( F ) Left panel : Immunoblot analysis of phospho - KIN - 1 substrates in young adult nematodes . Numbers correspond to band densitometries across 2 biological replicates . Right panel : immunoblot analysis of phospho - KIN - 1 substrates in age - 1 ; gas - 1 animals grown on bacteria carrying an empty vector ( EV ) or expressing kin - 1 RNAi . Two different kin - 1 RNAi clones ( g1 , b5 ) were used in this experiment . Numbers correspond to band densitometries of the presented immunoblot . Dashed rectangles : band corresponding to downregulated KIN - 1 phospho - substrate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate.


( G ) Representative lifespan assay of wt and age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and kin - 1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

(G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves.


ATP measurements in : ( H ) age - 1 ( hx546 ) ; gas - 1 ( fc21 ) ( a ; g ) and age - 1 ( hx546 ) ; aak - 2 ( ok524 ) ; gas - 1 ( fc21 ) ( a ; aa ; g ) mutant nematodes ;

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

ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes;


ATP measurements in : ( I ) age - 1 ; gas - 1 ( EV ) ( a ; g ( EV ) ) and age - 1 ; gas - 1 ; kin - 1 ( RNAi ) ( a ; g ; kin - 1 ( RNAi ) ) . Error bars : Mean ± SEM , n = 4 , * p value < 0 . 05 , Mann Whitney U test .

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

ATP measurements in: (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test.


( J ) Left panel : curves represent FRAP in age - 1 ; gas - 1 animals grown on RNAi from larval L1 ( or from egg for fzo - 1 RNAi ) to L4 stage . Each point : mean recovery of bleached regions ( 2 - 5 regions per animal , 10 - 20 animals per condition ) ± SEM . Right panel : Maximum ( total ) recovery per condition are shown , ± SEM . * p value < 0 . 05 , * * p value < 0 . 01 , * * * * p value < 0 . 0001 , Kruskal - Wallis test , Dunn ' s post - hoc correction .

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

(J) Left panel: curves represent FRAP in age-1; gas-1 animals grown on RNAi from larval L1 (or from egg for fzo-1 RNAi) to L4 stage. Each point: mean recovery of bleached regions (2-5 regions per animal, 10-20 animals per condition) ± SEM. Right panel: Maximum (total) recovery per condition are shown, ± SEM. *p value< 0.05, **p value< 0.01, ****p value< 0.0001, Kruskal-Wallis test, Dunn's post-hoc correction.


A ) Volcano plot of lipidomic profiling in gas - 1 versus age - 1 ; gas - 1 mutants . Red stars : significantly altered lipids in age - 1 ; gas - 1 versus gas - 1 ( 5 % FDR ; n = 5 ) .

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

A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5).


B ) NMR - 1D targeted metabolic profiling in age - 1 ; gas - 1 mutants ( normalized to gas - 1 ) . Dashed lines indicate fold change cutoff ( | log2 ( fold change ) | < 1 . 23 ) . Red bars : not significant , Light blue bars : FDR < 10 % , | log2 ( fold change ) | < 1 . 23 , Dark blue bars : FDR < 10 % , | log2 ( fold change ) | > 1 . 23 . Error bar : mean + SEM , n = 7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7.


D ) Left panel : qRT - PCR of xdh - 1 ( normalized to wt ) . Error bars : Mean ± SEM , n = 6 . * * * * p value < 0 . 0001 , * p value < 0 . 05 , ns = non - significant , Kruskal - Wallis with Dunn ' s post hoc test . Right : xanthine oxidase activity levels normalized to animal number . Error bars : mean ± SEM , n = 2 , ns = non - significant , Mann Whitney U test .

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

D) Left panel: qRT-PCR of xdh-1 (normalized to wt). Error bars: Mean ± SEM, n=6. ****p value< 0.0001, *p value< 0.05, ns= non-significant, Kruskal-Wallis with Dunn's post hoc test. Right: xanthine oxidase activity levels normalized to animal number. Error bars: mean ± SEM, n=2, ns= non-significant, Mann Whitney U test.


F ) Immunoblot analysis of phospho - Akt ( S473 ) , pan Akt , phospho - PRAS40 ( T246 ) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0 . 2 % DMSO ( - ) or AMP ( 2 μM ) , ENPF ( 2 μM ) and PTX ( 36 μM ) ( + ) for 24 h ( actin loading control ) .

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

F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control).


( G ) Immunoblot analysis of phospho - PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated ( actin loading control ) . For all immunoblot panels : numbers correspond to band densitometries across 2 - 3 biological replicates .

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

(G) Immunoblot analysis of phospho-PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated (actin loading control). For all immunoblot panels: numbers correspond to band densitometries across 2-3 biological replicates.


( A ) Effect of methylxanthines on wt lifespan . Average median lifespans ± SEM from all replicates indicated beneath representative curve .

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

(A) Effect of methylxanthines on wt lifespan. Average median lifespans ± SEM from all replicates indicated beneath representative curve.


( B ) Locomotory activity of 10 - day - old gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines . Values represent arbitrary units from 3 biological replicates with 4 technical replicates each , 35 worms per technical replicate . Error bars : mean ± SEM , * * p value < 0 . 01 , * * * p value < 0 . 001 , one - way ANOVA , Dunnett ' s post - hoc correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Locomotory activity of 10-day-old gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines. Values represent arbitrary units from 3 biological replicates with 4 technical replicates each, 35 worms per technical replicate. Error bars: mean ± SEM, **p value< 0.01, ***p value< 0.001, one-way ANOVA, Dunnett's post-hoc correction.


( C ) Effect of methylxanthines on the survival of gas - 1 mutants . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

O O O O 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) Effect of methylxanthines on the survival of gas-1 mutants. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Pharyngeal pumps / minute in young adult gas - 1 mutant nematodes treated with vehicle or methylxanthines . Error bars : mean ± SEM , n = 18 . * * * * p value < 0 . 0001 , one - way ANOVA , Dunnett ' s post - hoc correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pharyngeal pumps/minute in young adult gas-1 mutant nematodes treated with vehicle or methylxanthines. Error bars: mean ± SEM, n=18. ****p value< 0.0001, one-way ANOVA, Dunnett's post-hoc correction.


( E - F ) Immunoblot analysis of ( E ) KIN - 1 phospho - substrates in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) .

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

(E-F) Immunoblot analysis of (E) KIN-1 phospho-substrates in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).


( E - F ) Immunoblot analysis of ( F ) phospho - AAK - 2 in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) .

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

(E-F) Immunoblot analysis of (F) phospho-AAK-2 in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).


( G ) Left panel : FRAP in vehicle or PTX - treated gas - 1 animals . Each point : mean recovery of 14 bleached regions ( 2 regions per animal , 7 animals per condition ) ± SEM . Right panel : maximum ( total ) recovery per condition ± SEM . * * p value < 0 . 01 unpaired t test . For all immunoblot panels : numbers correspond to band densitometries across biological replicates and for all lifespan curves : the legend shows Mean median lifespan ± SEM across all biological replicates .

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

(G) Left panel: FRAP in vehicle or PTX-treated gas-1 animals. Each point: mean recovery of 14 bleached regions (2 regions per animal, 7 animals per condition) ± SEM. Right panel: maximum (total) recovery per condition ± SEM. **p value< 0.01 unpaired t test. For all immunoblot panels: numbers correspond to band densitometries across biological replicates and for all lifespan curves: the legend shows Mean median lifespan ± SEM across all biological replicates.


( A ) FAK + / + and FAK − / − murine SCC cells were lysed and FAK , Ret or Src immunoprecipitated and immunoblotting carried out using anti ‐ FAK , anti ‐ Ret , anti ‐ Src and anti ‐ actin .

O O O O O O O O O O O O O O O O O O O O O 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) FAK+/+ and FAK−/− murine SCC cells were lysed and FAK, Ret or Src immunoprecipitated and immunoblotting carried out using anti‐FAK, anti‐Ret, anti‐Src and anti‐actin.


( B ) Cells were fixed and stained with either anti ‐ Ret ( green in the upper left panels ) or anti ‐ Ret PY1062 ( green in the lower left panels ) and DAPI ( blue ) . Merged and zoomed images are shown . Solid arrows indicate localization at adhesions while broken arrows show localization in puncta . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . Lysates were immunoblotted with anti ‐ Ret PY1062 and anti ‐ actin ( lower right panels ) .

(B) Cells were fixed and stained with either anti‐Ret (green in the upper left panels) or anti‐Ret PY1062 (green in the lower left panels) and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate localization at adhesions while broken arrows show localization in puncta. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). Lysates were immunoblotted with anti‐Ret PY1062 and anti‐actin (lower right panels).


( C ) Cells were fixed and stained with anti ‐ Ret ( green ) and anti ‐ FAK ( red ) . Merged images are also shown . Solid arrows indicate co ‐ localization between Ret and FAK at adhesions , while broken arrows indicate its absence in puncta . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous cell carcinoma .

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

(C) Cells were fixed and stained with anti‐Ret (green) and anti‐FAK (red). Merged images are also shown. Solid arrows indicate co‐localization between Ret and FAK at adhesions, while broken arrows indicate its absence in puncta. Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma.


Ret localizes to Src ‐ positive autophagosomes in FAK ‐ deficient SCC cells . Cells were fixed and stained with either ( A ) anti ‐ Ret ( green ) and anti ‐ PY416 Src ( red ) or ( B ) anti ‐ PY416 Src ( red ) and anti ‐ LC3B ( green ) . Merged and zoomed images are shown . Solid arrows indicate co ‐ localization at adhesions , while broken arrows show co ‐ localization in intracellular puncta .

Ret localizes to Src‐positive autophagosomes in FAK‐deficient SCC cells. Cells were fixed and stained with either (A) anti‐Ret (green) and anti‐PY416 Src (red) or (B) anti‐PY416 Src (red) and anti‐LC3B (green). Merged and zoomed images are shown. Solid arrows indicate co‐localization at adhesions, while broken arrows show co‐localization in intracellular puncta.


( C ) Cells were transfected with scrambled , Atg5 or Atg7 siRNA , trypsinized and allowed to readhere before immunoblotting with anti ‐ Atg5 , anti ‐ Atg7 and anti ‐ actin ( right panels ) . Cells were fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Merged images are shown . Solid arrows indicate co ‐ localization between Ret and paxillin at adhesions , while broken arrows indicate its absence in puncta .

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

(C) Cells were transfected with scrambled, Atg5 or Atg7 siRNA, trypsinized and allowed to readhere before immunoblotting with anti‐Atg5, anti‐Atg7 and anti‐actin (right panels). Cells were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Merged images are shown. Solid arrows indicate co‐localization between Ret and paxillin at adhesions, while broken arrows indicate its absence in puncta.


( D ) Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) .

(D) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).


( E ) Cells , either untreated or treated with chloroquine for 24 h , were fixed and stained with anti ‐ Ret ( green ) and DAPI ( blue ) . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous cell carcinoma ; siRNA , short interfering RNA .

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

(E) Cells, either untreated or treated with chloroquine for 24 h, were fixed and stained with anti‐Ret (green) and DAPI (blue). Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma; siRNA, short interfering RNA.


( A ) FAK − / − cells stably re ‐ expressing FAK ‐ wt , FAK ‐ Y397F , FAK ‐ KD or FAK ‐ Y4F ‐ Y9F were immunoblotted using anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y861 , anti ‐ FAK Y925 , anti ‐ Src , anti ‐ Ret and anti ‐ actin . FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti ‐ Src , anti ‐ FAK or anti ‐ Ret .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FAK−/− cells stably re‐expressing FAK‐wt, FAK‐Y397F, FAK‐KD or FAK‐Y4F‐Y9F were immunoblotted using anti‐FAK, anti‐FAK Y397, anti‐FAK Y861, anti‐FAK Y925, anti‐Src, anti‐Ret and anti‐actin. FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti‐Src, anti‐FAK or anti‐Ret.


( B ) Cells were fixed and stained with anti ‐ Ret ( green ) and either anti ‐ FAK ( red in the upper panels ) or anti ‐ PY416 Src ( red in the lower panels ) . Merged images are shown . Solid arrows indicate Ret in adhesions , while broken arrows show Ret in puncta . Scale bars , 20 μM .

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

(B) Cells were fixed and stained with anti‐Ret (green) and either anti‐FAK (red in the upper panels) or anti‐PY416 Src (red in the lower panels). Merged images are shown. Solid arrows indicate Ret in adhesions, while broken arrows show Ret in puncta. Scale bars, 20 μM.


( C ) Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . FAK , focal adhesion kinase ; Ret , rearranged during transfection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). FAK, focal adhesion kinase; Ret, rearranged during transfection.


( A ) FAK + / + cells were treated with dasatinib , then lysates were immunoblotted with anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y925 , anti ‐ PY416 Src , anti ‐ Src and anti ‐ actin . FAK was immunoprecipitated and then immunoblotting performed using anti ‐ Src , anti ‐ FAK or anti ‐ Ret .

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

(A) FAK+/+ cells were treated with dasatinib, then lysates were immunoblotted with anti‐FAK, anti‐FAK Y397, anti‐FAK Y925, anti‐PY416 Src, anti‐Src and anti‐actin. FAK was immunoprecipitated and then immunoblotting performed using anti‐Src, anti‐FAK or anti‐Ret.


( B ) Cells were treated with dasatinib , then fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . ( C ) FAK − / − cells treated with dasatinib were fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions , while broken arrows show lack of co ‐ localization in puncta . Scale bars , 20 μM . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) .

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

(B) Cells were treated with dasatinib, then fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (C) FAK−/− cells treated with dasatinib were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions, while broken arrows show lack of co‐localization in puncta. Scale bars, 20 μM. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).


( D ) Lysates from untreated and dasatinib ‐ treated FAK − / − cells were immunoblotted with anti ‐ Ret , anti ‐ PY416 Src , anti ‐ Src and anti ‐ actin . Ret was also immunoprecipitated from cells and then immunoblotted using anti ‐ Src and anti ‐ Ret . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection .

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

(D) Lysates from untreated and dasatinib‐treated FAK−/− cells were immunoblotted with anti‐Ret, anti‐PY416 Src, anti‐Src and anti‐actin. Ret was also immunoprecipitated from cells and then immunoblotted using anti‐Src and anti‐Ret. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection.


( a ) Immunofluorescence labeling of wildtype mouse dorsal skin samples ( Dnmt1f / f , skin from P3 ) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte - specific knockout ( Dnmt1Δ / Δep ) results in the almost absence of a specific staining in the epidermis and in hair follicles . Dashed lines indicate the dermal - epidermal border and dotted lines indicate hair follicles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence labeling of wildtype mouse dorsal skin samples (Dnmt1f/f, skin from P3) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte-specific knockout (Dnmt1Δ/Δep) results in the almost absence of a specific staining in the epidermis and in hair follicles. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.


( d ) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5 - mC labeling of P3 skin . Dashed lines indicate the dermal - epidermal border and dotted lines indicate hair follicles .

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

(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.


( h ) Hematoxylin and Eosin ( H & E ) staining , immunolabeling for KERATIN 10 ( KRT10 ) , LORICRIN ( LOR ) and KERATIN 6 ( KRT6 ) expression of dorsal skin sections ( P5 ) of control and Dnmt1Δ / Δep mice .

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

(h) Hematoxylin and Eosin (H&E) staining, immunolabeling for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.


( c ) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ / Δep mice of different postnatal age . Cells are shown as percent of viable cells . Data are mean ± SEM , two - tailed t - test . ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 ( P3 ) , 4 ( P5 ) and 4 ( P6 + P7 ) . Data information : Scale bars , 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SEM, two-tailed t-test. ns, not significant, * p≤0.05, ** p≤0.01, **** p≤0.0001, n=3 (P3), 4 (P5) and 4 (P6 + P7). Data information: Scale bars, 50 µm.


( d ) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ / Δep mice at P3 ( n = 3 ) , P5 ( n = 5 ) and P7 ( n ≥ 3 ) . Data are mean ± SEM . P values are calculated using one - way ANOVA with post hoc Tukey multiple comparison test , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Data information : Scale bars , 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ/Δep mice at P3 (n=3), P5 (n=5) and P7 (n≥3). Data are mean ± SEM. P values are calculated using one-way ANOVA with post hoc Tukey multiple comparison test, * p≤0.05, ** p≤0.01, *** p≤0.001 , **** p≤0.0001. Data information: Scale bars, 50 µm.


( a ) Relative mRNA expression levels of epidermal Intracisternal A - type particle ( IAP ) during different postnatal time points ( P0 n ≥ 3 , P3 n = 3 , P5 n = 5 , P7 n ≥ 3 mice ) , which were compared using one way - ANOVA with post hoc Tukey multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative mRNA expression levels of epidermal Intracisternal A-type particle (IAP) during different postnatal time points (P0 n≥3, P3 n=3, P5 n=5, P7 n≥3 mice), which were compared using one way-ANOVA with post hoc Tukey multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01.


( c ) Relative expression of immune related genes of the epidermis from P7 of control ( n ≥ 7 ) , Dnmt1Δ / Δep , ( n ≥ 8 ) , MAVS knockout ( Mavs - / - , n = 7 ) and Dnmt1Δ / Δep Mavs - / - ( n = 6 ) mice were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

(c) Relative expression of immune related genes of the epidermis from P7 of control (n≥7), Dnmt1Δ/Δep, (n≥8), MAVS knockout (Mavs-/-, n=7) and Dnmt1Δ/Δep Mavs-/- (n=6) mice were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.


Immortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( c , d ) PicoGreen staining of PBS - and DAC - treated keratinocytes for 48h detected micronuclei ( arrow ) , which were analyzed in a blinded manner , and compared using two - tailed t - test . Data are mean ± SEM , * p ≤ 0 . 05 ( n = 3 biological replicates ) ( d ) . Data information : Scale bars , 20

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

Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). Data information: Scale bars, 20 µm


Immortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( e ) Co - labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes . Within enlarged sections on the right arrows point at DNA / cGAS double - positive blebs and micronuclei . ( f ) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way - ANOVA with post hoc Dunnett multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ( n = 4 biological replicates ) . Data information : Scale bars , , 50

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

Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (e) Co-labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes. Within enlarged sections on the right arrows point at DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates). Data information: Scale bars, , 50 µm


( c ) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f / f , Dnmt1Δ / Δep , and Dnmt1Δ / Δep Cgas - / - mice . Dashed lines indicate the dermal - epidermal border . ( d ) Quantification of CD45 - positve cells . Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ . Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . Data information : Scale bars , 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f/f, Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice. Dashed lines indicate the dermal-epidermal border. (d) Quantification of CD45-positve cells. Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ. Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ** p≤0.01, *** p≤0.001. Data information: Scale bars, 100 µm


( e ) Epidermis obtained from P7 control , Dnmt1Δ / Δep , Cgas - / - and Dnmt1Δ / Δep Cgas - / - mice was analyzed for relative mRNA expression levels of immune related genes using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 . At least 7 mice per genotype were used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epidermis obtained from P7 control, Dnmt1Δ/Δep, Cgas-/- and Dnmt1Δ/Δep Cgas-/- mice was analyzed for relative mRNA expression levels of immune related genes using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, **** p≤0.0001. At least 7 mice per genotype were used.


A ) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous , CAF1 - knockdown - induced ( through p60 or p150 KD ) 2 - cell - like cells . Fold - changes relative to ESCs were calculated based on bulk RNA - seq data

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous, CAF1-knockdown-induced (through p60 or p150 KD) 2-cell-like cells. Fold-changes relative to ESCs were calculated based on bulk RNA-seq data


A ) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous , CAF1 - knockdown - induced ( through p60 or p150 KD ) 2 - cell - like cells . Fold - changes relative to ESCs were calculated based on bulk RNA - seq

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) Oxygen consumption rate of ES ( blue line ) and 2 - cell - like cells ( green line ) . Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose , L - glutamine and pyruvate . Basal , maximal ( FCCP - induced ) and non - mitochondrial ( Rotenone and Antimycin - A mediated ) respiratory rates are indicated . A representative graph of 3 independent biological replicates performed on the Seahorse extracellular flux analyser is shown . Due to the low number of 2 - cell - like cells available , compared to ESCs , one technical replicate of the former was analysed per biological replicate , while three or more technical replicates were performed for the latter . Accordingly , mean ± S . D . of technical replicates is shown for ESCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oxygen consumption rate of ES (blue line) and 2-cell-like cells (green line). Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Basal, maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates are indicated. A representative graph of 3 independent biological replicates performed on the Seahorse extracellular flux analyser is shown. Due to the low number of 2-cell-like cells available, compared to ESCs, one technical replicate of the former was analysed per biological replicate, while three or more technical replicates were performed for the latter. Accordingly, mean ± S.D. of technical replicates is shown for ESCs.


D ) Basal oxygen consumption rate of ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cells ( green ) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser . Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose , L - glutamine and pyruvate . Boxes indicate the range between the first and third quantile , the band specifies the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Individual dots indicate the measurements obtained in each of the individual technical replicates .

D) Basal oxygen consumption rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser. Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Boxes indicate the range between the first and third quantile, the band specifies the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each of the individual technical replicates.


E ) Oxygen consumption rate of ES ( blue line ) , Zscan4 + ( red line ) and 2 - cell - like cells ( green line ) in glucose - free media and upon acute injection of sodium pyruvate or sodium L - lactate . Note that L - glutamine - but not glucose or pyruvate - was initially present in the assay medium . Maximal ( FCCP - induced ) and non - mitochondrial ( Rotenone and Antimycin - A mediated ) respiratory rates following pyruvate or lactate treatment are also are indicated . A graph including data from 3 independent biological replicates is presented , and the mean ± S . D . of technical replicates is shown .

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

E) Oxygen consumption rate of ES (blue line), Zscan4+ (red line) and 2-cell-like cells (green line) in glucose-free media and upon acute injection of sodium pyruvate or sodium L-lactate. Note that L-glutamine - but not glucose or pyruvate - was initially present in the assay medium. Maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates following pyruvate or lactate treatment are also are indicated. A graph including data from 3 independent biological replicates is presented, and the mean ± S.D. of technical replicates is shown.


F ) Representative electron microscopy micrographs of mitochondria from ES ( n = 49 sections ) and 2 - cell - like cells ( n = 57 sections ) generated across 2 independent technical and biological replicates . Scale bar , 1 μm . Representative images from 2 independent biological and technical replicates are shown . 162 and 99 mitochondria were analysed for 2 - cell - like and ES cells , respectively .

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

F) Representative electron microscopy micrographs of mitochondria from ES (n=49 sections) and 2-cell-like cells (n=57 sections) generated across 2 independent technical and biological replicates. Scale bar, 1 μm. Representative images from 2 independent biological and technical replicates are shown. 162 and 99 mitochondria were analysed for 2-cell-like and ES cells, respectively.


G ) Representative single section of CellROX - DeepRed fluorescence in ES and 2 - cell - like cells ( green arrow ) obtained using live cell microscopy . Scale bar , 10 μm . n , indicates the number of independent replicates . Representative images from 3 independent biological replicates are shown .

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

G) Representative single section of CellROX-DeepRed fluorescence in ES and 2-cell-like cells (green arrow) obtained using live cell microscopy. Scale bar, 10 μm. n, indicates the number of independent replicates. Representative images from 3 independent biological replicates are shown.


H ) FACS - assisted quantification of CellROX - DeepRed fluorescence intensity in ES and 2 - cell - like cells . Measurements were obtained from 2 independent biological replicates . * * * P < 0 . 005 ; Mann - Whitney U test .

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

H) FACS-assisted quantification of CellROX-DeepRed fluorescence intensity in ES and 2-cell-like cells. Measurements were obtained from 2 independent biological replicates. *** P < 0.005; Mann-Whitney U test.


A ) ATP content in ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cells ( green ) across 4 independent biological replicates .

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

A) ATP content in ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 4 independent biological replicates.


B ) Extracellular acidification rate of ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cells ( green ) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser .

O O O O O O O O O O O O O O O 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) Extracellular acidification rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser.


C ) Glucose uptake rates in Zscan4 + ( red ) and 2 - cell - like cells ( green ) were measured using a luciferase - based assay across 4 independent biological replicates and are represented relative to those of control ES cells ( blue ) .

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

C) Glucose uptake rates in Zscan4+ (red) and 2-cell-like cells (green) were measured using a luciferase-based assay across 4 independent biological replicates and are represented relative to those of control ES cells (blue).


F ) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES , Zscan4 + and 2 - cell - like cells . Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA . Shown are the mean ± s . d . of the indicated number of independent cell cultures , performed across 2 or more independent biological replicates each .

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

F) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4+ and 2-cell-like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each.


G ) Glucose - 6 - phosphate dehydrogenase activity was measured in ES ( blue ) , Zscan4 + ( red ) and 2 - cell - like cell ( green ) lysates using a fluorometric assay . Measurements were obtained from 3 independent biological replicates , performed in 3 technical replicates each . n . s . - not significant ; one - way ANOVA . Data information : In panels A - C and G , boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Individual dots indicate the measurements obtained in each individual technical replicate .

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

G) Glucose-6-phosphate dehydrogenase activity was measured in ES (blue), Zscan4+ (red) and 2-cell-like cell (green) lysates using a fluorometric assay. Measurements were obtained from 3 independent biological replicates, performed in 3 technical replicates each. n.s. - not significant; one-way ANOVA. Data information: In panels A-C and G, boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate.


B ) Heatmap showing the effect of the 20 metabolites tested on 2 - cell - like cell levels .

B) Heatmap showing the effect of the 20 metabolites tested on 2-cell-like cell levels.


Percentage of 2 - cell - like cells in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across 3 biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range .

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

Percentage of 2-cell-like cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


Percentage of Zscan4 + cells in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across 3 biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range .

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

Percentage of Zscan4+ cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


Percentage of Zscan4 + cells in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range .

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

Percentage of Zscan4+ cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


Percentage of 2 - cell - like cells in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range .

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

Percentage of 2-cell-like cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range.


A ) Immunofluorescence staining for OCT4 , ZSCAN4 and 2C : : tbGFP in control and acetate treated ESC cultures . Green arrows indicate 2 - cell - like cells , and inlets highlight their DAPI structure . Scale bar , 20 μm .

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

A) Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate treated ESC cultures. Green arrows indicate 2-cell-like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm.


B ) RT - qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h . Shown are the mean ± s . d . of 3 independent cell cultures , performed in 2 technical replicates .

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

B) RT-qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h. Shown are the mean ± s.d. of 3 independent cell cultures, performed in 2 technical replicates.


C ) Percentage of 2 - cell - like cells obtained upon transfection of control or Dux - targeting siRNAs in control conditions or in combination with sodium acetate treatment . Measurements were obtained from 2 independent cell cultures , performed across 3 independent biological replicates . Boxes indicate the range between the first and third quantile , the band depicts the median and the whiskers extend no further than 1 . 5 - times the interquartile range . Individual dots indicate the measurements obtained in each technical replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of 2-cell-like cells obtained upon transfection of control or Dux-targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from 2 independent cell cultures, performed across 3 independent biological replicates. Boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each technical replicate.


D ) Percentage of 2 - cell - like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment . Shown are the mean ± s . d . of 4 independent cell cultures , performed across 2 independent biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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) Percentage of 2-cell-like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of 4 independent cell cultures, performed across 2 independent biological replicates.


F ) Proportion of Zscan4 + cells at various timepoints during the time - lapse experiment . Shown are the mean ± s . d . of 3 independent experiments .

O O O O O O O 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) Proportion of Zscan4+ cells at various timepoints during the time-lapse experiment. Shown are the mean ± s.d. of 3 independent experiments.


Figure 1 . Breast cancer cells derived from tumor - draining lymph nodes gain aggressive malignancy ( A and B ) Inguinal LNs were harvested from 4T1 - injected mice at the indicated week after initial injection . LN tissues were fixed and paraffin - embedded . Immunoflurorescence was performed with anti - GFP antibody to visualize GFP + 4T1 cells . ( A ) Confocal micrographs of the representative field of the LN section . Scale bar : 10 μm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD .

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

Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (A) Confocal micrographs of the representative field of the LN section. Scale bar: 10 μm. Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.


Figure 1 . Breast cancer cells derived from tumor - draining lymph nodes gain aggressive malignancy ( A and B ) Inguinal LNs were harvested from 4T1 - injected mice at the indicated week after initial injection . LN tissues were fixed and paraffin - embedded . Immunoflurorescence was performed with anti - GFP antibody to visualize GFP + 4T1 cells . ( B ) Histograms illustrated that the dynamic changes of GFP + 4T1 cells metastasized to the inguinal LN . Cells were counted in whole sections ( 2 non - consecutive sections per LN per mouse ) . Each group was composed of at least two mice . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD .

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

Figure 1. Breast cancer cells derived from tumor-draining lymph nodes gain aggressive malignancy (A and B) Inguinal LNs were harvested from 4T1-injected mice at the indicated week after initial injection. LN tissues were fixed and paraffin-embedded. Immunoflurorescence was performed with anti-GFP antibody to visualize GFP+ 4T1 cells. (B) Histograms illustrated that the dynamic changes of GFP+ 4T1 cells metastasized to the inguinal LN. Cells were counted in whole sections (2 non-consecutive sections per LN per mouse). Each group was composed of at least two mice.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.


( C - I ) 4T1 - injected or EMT6 - injected mice were sacrificed at the indicated week after initial injection . Collagenase - digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment . The detailed process is described in the methods section . After several days selection / culture , viable 4T1PT , 4T1LN , EMT6PT , and EMT6LN cells were used for the following assays : ( C ) Soft - agar colony forming activity was examined in 4T1PT and 4T1LN cells ( 5x102 cells / well , n = 4 wells per group ) . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . In ( C ) , * * P = 0 . 000088 and * * P = 0 . 000004 for the 4T1LN cells at week 2 and week 3 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays: (C) Soft-agar colony forming activity was examined in 4T1PT and 4T1LN cells (5x102 cells/well, n = 4 wells per group). Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (C), **P = 0.000088 and **P = 0.000004 for the 4T1LN cells at week 2 and week 3, respectively.


( C - I ) 4T1 - injected or EMT6 - injected mice were sacrificed at the indicated week after initial injection . Collagenase - digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment . The detailed process is described in the methods section . After several days selection / culture , viable 4T1PT , 4T1LN , EMT6PT , and EMT6LN cells were used for the following assays : ( D and E ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1PT or 4T1LN cells . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) 4T1-injected or EMT6-injected mice were sacrificed at the indicated week after initial injection. Collagenase-digested specimens from the primary tumors or inguinal LN tissues were cultured in culture dish for cancer cell enrichment. The detailed process is described in the methods section. After several days selection/culture, viable 4T1PT, 4T1LN, EMT6PT, and EMT6LN cells were used for the following assays:(D and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.


( D and E ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1PT or 4T1LN cells . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD . In ( E ) , * * P = 0 . 000006 . In ( G ) * * P = 0 . 000734 . Level of significance was determined using two - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1PT or 4T1LN cells. Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD. In (E), **P = 0.000006. In (G) **P = 0.000734. Level of significance was determined using two-tailed unpaired t-test.


( F and G ) BALB / c mice ( n = 3 mice per group ) were injected with 2x105 4T1PT or 4T1LN cells via tail - vein . Lung colonization was examined by lung morphology ( F ) and the numbers of tumor nodule ( G ) on day 21 . Scale bar : 1 cm . Data information : In ( A - G ) , all experimental data were verified in at least two independent experiments . All values are presented as mean or mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) BALB/c mice (n = 3 mice per group) were injected with 2x105 4T1PT or 4T1LN cells via tail-vein. Lung colonization was examined by lung morphology (F) and the numbers of tumor nodule (G) on day 21. Scale bar: 1 cm.Data information: In (A-G), all experimental data were verified in at least two independent experiments. All values are presented as mean or mean ± SD.


( H and I ) NSG mice ( n = 5 mice per group ) were injected with 4T1PT ( 5x102 ) , 4T1LN ( 5x102 ) , EMT6PT ( 1x102 ) , or EMT6LN ( 1x102 ) cells . Distant organ metastasis was examined by bioluminscent images on day 28 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and I) NSG mice (n = 5 mice per group) were injected with 4T1PT (5x102), 4T1LN (5x102), EMT6PT (1x102), or EMT6LN (1x102) cells. Distant organ metastasis was examined by bioluminscent images on day 28. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.


( C and D ) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor - bearing mice after tumor resection with ( n = 13 mice ) or without ( n = 10 mice ) tumor - draining lymph node dissection on week 5 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . For the additional details , see the methods section . ( E and I ) Summary table of distant organ metastasis derived from each surgery group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Distant organ metastasis was examined by bioluminscent images of 4T1 tumor-bearing mice after tumor resection with (n=13 mice) or without (n=10 mice) tumor-draining lymph node dissection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section. (E and I) Summary table of distant organ metastasis derived from each surgery group.


( E and I ) Summary table of distant organ metastasis derived from each surgery group . ( G and H ) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre - removed tumor - bearing mice ( n = 9 mice ) or sham surgery tumor - bearing mice ( n = 5 mice ) after tumor resection on week 5 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image . For the additional details , see the methods section .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and I) Summary table of distant organ metastasis derived from each surgery group.(G and H) Distant organ metastasis was examined by bioluminscent images of inguinal lymph node pre-removed tumor-bearing mice (n=9 mice) or sham surgery tumor-bearing mice (n=5 mice) after tumor resection on week 5. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image. For the additional details, see the methods section.


Figure 3 . Up - regulation of Il - 17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor - draining lymph node ( A ) Gene expression profiles were shown at 4T1PT to 4T1LN cells . Five genes encoding cell surface proteins were identified among up - regulated genes .

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

Figure 3. Up-regulation of Il-17rb contributes to the aggressive malignancy phenotypes of breast cancer cell derived from tumor-draining lymph node (A) Gene expression profiles were shown at 4T1PT to 4T1LN cells. Five genes encoding cell surface proteins were identified among up-regulated genes.


( B ) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT - qPCR . Gapdh was used as an internal control .

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

(B) mRNA expression of each candidate gene in 4T1PT and 4T1LN cells was determined by RT-qPCR. Gapdh was used as an internal control.


( C and D ) Il - 17rb , Gpr56 , and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis .

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

(C and D) Il-17rb, Gpr56, and Scara5 expression in 4T1PT and 4T1LN cells were examined by Western blotting analysis.


( E ) Western blotting and RT - qPCR analysis of Il - 17rb , Gpr56 , and Scara5 expression in 4T1 cells transduced with Il - 17rb , Gpr56 , Scara5 , or control LacZ shRNA lentivirus respectively .

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

(E) Western blotting and RT-qPCR analysis of Il-17rb, Gpr56, and Scara5 expression in 4T1 cells transduced with Il-17rb, Gpr56, Scara5, or control LacZ shRNA lentivirus respectively.


( F ) Soft - agar colony forming activity was examined in lentivirus transduced shIl - 17rb , shGpr56 , shScara5 , or shLacZ 4T1 cells ( 5x102 cells / well , n = 6 wells per group ) .

(F) Soft-agar colony forming activity was examined in lentivirus transduced shIl-17rb, shGpr56, shScara5, or shLacZ 4T1 cells (5x102 cells/well, n = 6 wells per group).


( G ) Western blotting analysis was used to detect ectopic expression of Il - 17rb in 4T1 cells infected with either lentiviruses carrying Il - 17rb cDNA or empty vector .

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

(G) Western blotting analysis was used to detect ectopic expression of Il-17rb in 4T1 cells infected with either lentiviruses carrying Il-17rb cDNA or empty vector.


( H ) Soft - agar colony forming activity was examined using control or Il - 17rb over - expressing 4T1 cells ( 5x102 cells / well , n = 6 wells per group ) .

O O O O O O O O O O O O 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) Soft-agar colony forming activity was examined using control or Il-17rb over-expressing 4T1 cells (5x102 cells/well, n = 6 wells per group).


( I ) Western blotting analysis was used to examine Il - 17rb in a representative Il - 17rbDel 4T1 clone derived from Il - 17rb knocked - out 4T1 cells using a CRISPR / Cas9 system .

O O O O O O O O O O O O O O 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) Western blotting analysis was used to examine Il-17rb in a representative Il-17rbDel 4T1 clone derived from Il-17rb knocked-out 4T1 cells using a CRISPR/Cas9 system.


( J and K ) Tumorigenesis assays were determined in BALB / c mice ( n = 3 mice per group ) orthotopically injected with 5x102 4T1 cells from WT or Il - 17rbDel clone . Tumor mass ( J ) and weight ( K ) were measured on day 28 . Scale bar : 1 cm .

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

(J and K) Tumorigenesis assays were determined in BALB/c mice (n = 3 mice per group) orthotopically injected with 5x102 4T1 cells from WT or Il-17rbDel clone. Tumor mass (J) and weight (K) were measured on day 28. Scale bar: 1 cm.


( L and M ) BALB / c mice ( n = 3 mice per group ) were injected with 5x105 4T1 cells from WT or Il - 17rbDel clone via tail - vein . Lung colonization was examined by lung morphology ( L ) and the numbers of tumor nodule ( M ) on day 21 . Scale bar : 1 cm .

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

(L and M) BALB/c mice (n = 3 mice per group) were injected with 5x105 4T1 cells from WT or Il-17rbDel clone via tail-vein. Lung colonization was examined by lung morphology (L) and the numbers of tumor nodule (M) on day 21. Scale bar: 1 cm.


( B and C ) Inguinal lymph node - injected BALB / c mice were sacrificed at the indicated week after initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . Inguinal lymph node tissues came from un - injection BALB / c mice as control . After 5 - day co - culture , 4T1 cells at lower well were examined in the RT - qPCR ( B ) analyses of Il - 17rb expression . Gapdh was used as an internal control or as a loading control .

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

(B and C) Inguinal lymph node-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the RT-qPCR (B)analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.


( B and C ) 4T1 - injected BALB / c mice were sacrificed at the indicated week after initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . Inguinal lymph node tissues came from un - injection BALB / c mice as control . After 5 - day co - culture , 4T1 cells at lower well were examined in the Western blotting ( C ) analyses of Il - 17rb expression . Gapdh was used as an internal control or as a loading control .

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

(B and C) 4T1-injected BALB/c mice were sacrificed at the indicated week after initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. Inguinal lymph node tissues came from un-injection BALB/c mice as control. After 5-day co-culture, 4T1 cells at lower well were examined in the Western blotting (C) analyses of Il-17rb expression. Gapdh was used as an internal control or as a loading control.


( D ) Soft - agar colony forming activity was examined using co - cultured 4T1 cells at lower well ( 5x102 cells / well , n = 4 wells per group ) .

O O O O O O O O O O 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) Soft-agar colony forming activity was examined using co-cultured 4T1 cells at lower well (5x102 cells/well, n = 4 wells per group).


( E and F ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co - cultured with 4T1 cells . After 5 - day co - culture , 4T1 cells at lower well were used for the RT - qPCR ( E ) analyses of Il - 17rb expression . Gapdh was used as internal control or as a loading control .

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

(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the RT-qPCR (E)analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.


( E and F ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co - cultured with 4T1 cells . After 5 - day co - culture , 4T1 cells at lower well were used for the Western blotting ( F ) analyses of Il - 17rb expression . Gapdh was used as internal control or as a loading control .

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

(E and F) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Each lymphocyte subsets isolated from inguinal lymph node tissues by FACS sorting were transwell co-cultured with 4T1 cells. After 5-day co-culture, 4T1 cells at lower well were used for the Western blotting (F) analyses of Il-17rb expression. Gapdh was used as internal control or as a loading control.


( G ) Percentage of CD4 + Foxp3 + Tregs of total CD4 + cells in inguinal LN , spleen and peripheral blood of BALB / c mice ( n = 3 mice per group ) injected with 4T1 ( Blue ) or γ - irradiated 4T1 ( Red ) cancer cells was analyzed by FACS .

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

(G) Percentage of CD4+Foxp3+ Tregs of total CD4+ cells in inguinal LN, spleen and peripheral blood of BALB/c mice (n = 3 mice per group) injected with 4T1 (Blue) or γ-irradiated 4T1 (Red) cancer cells was analyzed by FACS.


( H ) 4T1 cells co - cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor - bearing mice by FACS sorting . After 5 - day co - culture , 4T1 cells at lower well were analyzed for Il - 17rb expression by RT - qPCR analysis . Gapdh was used as internal control .

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

(H) 4T1 cells co-cultured with the indicated lymphocyte populations isolated from inguinal LN of 4T1 tumor-bearing mice by FACS sorting. After 5-day co-culture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.


Figure 5 . Depletion of Tregs in the tumor - draining lymph node microenvironment abolishes Il - 17rb induction and enhanced malignancy in breast cancer cells . ( A ) 4T1 - injected BALB / c mice were treated with control IgG , anti - CD4 or anti - CD25 neutralizing antibody at the day 15 and day 18 after initial injection . Mice were sacrificed at three weeks after initial injection . Depletion of CD4 + Foxp3 + Tregs in inguinal LNs was confirmed by FACS analysis .

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

Figure 5. Depletion of Tregs in the tumor-draining lymph node microenvironment abolishes Il-17rb induction and enhanced malignancy in breast cancer cells. (A) 4T1-injected BALB/c mice were treated with control IgG, anti-CD4 or anti-CD25 neutralizing antibody at the day 15 and day 18 after initial injection. Mice were sacrificed at three weeks after initial injection. Depletion of CD4+Foxp3+ Tregs in inguinal LNs was confirmed by FACS analysis.


( B ) Western blotting analysis of Il - 17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice .

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

(B) Western blotting analysis of Il-17rb expression was performed in 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice.


( C and D ) BALB / c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . ( n = 3 mice per group ) . Tumor mass ( D ) and tumor weight ( E ) were measured on day 28 . Scale bar : 1 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) BALB/c mice were orthotopically injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. (n = 3 mice per group). Tumor mass (D) and tumor weight (E) were measured on day 28. Scale bar: 1 cm.


( E ) NSG mice ( n = 5 mice per group ) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG - , anti - CD4 , or anti - CD25 antibody - treated 4T1 tumor - bearing mice . Distant organ metastasis was examined by bioluminscent images on day 21 . The bioluminescent signal ( pseudocolor ) was recorded as photons per second per centimeter squared per steradian ( p / s / cm2 / sr ) and the luminescent image was overlaid on the photographic image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NSG mice (n = 5 mice per group) were injected with 5x102 4T1LN or 4T1PT cells collected from control IgG-, anti-CD4, or anti-CD25 antibody-treated 4T1 tumor-bearing mice. Distant organ metastasis was examined by bioluminscent images on day 21. The bioluminescent signal (pseudocolor) was recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr) and the luminescent image was overlaid on the photographic image.


Figure 6 . TGF - β1 secreted from Tregs induces Il - 17rb expression of breast cancer cells via Smad2 / 3 / 4 signaling . ( A ) CD4 + CD25 + Tregs were isolated from inguinal LN of BALB / c mice injected with 4T1 cells for three weeks , and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL - 10 or TGF - β1 . After 5 - day coculture , 4T1 cells at lower well were analyzed for Il - 17rb expression by RT - qPCR analysis . Gapdh was used as internal control .

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

Figure 6. TGF-β1 secreted from Tregs induces Il-17rb expression of breast cancer cells via Smad2/3/4 signaling. (A) CD4+CD25+ Tregs were isolated from inguinal LN of BALB/c mice injected with 4T1 cells for three weeks, and were transwell cocultured with 4T1 cells in the presence of control IgG or neutralizing antibodies against IL-10 or TGF-β1. After 5-day coculture, 4T1 cells at lower well were analyzed for Il-17rb expression by RT-qPCR analysis. Gapdh was used as internal control.


( B and C ) Western blotting analyzed Il - 17rb in 4T1 cells treated with recombinant TGF - β1 ( B ) proteins for 5 days .

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

(B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant TGF-β1 (B)proteins for 5 days.


( B and C ) Western blotting analyzed Il - 17rb in 4T1 cells treated with recombinant IL - 10 ( C ) proteins for 5 days .

(B and C) Western blotting analyzed Il-17rb in 4T1 cells treated with recombinant IL-10 (C) proteins for 5 days.


( D ) Western blotting analyzed Il - 17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF - β1 for 5 days .

O 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) Western blotting analyzed Il-17rb in shLacZ or shTgfbr1 4T1 cells treated with recombinant TGF-β1 for 5 days.


( E ) 4T1 - injected BALB / c mice were sacrificed at three weeks post initial injection . Total cells isolated from inguinal lymph node tissues were transwell co - cultured with 4T1 cells . After 5 - day coculture , soft - agar colony forming activity was determined in co - cultured shLacZ or shTgfbr1 4T1 cells at lower well . ( 5x102 cells / well , n = 6 wells per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 4T1-injected BALB/c mice were sacrificed at three weeks post initial injection. Total cells isolated from inguinal lymph node tissues were transwell co-cultured with 4T1 cells. After 5-day coculture, soft-agar colony forming activity was determined in co-cultured shLacZ or shTgfbr1 4T1 cells at lower well. (5x102 cells/well, n = 6 wells per group).


( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad2 ( F ) 4T1 cells treated with recombinant TGF - β1 for 5 days .

O O O O O O 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-H) Western blotting analyzed Il-17rb in shLacZ, shSmad2 (F)4T1 cells treated with recombinant TGF-β1 for 5 days.


( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad3 ( G ) 4T1 cells treated with recombinant TGF - β1 for 5 days .

(F-H) Western blotting analyzed Il-17rb in shLacZ,shSmad3 (G)4T1 cells treated with recombinant TGF-β1 for 5 days.


( F - H ) Western blotting analyzed Il - 17rb in shLacZ , shSmad4 ( H ) 4T1 cells treated with recombinant TGF - β1 for 5 days .

(F-H) Western blotting analyzed Il-17rb in shLacZ, shSmad4 (H) 4T1 cells treated with recombinant TGF-β1 for 5 days.


( I and J ) WT 4T1 ( I ) or Il - 17rbDel 4T1 ( J ) cells were treated with or without recombinant TGF - β1 for 5 days . After treatment , nuclear translocation of NF - κB p65 was assayed by Western blotting in cells treated with recombinant Il - 17b for 2 hours . Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts . f triplicate measurement . In ( B , C , D , F , G , H , I , and J ) , the intensity of each band was quantified using the Image J software . Gapdh , α - tubulin , or p84 was used as a loading control . Relative expression ( RE ) of protein levels in each sample to control 4T1 cells or Il - 17rbDel 4T1 clone is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and J) WT 4T1 (I) or Il-17rbDel 4T1 (J) cells were treated with or without recombinant TGF-β1 for 5 days. After treatment, nuclear translocation of NF-κB p65 was assayed by Western blotting in cells treated with recombinant Il-17b for 2 hours. Nuclear Matrix Protein p84 was used as a loading control of nuclear extracts.f triplicate measurement. In (B, C, D, F, G, H, I, and J), the intensity of each band was quantified using the Image J software. Gapdh, α-tubulin, or p84 was used as a loading control. Relative expression (RE) of protein levels in each sample to control 4T1 cells or Il-17rbDel 4T1 clone is indicated.


Figure 7 . IL - 17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor - draining lymph nodes of breast cancer patients . ( A and C ) Representative IHC images of IL - 17RB ( A ) and Foxp3 ( C ) were taken in breast specimens from primary tumors and their paired LN metastasis specimens . Scale bar : 10

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

Figure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μm


Figure 7 . IL - 17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor - draining lymph nodes of breast cancer patients . ( A and C ) Representative IHC images of IL - 17RB ( A ) and Foxp3 ( C ) were taken in breast specimens from primary tumors and their paired LN metastasis specimens . Scale bar : 10 μm .

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

Figure 7. IL-17RB expression is correlated with the elevated prevalence of regulatory T cells in the tumor-draining lymph nodes of breast cancer patients. (A and C) Representative IHC images of IL-17RB (A) and Foxp3 (C) were taken in breast specimens from primary tumors and their paired LN metastasis specimens. Scale bar: 10 μm.


Figure 8 . Elevated IL - 17RB expression in cancer cells derived from tumor - draining lymph nodes in breast cancer patients correlates with aggressive growth nature . ( A ) Freshly collected humanbreast cancer ( hBC ) specimens ( PT , primary tumors ; LN , paired LN metastasis from the same breast cancer patient ) were digested with collagenase - containing buffer overnight . Depletion of hematopoietic cells by CD45 Dyna beads ( Invitrogen ) enriched the EpCAM + cells for following xenografts . Percentages of EpCAM + cells in human primary breast cancer cells were confirmed by FACS analysis ( Left panel : before enrichment ; Right panel : after enrichment ) . All of the histograms were shown that they were EpCAM expressing ( black line ) , compared to isotype control ( gray filled ) .

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

Figure 8. Elevated IL-17RB expression in cancer cells derived from tumor-draining lymph nodes in breast cancer patients correlates with aggressive growth nature. (A) Freshly collected humanbreast cancer (hBC) specimens (PT, primary tumors; LN, paired LN metastasis from the same breast cancer patient) were digested with collagenase-containing buffer overnight. Depletion of hematopoietic cells by CD45 Dyna beads (Invitrogen) enriched the EpCAM+ cells for following xenografts. Percentages of EpCAM+ cells in human primary breast cancer cells were confirmed by FACS analysis (Left panel: before enrichment; Right panel: after enrichment). All of the histograms were shown that they were EpCAM expressing (black line), compared to isotype control (gray filled).


( B ) Tumorigenesis assay were determined in NOD / SCID / IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells . Representative data on day 180 were shown .

(B) Tumorigenesis assay were determined in NOD/SCID/IL2Rγnull mice orthotopically injected with hBCPT and hBCLN cells. Representative data on day 180 were shown.


( D and E ) H & E stainings and IHC stainings of pan - keratin were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 1 mm ( left panel ) , 100 μm ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E) H&E stainings and IHC stainings of pan-keratin were shown in the representative cases of fat-pad sections from (B). Scale bar: 1 mm (left panel), 100 μm (right panel).


( F ) IHC stainings of IL - 17RB were shown in the representative cases of fat - pad sections from ( B ) . Scale bar : 10 μm .

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

(F) IHC stainings of IL-17RB were shown in the representative cases of fat-pad sections from (B). Scale bar: 10 μm.


( G ) The correlation of tumor volumes and IL - 17RB expression ( composite IHC score ) in hBCLN cells - injected NOD / SCID / IL2Rγnull mice .

(G) The correlation of tumor volumes and IL-17RB expression (composite IHC score) in hBCLN cells-injected NOD/SCID/IL2Rγnull mice.


A Mass spectrometric analysis of LSD1 methylation . Immunoprecipitation ( IP ) was performed using anti - Flag antibody in HEK - 293T cells overexpressing Flag - tagged LSD1 , followed by liquid chromatography coupled tandem mass spectrometry ( LC - MS / MS ) analysis . The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Mass spectrometric analysis of LSD1 methylation. Immunoprecipitation (IP) was performed using anti-Flag antibody in HEK-293T cells overexpressing Flag-tagged LSD1, followed by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. The fragmentation of the LSD1 peptide IADQFLGAMYTLPR identified a dimethylated residue at R838.


B Interactions between LSD1 and CARM1 were detected by co - immunoprecipitation ( Co - IP ) assay with anti - Flag in HEK - 293T cells transfected with Flag - LSD1 or Flag - CARM1 , respectively .

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

B Interactions between LSD1 and CARM1 were detected by co-immunoprecipitation (Co-IP) assay with anti-Flag in HEK-293T cells transfected with Flag-LSD1 or Flag-CARM1, respectively.


C Interaction of endogenous LSD1 with CARM1 was analyzed by Co - IP assay using anti - CARM1 antibody in MDA - MB - 231 cells .

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

C Interaction of endogenous LSD1 with CARM1 was analyzed by Co-IP assay using anti-CARM1 antibody in MDA-MB-231 cells.


Purified GST - tagged CARM1 was pulled down with the Flag - LSD1 purified from HEK - 293T cells . The amounts of GST and GST - tagged CARM1 were visualized by Coomassie Blue staining ( asterisk : GST - CARM1 ) .

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

Purified GST-tagged CARM1 was pulled down with the Flag-LSD1 purified from HEK-293T cells. The amounts of GST and GST-tagged CARM1 were visualized by Coomassie Blue staining (asterisk: GST-CARM1).


E In vitro methylation assays . Purified fusion protein of GST - tagged LSD1 WT or the four methylated arginine mutants ( R - A ) , or four methylated arginine mutants in combination ( 4RA ) , were incubated with GST - CARM1 in the presence of 3H - SAM . Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining ( arrows : the position of LSD1 ; arrowheads : the position of CARM1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro methylation assays. Purified fusion protein of GST-tagged LSD1 WT or the four methylated arginine mutants (R-A), or four methylated arginine mutants in combination (4RA), were incubated with GST-CARM1 in the presence of 3H-SAM. Methylated proteins were detected via autoradiography and total amounts of proteins were visualized by Coomassie Blue staining (arrows: the position of LSD1; arrowheads: the position of CARM1).


F Increasing amounts of GST - CARM1 incubated with GST - LSD1 in presence of SAM at 30 ° C for 1 h . And methylation of LSD1 was analyzed by immunoblotting with anti - LSD1 R838me2a antibody .

F Increasing amounts of GST-CARM1 incubated with GST-LSD1 in presence of SAM at 30°C for 1 h. And methylation of LSD1 was analyzed by immunoblotting with anti-LSD1 R838me2a antibody.


G GST - CARM1 incubated with GST - LSD1 WT or GST - LSD1 R838A / R838K in presence of SAM at 30 ° C for 1 h , followed by immunoblotting with anti - LSD1 R838me2a to detect LSD1 methylation .

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

G GST-CARM1 incubated with GST-LSD1 WT or GST-LSD1 R838A/R838K in presence of SAM at 30°C for 1 h, followed by immunoblotting with anti-LSD1 R838me2a to detect LSD1 methylation.


A Methylation of LSD1 was assessed in MCF10A , MCF7 and MDA - MB - 231 cells by immunoblotting using anti - LSD1 R838me2a .

A Methylation of LSD1 was assessed in MCF10A, MCF7 and MDA-MB-231 cells by immunoblotting using anti-LSD1 R838me2a.


Protein expression levels of LSD1 in MDA - MB - 231 cells transfected with CARM1 shRNAs or control vector , were assessed by western blotting ( B ) ( Error bars represent the mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - test ) .

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

Protein expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by western blotting (B) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).


relative mRNA expression levels of LSD1 in MDA - MB - 231 cells transfected with CARM1 shRNAs or control vector , were assessed by qPCR analysis ( C ) ( Error bars represent the mean ± SD , n = 3 experimental replicates , ns = not significant , Student ' s t - test ) .

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

relative mRNA expression levels of LSD1 in MDA-MB-231 cells transfected with CARM1 shRNAs or control vector, were assessed by qPCR analysis (C) (Error bars represent the mean ± SD, n = 3 experimental replicates, ns = not significant, Student's t-test).


D MDA - MB - 231 cells were treated with the indicated amounts of MS049 for 48 h , LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies .

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

D MDA-MB-231 cells were treated with the indicated amounts of MS049 for 48 h, LSD1 R838me2a and LSD1 levels were assessed by western blotting using indicated antibodies.


E MDA - MB - 231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 ( 10 μM ) or DMSO , before LSD1 level was analyzed by immunoblotting .

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

E MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h in presence of MG132 (10 μM) or DMSO, before LSD1 level was analyzed by immunoblotting.


F MDA - MB - 231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h , and the cell lysates were analyzed by western blotting .

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

F MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 or DMSO for 10 h, and the cell lysates were analyzed by western blotting.


G MDA - MB - 231 cells were transfected with CARM1 shRNA # 2 or control vector , and then treated with CHX ( 100 μg / ml ) for the indicated time ; LSD1 protein level was examined by western blotting .

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

G MDA-MB-231 cells were transfected with CARM1 shRNA#2 or control vector, and then treated with CHX (100 μg/ml) for the indicated time; LSD1 protein level was examined by western blotting.


H MDA - MB - 231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h . Cell lysates were immunoprecipitated by anti - LSD1 antibody and then analysed by immunoblotting .

O O O O O O O O O O O O O O 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 MDA-MB-231 cells were transfected with control or CARM1 shRNAs and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-LSD1 antibody and then analysed by immunoblotting.


I MDA - MB - 231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h . The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti - LSD1 antibody .

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

I MDA-MB-231 cells were treated with increasing amounts of MS049 for 48 h and MG132 for 10 h. The ubiquitination level of LSD1 was assessed by immunoblotting after IP with anti-LSD1 antibody.