( F ) Representative immunoblot of Grk5 in fractions from left ventricle lysates of WT Cand2 KO mice after TAC surgery . Lamin B - marker , Gapdh - marker . Analyzed by One - way ANOVA , n = 3 biological replicates . Data information : Error bars indicate means ± . * - P ≤ 0 . 05 , * * * - P ≤ 0 . 001 .

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

(F) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery. Lamin B - nuclear marker, Gapdh - cytoplasmic marker. Analyzed by One-way ANOVA, n=3 biological replicates. Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.


( B ) mRNA of MEF2 - dependent genes Nr4a1 Xirp2 in NRCMs with Cand2 KD for indicated groups . PE treatment for 24h ( 50uM ) . Analyzed by One - way ANOVA , n = 6 biological replicates . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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

(B) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups. PE treatment for 24h (50uM). Analyzed by One-way ANOVA, n=6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( E ) Grk5 mRNA in NRCMs as well as MEF2 - luciferase activity after 24h of Torin 1 treatment . Analyzed by t - , n = 4 - 6 biological replicates per conditions . Torin1 activity measured by of phosphorylated ribosomal S6 ( S235 / 236rpS6 ) . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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

(E) Grk5 protein and mRNA levels in NRCMs as well as MEF2-luciferase activity after 24h of Torin 1 treatment. Analyzed by t-test, n=4-6 biological replicates per conditions. Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein (S235/236rpS6). Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( I ) Grk5 , Nppa , MEF2 targets Nr4a1 Xirp2 mRNA in NRCMs after Raptor KD . Analyzed by t - , n = 3 biological replicates per conditions . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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

(I) Grk5, Nppa, and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD. Analyzed by t-test, n=3 biological replicates per conditions. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( B ) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2 . NRCMs were treated with MLN4924 ( 100 nM ) to block Cul1 neddylation . of neddylated unnedylated Cul1 after MLN4924 treatment for 24h . t - , n = 4 biological replicates . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

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

(B) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2. NRCMs were treated with MLN4924 (100 nM) to block Cul1 neddylation. Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h. t-test, n=4 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( D ) Grk5 expression in NRCMs upon Cand2 KD Cul1 neddylation inhibition ( MLN ) analysed by immunoblot RT - qPCR . Analyzed by One - way ANOVA , n = 4 - 5 biological replicates . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition (MLN) analysed by immunoblot and RT-qPCR. Analyzed by One-way ANOVA, n=4-5 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( H ) mRNA of MEF2 - dependent genes Nr4a1 Xirp2 in NRCMs for indicated groups . Analyzed by One - way ANOVA , n = 6 biological replicates . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 , * * * * - P ≤ 0 .

O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA, n= 6 biological replicates. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001


( A ) of indicated mRNA 2 days after TAC measured by RT - qPCR . Analyzed by One - way ANOVA . n = 5 - 7 biological replicates per group . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of indicated mRNA levels 2 days after TAC measured by RT-qPCR. Analyzed by One-way ANOVA. n=5-7 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


( C ) Grk5 in vivo in LVs of WT Cand2 KO mice 2 days after sham TAC analyzed by immunoblotting as well as of mRNA analyzed by RT - qPCR . Analyzed by One - way ANOVA . n = 3 - 5 biological replicates per group . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

O O O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT-qPCR. Analyzed by One-way ANOVA. n=3-5 biological replicates per group. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


( D ) Representative immunoblot its of of neddylated unnedylated Cul1 in Cand2 KO mice compared to WT mice . Analyzed by t - . n = 5 - 8 mice per group . Cul1 neddylation was inhibited in C2C12 by MLN4924 ( - / + MLN ) for neddylated Cul1 band detection . Data information : Error bars indicate means ± ; * - P ≤ 0 . 05 , * * - P ≤ 0 . 01 , * * * - P ≤ 0 . 001 .

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

(D) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice. Analyzed by t-test. n=5-8 mice per group. Cul1 neddylation was inhibited in C2C12 by MLN4924 (-/+ MLN) for neddylated Cul1 band detection. Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.


Macrophage - conditioned medium induces MCF10A colony formation in soft agar as compared to control medium . Colonies ≥ 50 μm were counted at 5 . Error bars represent mean ± from 10 independent experiments ( n = 3 per condition ) . Macrophage donors are indicated as D1 - D16 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

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

Macrophage-conditioned medium induces MCF10A colony formation in soft agar as compared to control medium. Colonies ≥ 50 μm were counted at 5 weeks. Error bars represent mean ± SEM from 10 independent experiments (n=3 per condition). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.


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

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

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


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

O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O

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


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

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

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


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

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

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


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

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

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


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

O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

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


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

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

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


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

O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O

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


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

B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O

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


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

B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O

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


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

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

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


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

B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O

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


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

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

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


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

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

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


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

B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O 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-ORGANISM B-DISEASE I-DISEASE O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-ORGANISM B-DISEASE I-DISEASE O O O O

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


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

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

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


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

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

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


16 - days old MCF10A spheroids grown in matrigel / collagen mix were stimulated for 24h with either macrophage - conditioned or control medium in presence of amlexanox . Representative showing filling of spheroid lumen with nuclei . spheroids were stained for ( Hoechst 33342 in blue ) 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 B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 spheroid lumen with nuclei . Filling of spheroid lumen with nuclei categorized into 4 groups ( clear , partially filled , almost filled filled ) . Error bars represent mean ± from 7 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled filled spheroids were combined together ( non - empty spheroids ) for statistical . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-TISSUE O B-SUBCELLULAR O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 spheroid lumen with nuclei . Filling of spheroid lumen with nuclei . Partially filled , almost filled filled spheroids were combined together ( non - empty spheroids ) . Error bars represent mean ± from 7 independent experiments ( n = 2 per condition ; 50 spheroids each ) . Partially filled , almost filled filled spheroids were combined together ( non - empty spheroids ) for statistical . Macrophage donors are indicated as D1 - D25 . M1D - M1 differentiated , M1A - M1 activated , M2D - M2 differentiated , M2A - M2 activated macrophages .

O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-TISSUE O B-SUBCELLULAR O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 number of spheroids with invasive protrusions . Error bars represent mean ± 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 B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 old C57Bl / 6 mice on normal ( ND ) Organoids isolated from mice on ND were cultured in matrigel / collagen mix for 2 days then stimulated with macrophage - conditioned or control medium in presence of amlexanox . Amlexanox reduces number of invasive organoids . Error bars represent mean ± 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 .

B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 old C57Bl / 6 mice on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were cultured in collagen for 2 days after which number of invasive protrusions per each organoid was determined microscopically . Amlexanox reduces 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 ) .

B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 luminal isolated from breast specimens were cultured in collagen gels for 14 days to reform structures with luminal compartment . Reformed were then cultured for 7 days in macrophage - conditioned or control medium in presence of amlexanox . Representative showing filling of lumen with nuclei . were stained for ( DAPI in blue ) F - actin ( Phalloidin in red ) . Scale bar : 20 μm . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O B-SUBCELLULAR O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 luminal isolated from breast specimens were cultured in collagen gels for 14 days to reform structures with luminal compartment . Reformed were then cultured for 7 days in macrophage - conditioned or control medium in presence of amlexanox . Amlexanox inhibits filling of lumen with nuclei . Error bars represent mean ± ( A total of 24 structures was counted per each condition : 2 structures ( n = 2 ) , each labelled with a different symbol shape , were cultured with macrophage - conditioned media from either donor 17 or donor 18 , 3 each ) . Filling of lumen was determined as % of luminal space filled with . Macrophage donors are indicated as M1A - M1 activated , M2 activated macrophages .

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 via CRISPR - Cas9 technology . 3 knockout clones were combined for each . IKKε CRISPR - Cas9 knockout MCF10A form less colonies when grown in soft agar with macrophage - conditioned medium for 5 compared to CRISPR - Cas9 control ( nontargeting crRNA ) . Colonies ≥ 50 μm were counted . Error bars represent mean ± from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 via CRISPR - Cas9 technology . 3 knockout clones were combined for each . 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 are less invasive compared CRISPR Control ( nontargeting crRNA ) . Error bars represent mean ± from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 ; " 1 " : weak ; " 2 " : moderate ; " 3 " : strong ) on human breast cancer sections using immunohistochemistry . Representative according to PSAT1 grade are shown . Scale bar : 50 μm .

B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O 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-ORGANISM B-DISEASE I-DISEASE O O B-EXP_ASSAY O O O O B-GENEPROD 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 of SBP enzyme genes PSAT1 ( E ) , PHGDH ( F ) , PSPH ( G ) with immune signature ( Yoshihara et al , 2013 ) in METABRIC transcriptomic dataset from 1981 breast cancer ( Curtis et al , 2012 ) .

O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE 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 grown in soft agar with macrophage - conditioned medium for 5 compared to controls . Colonies ≥ 50 μm were counted . Error bars represent mean ± 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-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 showing filling of spheroid lumen with nuclei . spheroids were stained for ( Hoechst 33342 in blue ) 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 B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O B-SUBCELLULAR O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 filling of spheroid lumen with nuclei . Filling of spheroid lumen with nuclei categorized into 4 groups ( clear , partially filled , almost filled 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 B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O B-SUBCELLULAR O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 filling of spheroid lumen with nuclei . Filling of spheroid lumen with nuclei . Partially filled , almost filled 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 B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O B-SUBCELLULAR O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 number of spheroids with invasive protrusions ( n = 2 per condition ) . Error bars represent mean ± 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 B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 old C57Bl / 6 mice that were either on normal ( ND ) or high fat diet ( HFD ) ( n = 3 ) . Organoids were isolated from mice on ND 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 number of invasive organoids . Error bars represent mean ± 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 .

B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 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 number of invasive protrusions per each organoid was determined microscopically . NCT502 ( 10 μM ) reduces 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 ) .

B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 luminal isolated from breast specimens were cultured in collagen gels for 14 days to reform structures with luminal compartment . Reformed were then cultured for 7 days in macrophage - conditioned or control medium in presence of NCT502 ( 10μM ) . Representative showing filling of lumen with nuclei . were stained for ( DAPI in blue ) F - actin ( Phalloidin in red ) . Scale bar : 20 μm . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

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

B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-TISSUE O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 via CRISPR - Cas9 technology . 3 knockout clones were combined for each . PHGDH CRISPR - Cas9 knockout MCF10A form less colonies when grown in soft agar with macrophage - conditioned medium for 5 compared to CRISPR - Cas9 control ( nontargeting crRNA ) . Colonies ≥ 50 μm were counted . Error bars represent mean ± from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 in each experiment . Macrophage donors are indicated as , M1A - M1 activated , M2A - M2 activated macrophages .

B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 via CRISPR - Cas9 technology . 3 knockout clones were combined for each . 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 are less invasive compared CRISPR Control ( nontargeting crRNA ) . Error bars represent mean ± from 3 independent experiments ( n = 3 per condition ) . Media from two macrophage donors were combined in 1 : 1 in each experiment . Macrophage donors are indicated as M1A - M1 activated , M2A - M2 activated macrophages .

B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 C57Bl / 6 mice heterozygous for PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Wild - type mice on HFD gain more compared to ND mice . * p < 0 . 05 by one - students t - .

O O O O O O O O O O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM 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 C57Bl / 6 mice heterozygous for PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 old ( prior of mammary tumour development ) together with control mice on normal diet ( ND ; n = 10 ) . Average adipocytes ( major diameter ) in mammary fat pads is larger in HFD mice compared to ND mice on of at least 100 adipocytes per mice on H & E stained sections . * p < 0 . 05 by two - students t - .

O O O O O O O O O O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-ORGANISM O O O O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O O O O B-ORGANISM O O O B-ORGANISM O O O O O B-CELL_TYPE O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

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

O O O O O O O O O O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O O O O B-ORGANISM O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

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 C57Bl / 6 mice heterozygous for PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 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 efficacy of diet to control for possible unpredicted interactions of MMTV - PyMT gene . PyMT - MMTV + / - mice on HFD gain more compared to ND mice . * p < 0 . 05 by one - students t - . Amlexanox does not affect gain either in mice on F ) ND or G ) HFD .

O O O O O O O O O O B-ORGANISM O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM 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 C57Bl / 6 mice heterozygous for PyMT - MMTV started high fat diet ( HFD ) when 6 - 7 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 efficacy of diet to control for possible unpredicted interactions of 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 B-ORGANISM O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O B-DISEASE I-DISEASE O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM 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 of Bax / Bak − / − MEFs treated with etoposide . Bax / Bak − / − MEFs were treated with 20 μM etoposide in presence of PI ( 2 μM ) for indicated times , were analysed under a phase - contrast ( PI ) microscope .

O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 ) Bax / Bak − / − ( squares ) MEFs were treated with 20 μM etoposide in presence ( closed symbols ) or absence ( open symbols ) of 100 μM zVAD - fmk . Then viability was measured by PI ( b ) CTB ( c ) , expressed as a of initial value without etoposide . Data are shown as mean ± s . d . ( n = 4 ) .

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

(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 . Bax / Bak − / − MEFs were either not treated ( 0 h ) or were treated with 20 μM etoposide for 12 24 h , then were recovered 5 , 000 were reseeded . Viable numbers were measured on indicated days by CTB . Results were normalized by adjusting day 0 value . n = 4 .

O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 staurosporine , as assessed by clonogenicity . MEFs were treated with etoposide ( 20 μM ) or staurosporine ( 1 μM ) at indicated times , collected , 2 , 000 were seeded in normal medium . After 1 week , colonies were counted . n =

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

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

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

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

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

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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 absence or presence of 10 mM 3 - MA for 24 h , then were examined by phase - contrast microscopy ( a ) electron microscopy ( × 8 , 400 ) ( b ) . Autolysosomes / autophagosomes were hardly observed . (

O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR 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 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 presence or absence of 10 mM 3 - MA for 24 h , then were examined by confocal microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in presence of 3 - MA . ( d ) of with punctate GFP - LC3 was calculated relative to GFP - LC3 - positive . Data are shown as mean ± s . d . ( n = 4 ) .

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

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 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 presence or absence of 10 mM 3 - MA for 24 h , then were examined by confocal microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in presence of 3 - MA . ( d ) of with punctate GFP - LC3 was calculated relative to GFP - LC3 - positive . Data are shown as mean ± s . d . ( n = 4 ) .

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

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

O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 indicated siRNAs ( 10 μg ) for 24 h then incubated with 20 μM etoposide for indicated times . Expression of APG5 - APG12 ( a ) , Beclin 1 ( b ) VDAC ( loading control ) was analysed by Western blot .

O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 were analysed by electron microscopy ( × 8 , 500 ) .

O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 in Bax / Bak − / − MEFs by silencing of Beclin 1 APG5 . Bax / Bak − / − MEFs that were transfected with GFP - LC3 together with indicated siRNAs , were incubated without ( NT ) or with 20 μM etoposide for 24 h , then examined by confocal microscopy . of with punctate GFP - LC3 was calculated relative to GFP - LC3 - positive . Data are shown as mean ± s . d . ( n = 4 ) .

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

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

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

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

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

(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 APG5 - APG12 in Bcl - 2 - transfected MEFs after etoposide treatment . WT MEFs were transfected with indicated plasmids , were incubated without ( NT ) or with 20 μM etoposide for 24 h .

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

(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 in Bcl - xL - overexpressing APG5 + / + MEFs , but not APG5 − / − MEFs . APG5 + / + ( open columns ) APG5 − / − ( closed columns ) MEFs were transfected with indicated plasmids ( 2 μg ) . After 24 h , transfected were incubated with 20 μM of etoposide in presence or absence of 100 μM of zVAD or 10 mM of 3 - MA for 24 h , viability was measured by CTB ( d ) apoptotic ( 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 . WT MEFs with silencing of control GFP ( open columns ) or Beclin 1 ( closed columns ) were transfected with indicated plasmids ( 1 μg ) . After 24 h , transfected were incubated with 20 μM of etoposide in presence or absence of 10 mM of 3 - MA for 24 h , viability was measured by CTB . n = 3 .

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

(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 by overexpression of human Bcl - 2 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 indicated plasmids ( 1 μg ) . After 24 h , transfected were incubated with 20 μM etoposide for 24 h , viability was assessed by CTB . Data are shown as mean ± s . d . ( n = 3 ) .

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

(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 of Bcl - 2 , Bcl - xL Lamin B ( loading control ) were analysed by Western blot .

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

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

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

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

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

(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 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 absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA , viability was assessed by CTB at 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.