .

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(d, e) Occurrence of etoposide-induced 3-MA-inhibitable non-apoptotic death in Bcl-xL-overexpressing APG5+/+ MEFs, but not APG5−/− MEFs. APG5+/+ (open columns) and APG5−/− (closed columns) MEFs were transfected with the indicated plasmids (2 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay (d) and apoptotic nuclear morphology (e). Data are shown as mean ± s.d. (n = 4).


( f ) Overexpression of Bcl - xL does not sensitize Beclin 1 - silenced MEFs to etoposide - induced 3 - MA - inhibitable non - apoptotic death . WT MEFs with silencing of control GFP ( open columns ) or Beclin 1 ( closed columns ) were transfected with the indicated plasmids ( 1 μg ) . After 24 h , transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3 - MA for 24 h , and cell viability was measured by the CTB assay . n = 3 .

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(f) Overexpression of Bcl-xL does not sensitize Beclin 1-silenced MEFs to etoposide-induced 3-MA-inhibitable non-apoptotic death. WT MEFs with silencing of control GFP (open columns) or Beclin 1 (closed columns) were transfected with the indicated plasmids (1 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay. n = 3.


( a ) Enhancement of etoposide - induced non - apoptotic death by overexpression of human Bcl - 2 and Bcl - xL in Bax / Bak − / − MEFs in a Beclin 1 - dependent manner . Bax / Bak − / − MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids ( 1 μg ) . After 24 h , the transfected cells were incubated with 20 μM etoposide for 24 h , and cell viability was assessed by the CTB assay . Data are shown as mean ± s . d . ( n = 3 ) .

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(a) Enhancement of etoposide-induced non-apoptotic death by overexpression of human Bcl-2 and Bcl-xL in Bax/Bak−/− MEFs in a Beclin 1-dependent manner. Bax/Bak−/− MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids (1 μg). After 24 h, the transfected cells were incubated with 20 μM etoposide for 24 h, and cell viability was assessed by the CTB assay. Data are shown as mean ± s.d. (n = 3).


( b ) Bax / Bak − / − MEFs were transiently transfected with 10 μg of siRNA for Bcl - 2 , Bcl - x , or Bax ( negative control ) for 24 h . Expression levels of Bcl - 2 , Bcl - xL and Lamin B ( loading control ) were analysed by Western blot analysis .

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(b) Bax/Bak−/− MEFs were transiently transfected with 10 μg of siRNA for Bcl-2, Bcl-x, or Bax (negative control) for 24 h. Expression levels of Bcl-2, Bcl-xL and Lamin B (loading control) were analysed by Western blot analysis.


( c - e ) Inhibition of etoposide - induced autophagy in Bax / Bak − / − cells treated with siRNA for Bcl - x . ( c ) Bax / Bak − / − MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h , then examined by phase - contrast microscopy .

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(c-e) Inhibition of etoposide-induced autophagy in Bax/Bak−/− cells treated with siRNA for Bcl-x. (c) Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h, then examined by phase-contrast microscopy.


( d ) Bax / Bak − / − MEFs that were transfected with siRNA for Bcl - x and then treated with etoposide for 18 h were analysed by electron microscopy ( × 8 , 500 )

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(d) Bax/Bak−/− MEFs that were transfected with siRNA for Bcl-x and then treated with etoposide for 18 h were analysed by electron microscopy (×8,500)


( e ) Reduction of punctate GFP - LC3 fluorescence in etoposide - treated Bax / Bak − / − MEFs by silencing of Bcl - x . Bax / Bak − / − MEFs that were transfected with both GFP - LC3 and the indicated siRNAs were incubated without ( NT ) or with etoposide for 24 h , and then were examined by confocal fluorescent microscopy . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . n = 4 .

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(e) Reduction of punctate GFP-LC3 fluorescence in etoposide-treated Bax/Bak−/− MEFs by silencing of Bcl-x. Bax/Bak−/− MEFs that were transfected with both GFP-LC3 and the indicated siRNAs were incubated without (NT) or with etoposide for 24 h, and then were examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. n = 4.


f ) Inhibition of etoposide - induced cell death by silencing of Bcl - x . Bax / Bak − / − MEFs were transfected with siRNA ( circles , siRNA for Bax ; squares , siRNA for Bcl - x ) were treated with 20 μM etoposide in the absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA , and cell viability was assessed by the CTB assay at the indicated times . n = 4 .

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f) Inhibition of etoposide-induced cell death by silencing of Bcl-x. Bax/Bak−/− MEFs were transfected with siRNA (circles, siRNA for Bax; squares, siRNA for Bcl-x) were treated with 20 μM etoposide in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA, and cell viability was assessed by the CTB assay at the indicated times. n = 4.


( g ) Clonogenicity assay of Bax / Bak − / − MEFs after exposure to etoposide and staurosporine . Bax / Bak − / − MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h . After collection , 2 , 000 cells were seeded in the normal medium . After one week , colonies were counted .

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(g) Clonogenicity assay of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine. Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h. After collection, 2,000 cells were seeded in the normal medium. After one week, colonies were counted.


h , i ) Inhibition of APG5 - APG12 induction by silencing of Bcl - x or Beclin 1 . Bax / Bak − / − MEFs were treated with the indicated siRNAs ( 10 μg ) for 24 h and then incubated with 20 μM etoposide for the indicated times . Expression of APG5 - APG12 complex , Beclin 1 and GAPDH / VDAC ( loading control ) was analysed by Western blot analysis .

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h, i) Inhibition of APG5-APG12 induction by silencing of Bcl-x or Beclin 1. Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex, Beclin 1 and GAPDH/VDAC (loading control) was analysed by Western blot analysis.


Figure 1 . Ret - eGFP is Expressed in Multiple Sensory Neuron Subsets ( A − L ) Ret - positive neurons largely overlap with markers for myelinated neurons ( NF200 ) , non - peptidergic nociceptors ( IB4 ) and C - fiber low - threshold mechanoreceptors ( TH ) . However some Ret positive neurons are negative for these markers ( indicated by the arrows in D , H and L ) suggesting the existence of a further subset of Ret + neurons . ( M ) Quantification of the proportion of Ret - eGFP positive neurons in DRG . ( N ) Quantification of Ret - eGFP co - expression with other markers . ( n = 2278 cells from 3 mice ) Scale bars , 50 μm .

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Figure 1. Ret-eGFP is Expressed in Multiple Sensory Neuron Subsets(A−L) Ret-positive neurons largely overlap with markers for myelinated neurons (NF200), non-peptidergic nociceptors (IB4) and C-fiber low-threshold mechanoreceptors (TH). However some Ret positive neurons are negative for these markers (indicated by the arrows in D,H and L) suggesting the existence of a further subset of Ret+ neurons. (M) Quantification of the proportion of Ret-eGFP positive neurons in DRG . (N) Quantification of Ret-eGFP co-expression with other markers. (n=2278 cells from 3 mice) Scale bars, 50 μm.


( A ) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret - eGFP expression and IB4 binding ( also indicated in Figure EV1 ) . There are 5 well - defined subsets , 2 of which do not bind to IB4 but display a differential level of eGFP intensity ( termed Ret - eGFPLo : IB4Neg , A , and Ret - eGFPHi : IB4Neg , B , respectively ) . The other 3 subsets bind IB4 and display a range of eGFP intensities , termed Ret - eGFPLo : IB4Lo , C , Ret - eGFPHi : IB4Lo , D , and Ret - eGFPHi : IB4Hi , E . Percent of total events are indicated in each box .

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(A) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret-eGFP expression and IB4 binding (also indicated in Figure EV1). There are 5 well-defined subsets, 2 of which do not bind to IB4 but display a differential level of eGFP intensity (termed Ret-eGFPLo:IB4Neg, A, and Ret-eGFPHi:IB4Neg ,B, respectively). The other 3 subsets bind IB4 and display a range of eGFP intensities, termed Ret-eGFPLo:IB4Lo, C, Ret-eGFPHi:IB4Lo, D, and Ret-eGFPHi:IB4Hi, E. Percent of total events are indicated in each box.


( B ) No correlation between the median cell size of different Ret + subsets and eGFP intensity or IB4 binding . The graph shows the forward scatter values plotted against the normalized number of cells , displayed as the percent of Max . ( C − D ) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy .

(B) No correlation between the median cell size of different Ret+ subsets and eGFP intensity or IB4 binding. The graph shows the forward scatter values plotted against the normalized number of cells, displayed as the percent of Max. (C−D) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy.


( C ) Cultured neurons and ( D ) DRG section from Avil - Cre : : Ret + / eGFPmice displaying native eGFPfluorescence and stained with IB4 . Different levels of eGFP intensity ( high - indicated by arrows and low - indicated by arrowheads ) are detected across IB4 + and IB4 - cells . Scale bar , 50 μm .

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(C) Cultured neurons and (D) DRG section from Avil-Cre::Ret+/eGFPmice displaying native eGFPfluorescence and stained with IB4. Different levels of eGFP intensity (high -indicated by arrows and low -indicated by arrowheads) are detected across IB4+ and IB4- cells. Scale bar, 50 μm.


( A ) Ret - eGFPHi : IB4Neg and Ret - eGFPLo : IB4Negneurons display distinct expression profiles . A volcano plot of fold change expression in Ret - eGFPLo : IB4Neg versus Ret - eGFPHi : IB4Neg against probability . Ret has higher expression in the Ret - eGFPHi : IB4Neg subset , which also shows an up regulation of the Ret co - receptor Gfra2 and Fam38b , encoding for the mechanosensitive ion channel Piezo2 . The Ret - eGFPLo : IB4Neg subset , displays an array of molecules previously associated with itch ( marked in red ) .

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(A) Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Negneurons display distinct expression profiles. A volcano plot of fold change expression in Ret-eGFPLo:IB4Neg versus Ret-eGFPHi:IB4Neg against probability. Ret has higher expression in the Ret-eGFPHi:IB4Neg subset, which also shows an up regulation of the Ret co-receptor Gfra2 and Fam38b, encoding for the mechanosensitive ion channel Piezo2. The Ret-eGFPLo:IB4Neg subset, displays an array of molecules previously associated with itch (marked in red).


( B ) Itch - associated transcripts are enriched in the Ret - eGFPLo : IB4Neg population compared to all DRGneurons . Volcano plot of fold change expression in Ret - eGFPLo : IB4Negneurons versus sorted neurons from Avil - Cre : : R26tdRFPmice against probability . Molecules linked to itch perception are significantly upregulated in the Ret - eGFPLo : IB4Neg subset .

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(B) Itch-associated transcripts are enriched in the Ret-eGFPLo:IB4Neg population compared to all DRGneurons. Volcano plot of fold change expression in Ret-eGFPLo:IB4Negneurons versus sorted neurons from Avil-Cre::R26tdRFPmice against probability. Molecules linked to itch perception are significantly upregulated in the Ret-eGFPLo:IB4Neg subset.


( C ) Gene expression within the Ret - eGFPLo : IB4Neg population is confirmed by triple comparison between Ret - eGFPLo : IB4Neg , Ret - eGFPHi : IB4Neg and Avil - Cre : : R26tdRFPdatasets .

(C) Gene expression within the Ret-eGFPLo:IB4Neg population is confirmed by triple comparison between Ret-eGFPLo:IB4Neg, Ret-eGFPHi:IB4Neg and Avil-Cre::R26tdRFPdatasets.


( D ) Validation of differential microarray screening by quantitative RT - PCR . Transcripts encoding ion channels , receptors , neuropeptides , signaling molecules and transcription factors were selected for quantitative RT - PCR . Differential expression between Ret - eGFPLo : IB4Neg and Ret - eGFPHi : IB4Neg populations correlates with microarray analysis ( n = 3 ) .

(D) Validation of differential microarray screening by quantitative RT-PCR. Transcripts encoding ion channels, receptors, neuropeptides, signaling molecules and transcription factors were selected for quantitative RT-PCR. Differential expression between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations correlates with microarray analysis (n=3).


( A − H ) SstCre mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co - express NF200 or TH . Triple immunostaining of DRG from Sst - Cre : : ReteGFP / + mice with RetGFP ( A ) , IB4 ( B ) and NF200 ( C ) , and RetGFP ( E ) , IB4 ( F ) and TH ( G ) .

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(A−H) SstCre mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co-express NF200 or TH. Triple immunostaining of DRG from Sst-Cre::ReteGFP/+mice with RetGFP (A), IB4 (B) and NF200 (C), and RetGFP (E), IB4 (F) and TH (G).


( I ) Quantification of SstCre : : Ret - eGFP expression in DRG . ( J ) Quantification of co - expression of SstCre : : Ret - eGFP with neuronal markers ( n = 8827 cells from 3 mice ) .

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(I) Quantification of SstCre::Ret-eGFP expression in DRG. (J) Quantification of co-expression of SstCre::Ret-eGFP with neuronal markers (n=8827 cells from 3 mice).


( K − M ) Peripheral projections of sensory neurons from Sst - Cre : : Rosa26SnapCaaXmice terminate in the hairy skin as free nerve endings . Non - innervated hair follicles are indicated by arrows . Double labelling using TMRStar to label SNAP - tag ( K ) and DAPI ( L ) . Scale bars , 50 μm .

(K−M) Peripheral projections of sensory neurons from Sst-Cre::Rosa26SnapCaaXmice terminate in the hairy skin as free nerve endings. Non-innervated hair follicles are indicated by arrows. Double labelling using TMRStar to label SNAP-tag (K) and DAPI (L). Scale bars, 50 μm.


( A - B ) Contour diagram of dissociated sensory neurons from Sst - Cre : : ReteGFP / + ( A ) and Avil - Cre : : ReteGFP / + neurons ( B ) , plotted according to their native eGFP expression and IB4 binding . Only one subset of Ret - eGFP cells can be defined within the Sst - Cre : : ReteGFP / + dissociated DRGneurons . This displays the same range of eGFPfluorescence as the Ret - eGFPLo : IB4Neg population and doesn ' t bind to IB4 .

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(A-B) Contour diagram of dissociated sensory neurons from Sst-Cre::ReteGFP/+ (A) and Avil-Cre::ReteGFP/+neurons (B), plotted according to their native eGFP expression and IB4 binding. Only one subset of Ret-eGFP cells can be defined within the Sst-Cre::ReteGFP/+ dissociated DRGneurons. This displays the same range of eGFPfluorescence as the Ret-eGFPLo:IB4Neg population and doesn't bind to IB4.


( C ) Quantitative RT - PCR analysis of selected transcripts in the Sst - Cre : : ReteGFP ( darker colors ) and Ret - eGFPHi : IB4Neg ( lighter colors ) subsets displayed as differential expression compared to the Ret - eGFPHi : IB4Neg subset . Almost all the genes that are up - regulated in the Ret - eGFPLo : IB4Neg compared to the Ret - eGFPHi : IB4Neg subset are also enriched in the Sst - Cre : : ReteGFP population ( n = 3 )

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(C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)


( A - G ) Ablation of Sst - Cre : : AviliDTR positive sensory neurons by injection of diphtheria toxin ( DTX ) . Immunostaining of DRG from Sst - Cre : : AviliDTRmice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin ( A - C ) and after systemic injection of diphtheria toxin ( D - F ) .

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(A-G) Ablation of Sst-Cre::AviliDTR positive sensory neurons by injection of diphtheria toxin (DTX). Immunostaining of DRG from Sst-Cre::AviliDTRmice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin (A-C) and after systemic injection of diphtheria toxin (D-F).


( G ) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin .

(G) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin.


( H - K ) Examples of calcium flux in dissociated DRG upon application of histamine ( H ) , chloroquine ( I ) , IL -

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(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31


( H - K ) Examples of calcium flux in dissociated DRG upon application of histamine ( H ) , chloroquine ( I ) , IL - 31 ( J ) and 5HT1F agonist Ly344864 ( K ) .

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(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (J) and 5HT1F agonist Ly344864 (K).


( L ) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst - Cre : : AviliDTRmice . Scale bars , 50 μm , asterisk denotes p < 0 . 05 , n = 4 - 6 mice , Student ' s t - test , error bars indicate SEM .

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(L) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst-Cre::AviliDTRmice. Scale bars, 50 μm, asterisk denotes p<0.05, n=4-6 mice, Student's t-test, error bars indicate SEM.


( A - B ) Nociceptive withdrawal thresholds in control ( AviliDTR without the Cre ) and Sst - Cre : : AviliDTRmice after systemic diphtheria toxin injection . No significant differences in thermal ( A ) or mechanical ( B ) withdrawal reflexes after ablation of SstCre positive neurons .

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(A-B) Nociceptive withdrawal thresholds in control (AviliDTR without the Cre) and Sst-Cre::AviliDTRmice after systemic diphtheria toxin injection. No significant differences in thermal (A) or mechanical (B) withdrawal reflexes after ablation of SstCre positive neurons.


( C - F ) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour . All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control ( AviliDTR ) or Sst - Cre : : AviliDTRmice . Diphtheria toxin injection in control mice ( AviliDTR ) had no effect on scratching behavior evoked by histamine ( C ) , chloroquine ( D ) , IL - 31 ( E ) or 5HT1F agonist Ly344864 ( F ) but significantly reduced responses to IL31 , or Ly344864 in Sst - Cre : : AviliDTRmice . n - numbers indicated in brackets , asterisk denotes p < 0 . 05 , two way RM ANOVA , Holm - Sidak multiple comparison . Error bars indicate SEM .

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(C-F) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour. All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control (AviliDTR) or Sst-Cre::AviliDTRmice. Diphtheria toxin injection in control mice (AviliDTR) had no effect on scratching behavior evoked by histamine (C), chloroquine (D), IL-31 (E) or 5HT1F agonist Ly344864 (F) but significantly reduced responses to IL31, or Ly344864 in Sst-Cre::AviliDTRmice. n-numbers indicated in brackets, asterisk denotes p<0.05, two way RM ANOVA, Holm-Sidak multiple comparison. Error bars indicate SEM.


A ) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc . At 3 days post - infection , cells prestained with MitoTracker ( Mito , red ) were immunostained with anti - Parkin ( green ) and HCV core ( orange ) antibodies . Nuclei were stained with DAPI ( blue ) . Infected ( + ) and uninfected cells ( − ) are marked . The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells ( gray ) . In the zoomed images , the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV - infected cells ( yellow ) .

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A) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc. At 3 days post-infection, cells prestained with MitoTracker (Mito, red) were immunostained with anti-Parkin (green) and HCV core (orange) antibodies. Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked. The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells (gray). In the zoomed images, the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV-infected cells (yellow).


( B ) Ultrastructure of HCV - infected cells showing perinuclear clustering of damaged mitochondria . Control naïve Huh7 cells ( left ) and stable cells harboring HCV full - length replicon FLR - JFH1 ( right ) were examined by electron microscopy . In the zoomed images , typical ultrastructure of mitochondria in naïve cells and ultrastructural abnormalities of mitochondria in HCV replicon cells are shown . Organelle mark : N , nucleus ; M and white arrow , mitochondria . Scale bar = 1 µM . Fluorescent images ( below ) indicate the expression of HCV core protein in HCV replicon cells . Cells were immunostained with anti - HCV core antibody ( red ) . Nuclei were stained with DAPI ( blue ) .

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(B) Ultrastructure of HCV-infected cells showing perinuclear clustering of damaged mitochondria. Control naïve Huh7 cells (left) and stable cells harboring HCV full-length replicon FLR-JFH1 (right) were examined by electron microscopy. In the zoomed images, typical ultrastructure of mitochondria in naïve cells and ultrastructural abnormalities of mitochondria in HCV replicon cells are shown. Organelle mark: N, nucleus; M and white arrow, mitochondria. Scale bar = 1 µM. Fluorescent images (below) indicate the expression of HCV core protein in HCV replicon cells. Cells were immunostained with anti-HCV core antibody (red). Nuclei were stained with DAPI (blue).


( C ) Quantification of the number of mitochondria in the perinuclear region ( mean ± SEM ; n ≥ 5 cells ; * p < 0 . 05 ) .

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(C) Quantification of the number of mitochondria in the perinuclear region (mean ± SEM; n≥5 cells; *p<0.05).


( D ) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP - treated ( see Figure S1 ) or HCV - infected cells ( A ) and those treated with 3 - MA or BafA1 , respectively ( see Figure 4A ) ( mean ± SEM ; n = 10 cells ; * p < 0 . 01 ) . P values were calculated by using an unpaired Student ' s t - test .

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(D) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP-treated (see Figure S1) or HCV-infected cells (A) and those treated with 3-MA or BafA1, respectively (see Figure 4A) (mean ± SEM; n = 10 cells; *p<0.01). P values were calculated by using an unpaired Student's t-test.


( E ) Western blot analysis showing endogenous Parkin recruitment to mitochondria in HCV - infected cells . Huh7 cells were infected with HCVcc . At 5 days post - infection , pure cytoplasm and mitochondria fractions were isolated by ultracentrifugation as described in Materials and Methods . Cellular fractions of HCV - infected cells were analyzed by immunoblotting using antibodies specific for the indicated proteins . Fractions : whole cell lysates , WCL ; purified cytoplasm , pCyto ; purified mitochondria , pMito . Organelle markers : VDAC1 , mitochondria ; Calreticulin , ER ; GAPDH , cytoplasm .

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(E) Western blot analysis showing endogenous Parkin recruitment to mitochondria in HCV-infected cells. Huh7 cells were infected with HCVcc. At 5 days post-infection, pure cytoplasm and mitochondria fractions were isolated by ultracentrifugation as described in Materials and Methods. Cellular fractions of HCV-infected cells were analyzed by immunoblotting using antibodies specific for the indicated proteins. Fractions: whole cell lysates, WCL; purified cytoplasm, pCyto; purified mitochondria, pMito. Organelle markers: VDAC1, mitochondria; Calreticulin, ER; GAPDH, cytoplasm.


( A ) Parkin ubiquitination in the purified mitochondria isolated from HCV - infected cells . Parkin protein in pCyto and pMito fractions , respectively , was immunoprecipitated by anti - Parkin antibody , followed by immunoblotting with anti - ubiquitin ( Ub ) antibody . Normal rabbit IgG was used as a negative control for immunoprecipitation ( IP ) .

(A) Parkin ubiquitination in the purified mitochondria isolated from HCV-infected cells. Parkin protein in pCyto and pMito fractions, respectively, was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a negative control for immunoprecipitation (IP).


( B , E , and F ) At the indicated time points post - infection , WCL were extracted from HCV - infected Huh7 cells . ( B ) Parkin ubiquitination in WCL extracted from HCV - infected cells . All ubiquitinated proteins were immunoprecipitated with anti - Ub antibody and analyzed by immunoblotting with anti - Parkin antibody . HCV infection was verified by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control .

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(B, E, and F) At the indicated time points post-infection, WCL were extracted from HCV-infected Huh7 cells. (B) Parkin ubiquitination in WCL extracted from HCV-infected cells. All ubiquitinated proteins were immunoprecipitated with anti-Ub antibody and analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( C ) Confocal microscopy showing Parkin ubiquitination in HCV - infected cells . HCV - infected cells prestained with MitoTracker ( Mito ) were immunostained with anti - Parkin and ubiquitin ( Ub ) antibodies . In the zoomed images , the arrows ( white spots ) indicate the merge of Mito ( green ) , Parkin ( red ) , and Ub ( blue ) . Nuclei are demarcated with white dot circles . Fluorescence image of HCV E2 verifies HCV - infected cells ( light gray ) .

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(C) Confocal microscopy showing Parkin ubiquitination in HCV-infected cells. HCV-infected cells prestained with MitoTracker (Mito) were immunostained with anti-Parkin and ubiquitin (Ub) antibodies. In the zoomed images, the arrows (white spots) indicate the merge of Mito (green), Parkin (red), and Ub (blue). Nuclei are demarcated with white dot circles. Fluorescence image of HCV E2 verifies HCV-infected cells (light gray).


( D ) Quantitative analysis of the ubiquitination of endogenous Parkin colocalized with mitochondria in the panels ( C ) ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 05 ) .

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(D) Quantitative analysis of the ubiquitination of endogenous Parkin colocalized with mitochondria in the panels (C) (mean ± SEM; n≥10 cells; *p<0.05).


( E ) Western blot analyses of Mfn2 and VDAC1 expression , mitochondrial substrate of Parkin , in HCV - infected cells . Intracellular HCV RNA levels were analyzed by real - time qRT - PCR , as described in Materials and Methods ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . WCL extracted from HCV - infected cells were analyzed by immunoblotting with anti - Parkin , Mfn2 , and VDAC1 antibodies , respectively . HCV infection was verified by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control .

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(E) Western blot analyses of Mfn2 and VDAC1 expression, mitochondrial substrate of Parkin, in HCV-infected cells. Intracellular HCV RNA levels were analyzed by real-time qRT-PCR, as described in Materials and Methods (mean ± SD; n = 3; *p<0.01). WCL extracted from HCV-infected cells were analyzed by immunoblotting with anti-Parkin, Mfn2, and VDAC1 antibodies, respectively. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( F ) Western blot analysis of p62 expression , the autophagy - associated factor , in HCV - infected cells . WCL extracted from HCV - infected cells were analyzed by immunoblotting with anti - p62 antibody . β - actin was used as an internal loading control . ( E and F ) The relative intensity of Mfn2 , VDAC1 , and p62 expression normalized to β - actin was analyzed by ImageJ .

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(F) Western blot analysis of p62 expression, the autophagy-associated factor, in HCV-infected cells. WCL extracted from HCV-infected cells were analyzed by immunoblotting with anti-p62 antibody. β-actin was used as an internal loading control. (E and F) The relative intensity of Mfn2, VDAC1, and p62 expression normalized to β-actin was analyzed by ImageJ.


( G ) Parkin - mediated ubiquitination of Mfn2 and VDAC1 in HCV - infected cells . The ubiquitinated Mfn2 and VDAC1 proteins were analyzed by immunoprecipitation with anti - Mfn2 and VDAC1 antibodies , respectively , followed by immunoblotting with anti - Ub antibody . The protein expression levels of Parkin were analyzed by immunoblotting with anti - Parkin antibody . HCV infection was verified by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control . Normal mouse IgG was used as a negative control for immunoprecipitation ( IP ) . ( D and E ) P values were calculated by using an unpaired Student ' s t - test .

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(G) Parkin-mediated ubiquitination of Mfn2 and VDAC1 in HCV-infected cells. The ubiquitinated Mfn2 and VDAC1 proteins were analyzed by immunoprecipitation with anti-Mfn2 and VDAC1 antibodies, respectively, followed by immunoblotting with anti-Ub antibody. The protein expression levels of Parkin were analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control. Normal mouse IgG was used as a negative control for immunoprecipitation (IP). (D and E) P values were calculated by using an unpaired Student's t-test.


( A and B ) Quantitative analyses of ATF4 , Parkin , and PINK1 gene expression in HCV - infected cells . Huh7 cells infected with HCVcc for 5 days or treated with CCCP ( 10 or 20 µM ) for 24 h were used for analysis of ATF4 , Parkin , and PINK1 gene expression . ( A ) Intracellular mRNA levels of ATF4 , Parkin , and PINK1 were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in Parkin and PINK1 gene expression ( mean ± SD ; n = 3 ; * p < 0 . 01 , * * p < 0 . 05 ) . P values were calculated by using an unpaired Student ' s t - test .

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(A and B) Quantitative analyses of ATF4, Parkin, and PINK1 gene expression in HCV-infected cells. Huh7 cells infected with HCVcc for 5 days or treated with CCCP (10 or 20 µM) for 24 h were used for analysis of ATF4, Parkin, and PINK1 gene expression. (A) Intracellular mRNA levels of ATF4, Parkin, and PINK1 were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin and PINK1 gene expression (mean ± SD; n = 3; *p<0.01, **p<0.05). P values were calculated by using an unpaired Student's t-test.


B ) The protein expression levels of Parkin and PINK1 were analyzed by immunoblotting with anti - Parkin and PINK1 antibodies . The expression of HCV core protein was analyzed by immunoblotting with anti - HCV core antibody . β - actin was used as an internal loading control .

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B) The protein expression levels of Parkin and PINK1 were analyzed by immunoblotting with anti-Parkin and PINK1 antibodies. The expression of HCV core protein was analyzed by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( C ) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients . Samples 1 and 2 represent liver biopsies of HCV - negative patients and samples 3 - 7 represent HCV - positive chronic hepatitis patients . ( B and C ) The relative intensity of Parkin and PINK1 expression normalized to β - actin analyzed by ImageJ .

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(C) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients. Samples 1 and 2 represent liver biopsies of HCV-negative patients and samples 3-7 represent HCV-positive chronic hepatitis patients. (B and C) The relative intensity of Parkin and PINK1 expression normalized to β-actin analyzed by ImageJ.


( D ) Confocal microscopy showing Parkin overexpression in HCV - infected cells . Huh7 cells infected with HCVcc were immunostained with anti - Parkin antibody ( red ) . Fluorescence image of HCV E2 verifies HCV - infected cells ( light gray ) . Nuclei were stained with DAPI ( blue ) . Infected ( + ) and uninfected cells ( − ) are marked .

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(D) Confocal microscopy showing Parkin overexpression in HCV-infected cells. Huh7 cells infected with HCVcc were immunostained with anti-Parkin antibody (red). Fluorescence image of HCV E2 verifies HCV-infected cells (light gray). Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked.


Confocal microscopy showing mitophagosome formation associated with Parkin in HCV - infected cells . ( A ) Huh7 cells transiently expressing GFP - LC3 protein were infected with HCVcc in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 12 h before fixation . At 3 day post - infection , cells were immunostained with antibodies against HCV E2 ( orange ) , TOM20 ( blue ) , and Parkin ( red ) . In the zoom images , the arrows ( white puncta ) indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and Parkin . Nuclei are demarcated with white dot circles . (

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Confocal microscopy showing mitophagosome formation associated with Parkin in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 day post-infection, cells were immunostained with antibodies against HCV E2 (orange), TOM20 (blue), and Parkin (red). In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. Nuclei are demarcated with white dot circles. (


( B ) Quantification of the number of GFP - LC3 puncta colocalized with TOM20 in the panel ( A ) ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 01 ) .

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(B) Quantification of the number of GFP-LC3 puncta colocalized with TOM20 in the panel (A) (mean ± SEM; n≥10 cells; *p<0.01).


( C ) Quantitative analysis of the area of GFP - LC3 puncta ( white ) representing merge of GFP - LC3 puncta , TOM20 , and Parkin in the panel ( A ) ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 001 ) . ( B and C ) P values were calculated by using an unpaired Student ' s t - test .

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(C) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A) (mean ± SEM; n≥10 cells; *p<0.001). (B and C) P values were calculated by using an unpaired Student's t-test.