I - K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h ( I ; n = 3 ) , 50 nM CPT for 18 h ( J ; n = 3 ) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug ( K ; n = 3 ) . Graphs represent averaged median values ; error bars indicate SEM . Statistical analysis according to one - way ANOVA with Bonferroni post - test ; * * * P ≤ 0 . 001 ; * * P ≤ 0 . 01 ; ns , not significant . Representative immunofluorescence images are shown in ( H ) , Figures EV6G and EV6H , respectively .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.


B - D Strand exchange assay with ssDNA and RAD51 , and with ( C ) or without ( B ) the addition of MMS22L - TONSL ( 75 nM ) . D Quantification shows averages , n = 2 ; error bars , SEM .

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

B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM.


E Averages of quantified strand exchange assays with ssDNA , RAD51 ( 270 nM ) and varying concentrations of MMS22L - TONSL or MMS22L . n = 2 ; error bars , SEM .

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

E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM.


F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA , RAD51 and MMS22L - TONSL or MMS22L . Averages shown , n = 2 ; error bars , SEM .

O O O O O O O O B-CONTROLLED_VAR 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

F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.


H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA ( 1 nM ) .

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

H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).


I A representative immunoblot showing effects of MMS22L - TONSL and MMS22L titration into reactions containing RAD51 , ssDNA ( 1 nM ) and dsDNA ( 10 nM ) , as indicated .

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

I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.


a , alpha - KG extends the lifespan of adult worms in the metabolite longevity screen . All metabolites were given at a concentration of 8 mM .

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

a, alpha-KG extends the lifespan of adult worms in the metabolite longevity screen. All metabolites were given at a concentration of 8 mM.


c , Dose - response curve of the α - KG effect on longevity .

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

c, Dose-response curve of the α-KG effect on longevity.


d , e , α - KG extends the lifespan of worms fed bacteria that have been ampicillin arrested , mean lifespan ( days of adulthood ) with vehicle treatment ( mveh ) = 19 . 4 ( n = 80 animals tested ) , mα - KG = 25 . 1 ( n = 91 ) , P 0 . 0001 ( log - rank test ) ( d ) ; or γ - irradiation - killed , mveh = 19 . 0 ( n = 88 ) , mα - KG = 23 . 0 ( n = 46 ) , P 0 . 0001 ( log - rank test ) ( e ) . OP50 , E . coli OP50 strain .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, e, α-KG extends the lifespan of worms fed bacteria that have been ampicillin arrested, mean lifespan (days of adulthood) with vehicle treatment (mveh) = 19.4 (n = 80 animals tested), mα-KG = 25.1 (n = 91), P  0.0001 (log-rank test) (d); or γ-irradiation-killed, mveh = 19.0 (n = 88), mα-KG = 23.0 (n = 46), P  0.0001 (log-rank test) (e). OP50, E. coli OP50 strain.


f alpha - KG does not further extend the lifespan of ogdh - 1 RNAi worms , mveh = 21 . 2 ( n = 98 ) , mα - KG = 21 . 1 ( n = 100 ) , P = 0 . 65 ( log - rank test ) .

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

f alpha-KG does not further extend the lifespan of ogdh-1 RNAi worms, mveh = 21.2 (n = 98), mα-KG = 21.1 (n = 100), P = 0.65 (log-rank test).


a , DARTS identifies ATP5B as an alpha - KG - binding protein . Red arrowhead , protected band .

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

a, DARTS identifies ATP5B as an alpha-KG-binding protein. Red arrowhead, protected band.


b , DARTS confirms alpha - KG binding specifically to ATP5B . IB , immunoblot .

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

b, DARTS confirms alpha-KG binding specifically to ATP5B. IB, immunoblot.


c , Inhibition of ATP synthase by alpha - KG ( released from octyl alpha - KG ; Supplementary Notes ) . This inhibition was reversible ( data not shown ) .

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

c, Inhibition of ATP synthase by alpha-KG (released from octyl alpha-KG; Supplementary Notes). This inhibition was reversible (data not shown).


d , e , Reduced ATP levels in octyl alpha - KG - treated normal humanfibroblasts ( * * P = 0 . 0016 , * * * * P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( d )

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

d, e, Reduced ATP levels in octyl alpha-KG-treated normal humanfibroblasts (**P = 0.0016, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (d)


d , e , Reduced ATP levels in octyl alpha - KG - treated worms ( day 2 , P = 0 . 969 ; day 8 , * P = 0 . 012 ; by t - test , two - tailed , two - sample unequal variance ) ( e ) . RLU , relative luminescence units .

O O O O O B-MEASURED_VAR O 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 O 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, e, Reduced ATP levels in octyl alpha-KG-treated worms (day 2, P = 0.969; day 8, *P = 0.012; by t-test, two-tailed, two-sample unequal variance) (e). RLU, relative luminescence units.


f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated cells ( * * * P = 0 . 0004 , * * * * P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( f )

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

f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated cells (***P = 0.0004, ****P  0.0001; by t-test, two-tailed, two-sample unequal variance) (f)


f , g , Decreased oxygen consumption rate ( OCR ) in octyl alpha - KG - treated worms ( P 0 . 0001 ; by t - test , two - tailed , two - sample unequal variance ) ( g ) .

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

f, g, Decreased oxygen consumption rate (OCR) in octyl alpha-KG-treated worms (P  0.0001; by t-test, two-tailed, two-sample unequal variance) (g).


h , alpha - KG , released from octyl alpha - KG ( 800 µM ) , decreases state 3 , but not state 4o or 3u ( P = 0 . 997 ) , respiration in mitochondria isolated from mouse liver . The respiratory control ratio is decreased in the octyl alpha - KG - ( 3 . 1 ± 0 . 6 ) versus vehicle - treated mitochondria ( 5 . 2 ± 1 . 0 ) ( * P = 0 . 015 ; by t - test , two - tailed , two - sample unequal variance ) . Oligo , oligomycin ; FCCP , carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ; AA , antimycin A .

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

h, alpha-KG, released from octyl alpha-KG (800 µM), decreases state 3, but not state 4o or 3u (P = 0.997), respiration in mitochondria isolated from mouse liver. The respiratory control ratio is decreased in the octyl alpha-KG- (3.1 ± 0.6) versus vehicle-treated mitochondria (5.2 ± 1.0) (*P = 0.015; by t-test, two-tailed, two-sample unequal variance). Oligo, oligomycin; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A.


i , Eadie - Hofstee plot of steady - state inhibition kinetics of ATP synthase by alpha - KG ( produced by in situ hydrolysis of octyl alpha - KG ) . [ S ] is the substrate ( ADP ) concentration , and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol ( vehicle control ) or octyl alpha - KG . alpha - KG ( produced from octyl alpha - KG ) decreases the apparent Vmax ( 53 . 9 to 26 . 7 ) and Km ( 25 . 9 to 15 . 4 ) , by nonlinear regression least - squares fit . c - i , Results were replicated in two independent experiments . Mean ± standard deviation ( s . d . ) is plotted in all cases .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR 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 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 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 O O O O O O O O O O O O O O O O O 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, Eadie-Hofstee plot of steady-state inhibition kinetics of ATP synthase by alpha-KG (produced by in situ hydrolysis of octyl alpha-KG). [S] is the substrate (ADP) concentration, and V is the initial velocity of ATP synthesis in the presence of 200 µM octanol (vehicle control) or octyl alpha-KG. alpha-KG (produced from octyl alpha-KG) decreases the apparent Vmax (53.9 to 26.7) and Km (25.9 to 15.4), by nonlinear regression least-squares fit. c-i, Results were replicated in two independent experiments. Mean ± standard deviation (s.d.) is plotted in all cases.


O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-g, Effect of alpha-KG on the lifespan of mutant or RNAi worms. a, atp-2 RNAi, mveh = 22.8 (n = 97), mα-KG = 22.5 (n = 94), P = 0.35; or RNAi control, mveh = 18.6 (n = 94), mα-KG = 23.4 (n = 91), P  0.0001. b, daf-2(e1370), mveh = 38.0 (n = 72), mα-KG = 47.6 (n = 69), P  0.0001. c, eat-2(ad1116), mveh = 22.8 (n = 59), mα-KG = 22.9 (n = 40), P = 0.79. d, let-363 RNAi, mveh = 25.1 (n = 96), mα-KG = 25.7 (n = 74), P = 0.95; or gfp RNAi control, mveh = 20.2 (n = 99), mα-KG = 27.7 (n = 81), P  0.0001. e, daf-16(mu86), mveh = 13.4 (n = 71), mα-KG = 17.4 (n = 72), P  0.0001; or N2, mveh = 13.2 (n = 100), mα-KG = 22.3 (n = 104), P  0.0001. f, pha-4(zu225), mveh = 14.2 (n = 94), mα-KG = 13.5 (n = 109), P = 0.55. g, hif-1(ia4), mveh = 20.5 (n = 85), mα-KG = 26.0 (n = 71), P  0.0001; or N2, mveh = 21.5 (n = 101), mα-KG = 24.6 (n = 102), P  0.0001. P values were determined by the log-rank test. Number of independent experiments: RNAi control (6), atp-2 (2), let-363 (3), N2 (5), daf-2 (2), eat-2 (2), pha-4 (2), daf-16 (2), hif-1 (5).


a , Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl alpha - KG or oligomycin . Similar results were obtained in HEK - 293 cells , normal human fibroblasts and mouse embryonic fibroblasts ( data not shown ) . P , phospho .

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 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

a, Decreased phosphorylation of mammalian TOR substrates in U87 cells treated with octyl alpha-KG or oligomycin. Similar results were obtained in HEK-293 cells, normal human fibroblasts and mouse embryonic fibroblasts (data not shown). P, phospho.


b , Increased autophagy in animals treated with alpha - KG or RNAi for atp - 2 or let - 363 . Photographs were taken at × 100 magnification . c , GFP : : LGG - 1 puncta quantified using ImageJ ( Methods ) . Data show results of 2 - 3 independent experiments . Bars indicate the mean . * * * * P 0 . 0001 ; NS , not significant ( t - test , two - tailed , two - sample unequal variance ) .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Increased autophagy in animals treated with alpha-KG or RNAi for atp-2 or let-363. Photographs were taken at ×100 magnification. c, GFP::LGG-1 puncta quantified using ImageJ (Methods). Data show results of 2-3 independent experiments. Bars indicate the mean. ****P  0.0001; NS, not significant (t-test, two-tailed, two-sample unequal variance).


d , alpha - KG levels are increased in starved worms . * * P 0 . 01 ( t - test , two - tailed , two - sample unequal variance ) . Mean ± s . d . is plotted .

O O B-MEASURED_VAR I-MEASURED_VAR I-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

d, alpha-KG levels are increased in starved worms. **P  0.01 (t-test, two-tailed, two-sample unequal variance). Mean ± s.d. is plotted.


G . Purified recombinant FLAG - FAM111A proteins were incubated at indicated temperatures for 4 h , and FAM111A auto - proteolytic activity was analyzed by immunoblotting with FLAG antibody . H . As in ( G ) , using recombinant human FLAG - FAM111B proteins .

O O O O O O O O O O O O O O O O O O O O O O O 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. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting with FLAG antibody. H. As in (G), using recombinant human FLAG-FAM111B proteins.


I . U2OS cell lines conditionally expressing indicated GFP - FAM111A alleles were fixed at the indicated times after treatment with Doxycycline ( DOX ) to induce expression of the transgenes and stained with crystal violet .

O O O O O O O O O O O O O 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. U2OS cell lines conditionally expressing indicated GFP-FAM111A alleles were fixed at the indicated times after treatment with Doxycycline (DOX) to induce expression of the transgenes and stained with crystal violet.


A . Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP - FAM111A .

O O O O O O O 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. Immunoblot analysis of stable U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of WT or mutant forms of GFP-FAM111A.


B . DNA replication rates in U2OS / GFP - FAM111A cells treated with DOX for the indicated times , pulse - labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image - based cytometry ( QIBC ) ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) . See also Appendix Figure S6A .

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

B. DNA replication rates in U2OS/GFP-FAM111A cells treated with DOX for the indicated times, pulse-labeled with EdU and stained with DAPI were analyzed by quantifying EdU signal intensity in S phase cells using quantitative image-based cytometry (QIBC) (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). See also Appendix Figure S6A.


C . Cells treated as in ( B ) were pre - extracted , fixed and stained with PCNA or MCM2 antibody , and subsequently analyzed by QIBC ( n > 2000 cells per condition ) .

C. Cells treated as in (B) were pre-extracted, fixed and stained with PCNA or MCM2 antibody, and subsequently analyzed by QIBC (n>2000 cells per condition).


D - F . Quantification of data in ( C ) ( red bars , mean ) . Cells in S phase were identified based on EdU positivity . See also Appendix Figure S6B - D .

D-F. Quantification of data in (C) (red bars, mean). Cells in S phase were identified based on EdU positivity. See also Appendix Figure S6B-D.


G . Analysis of FAM111A interactors . U2OS / GFP - FAM111A WT cells were treated or not with DOX for 4 h , subjected to GFP immunoprecipitation ( IP ) and analyzed by mass spectrometry . Volcano plot shows enrichment of individual proteins ( + DOX / - DOX ratio ) plotted against the P value . Dashed lines indicate the significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of FAM111A interactors. U2OS/GFP-FAM111A WT cells were treated or not with DOX for 4 h, subjected to GFP immunoprecipitation (IP) and analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (+DOX/-DOX ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


H . U2OS or U2OS / ∆ FAM111A cells were subjected to IP with IgG ( control ) or RFC1 antibody followed by immunoblotting with indicated antibodies .

O O O O 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. U2OS or U2OS/∆FAM111A cells were subjected to IP with IgG (control) or RFC1 antibody followed by immunoblotting with indicated antibodies.


I . U2OS cells transfected with empty vector ( EV ) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies .

I. U2OS cells transfected with empty vector (EV) or indicated RFC subunit expression plasmids were subjected to FLAG IP and immunoblotted with indicated antibodies.


J . As in ( C ) , except that cells were stained with RFC1 antibody ( n > 2000 cells per condition ) .

O 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. As in (C), except that cells were stained with RFC1 antibody (n>2000 cells per condition).


K . Quantification of data in ( J ) for S phase ( EdU - positive ) cells ( red bars , mean ) .

K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).


A . Immunoblot analysis of U2OS / GFP - FAM111A WT cells treated with DOX for the indicated times .

A. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated with DOX for the indicated times.


B . Representative images of U2OS / GFP - FAM111A cell lines that were treated or not with DOX , fixed and stained with γ - H2AX antibody and DAPI .

B. Representative images of U2OS/GFP-FAM111A cell lines that were treated or not with DOX, fixed and stained with γ-H2AX antibody and DAPI.


C . Cells in ( B ) were subjected to QIBC analysis of γ - H2AX signal intensity ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

O O O O O O O O O O O O O O O O O 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 in (B) were subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


D . Immunoblot analysis of U2OS / GFP - FAM111A WT cells treated or not with DOX and the pan - Caspase inhibitor Z - VAD - FMK as indicated .

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

D. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated or not with DOX and the pan-Caspase inhibitor Z-VAD-FMK as indicated.


Transcriptional activity in U2OS / GFP - FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC ( red bars , mean ; n > 2000 cells per condition ) .

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

Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


Transcriptional activity in U2OS / GFP - FAM111A cells treated or not with DOX pulse - labeled with EU were analyzed by QIBC ( red bars , mean ; n > 2000 cells per condition ) .

Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


Transcriptional activity in U2OS / GFP - FAM111A cells treated or not with DOX pulse - labeled with EU and stained with DAPI were analyzed by QIBC ( red bars , mean ; n > 2000 cells per condition ) .

Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU and stained with DAPI were analyzed by QIBC (red bars, mean; n>2000 cells per condition).


H . U2OS / GFP - FAM111A cell lines treated or not with DOX were pre - extracted , fixed and stained with RPB1 antibody , and analyzed by QIBC ( n > 2000 cells per condition ) . I . Quantification of data in ( H ) ( red bars , mean ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, and analyzed by QIBC (n>2000 cells per condition). I. Quantification of data in (H) (red bars, mean).


U2OS / GFP - FAM111A cell lines treated or not with DOX were pre - extracted , fixed and stained with RPB1 antibody , J . Representative images from the experiment in ( H ) . Scale bars , 10 μm .

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

U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, J. Representative images from the experiment in (H). Scale bars, 10 μm.


B . Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP - FAM111A alleles . U2OS / GFP - FAM111A WT ( low ) cells express the transgene at a lower level than U2OS / GFP - FAM111A WT cells used in Figure 1 and 2 ( see Figure EV4A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of U2OS cell lines left untreated or incubated in the presence of DOX to induce expression of the indicated GFP-FAM111A alleles. U2OS/GFP-FAM111A WT (low) cells express the transgene at a lower level than U2OS/GFP-FAM111A WT cells used in Figure 1 and 2 (see Figure EV4A).


C . U2OS / GFP - FAM111A cell lines treated or not with DOX , pulse - labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC .

O O O O O O O O O O O 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. U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC.


U2OS / GFP - FAM111A cell lines treated or not with DOX , pulse - labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC . D . Quantification of data in ( C ) for S phase ( EdU - positive ) cells ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

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

U2OS/GFP-FAM111A cell lines treated or not with DOX, pulse-labeled with EdU and stained with DAPI were analyzed for DAPI and EdU signal intensity using QIBC. D. Quantification of data in (C) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


U2OS / GFP - FAM111A cell lines treated or not with DOX , and stained with DAPI were analyzed for DAPI signal intensity using QIBC . cells were pulse - labeled with EU .

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

U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. cells were pulse-labeled with EU.


U2OS / GFP - FAM111A cell lines treated or not with DOX , and stained with DAPI were analyzed for DAPI signal intensity using QIBC . Quantification of EU incorporation in cells in ( E ) ( red bars , mean ; n > 2000 cells per condition ) .

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

U2OS/GFP-FAM111A cell lines treated or not with DOX, and stained with DAPI were analyzed for DAPI signal intensity using QIBC. Quantification of EU incorporation in cells in (E) (red bars, mean; n>2000 cells per condition).


G . U2OS / GFP - FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody , pre - extracted and fixed , and stained with DAPI . RFC1 and DAPI signal intensities were analyzed by QIBC .

O O O O O O O O O O O O O O O O O O O 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. U2OS/GFP-FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC.


U2OS / GFP - FAM111A cell lines that were treated or not with DOX were pre - extracted and fixed , and stained with DAPI . DAPI signal intensities were analyzed by QIBC . H . cells were stained with RPB1 antibody .

U2OS/GFP-FAM111A cell lines that were treated or not with DOX were pre-extracted and fixed, and stained with DAPI. DAPI signal intensities were analyzed by QIBC. H. cells were stained with RPB1 antibody.


I . Immunoblot analysis of U2OS / GFP - FAM111A cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK .

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

I. Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK . J . using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles .

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

Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK. J. using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles.


K . Purified recombinant FLAG - FAM111A proteins were incubated at indicated temperatures for 4 h , and FAM111A auto - proteolytic activity was analyzed by immunoblotting .

K. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting.


L . GFP IPs from U2OS cell lines expressing GFP - FAM111A WT or D528G mutant were analyzed by mass spectrometry . Volcano plot shows enrichment of individual proteins ( WT / D528G ratio ) plotted against the P value . Dashed lines indicate the significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GFP IPs from U2OS cell lines expressing GFP-FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


B . U2OS / GFP - FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies . C . As in ( B ) , using U2OS / GFP - FAM111B WT cells .

B. U2OS/GFP-FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies. C. As in (B), using U2OS/GFP-FAM111B WT cells.


D . Purified recombinant FLAG - FAM111A and FLAG - FAM111B proteins were incubated separately or mixed , subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody .

O O O O O O 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. Purified recombinant FLAG-FAM111A and FLAG-FAM111B proteins were incubated separately or mixed, subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody.


E . Immunoblot analysis of U2OS / GFP - FAM111B cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK .

E. Immunoblot analysis of U2OS/GFP-FAM111B cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


F . Cells in ( E ) were pulse - labeled with EdU , stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC .

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

F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.


Cells analyzed using QIBC . G . Quantification of data in ( F ) for S phase ( EdU - positive ) cells ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

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

Cells analyzed using QIBC. G. Quantification of data in (F) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC . H . cells were pulse - labeled with EU .

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

Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC. H. cells were pulse-labeled with EU.


Cells analyzed using QIBC . I . Quantification of EU incorporation in cells in ( H ) ( red bars , mean ; n > 2000 cells per condition ) .

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

Cells analyzed using QIBC. I. Quantification of EU incorporation in cells in (H) (red bars, mean; n>2000 cells per condition).


J . U2OS / GFP - FAM111B cell lines ( Figure 5E ) were fixed at the indicated times after DOX treatment and stained with crystal violet .

J. U2OS/GFP-FAM111B cell lines (Figure 5E) were fixed at the indicated times after DOX treatment and stained with crystal violet.


K . Cells in ( E ) were fixed , immunostained with γ - H2AX antibody and subjected to QIBC analysis of γ - H2AX signal intensity ( red bars , mean ; n > 2000 cells per condition ) .

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

K. Cells in (E) were fixed, immunostained with γ-H2AX antibody and subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean; n>2000 cells per condition).


L . Immunoblot analysis of purified recombinant FLAG - FAM111B proteins and their auto - cleavage products .

L. Immunoblot analysis of purified recombinant FLAG-FAM111B proteins and their auto-cleavage products.


A , miR - 203 expression , as determined by qPCR , in five temporal different stages of normal early development : oocyte , 2 - cell embryo , morula , compacted morula and blastocyst . RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR . RNA expression is normalized by a housekeeping miRNA ( miR - 16 ) that maintained invariable during early embryogenesis . Data represent the mean of 6 different qPCR measures ( red bars ) . P = 0 . 05 ( Student ' s t - test ) comparing 2C / morula versus compacted morula / blastocyst .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, miR-203 expression, as determined by qPCR, in five temporal different stages of normal early development: oocyte, 2-cell embryo, morula, compacted morula and blastocyst. RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR. RNA expression is normalized by a housekeeping miRNA (miR-16) that maintained invariable during early embryogenesis. Data represent the mean of 6 different qPCR measures (red bars). P=0.05 (Student's t-test) comparing 2C/morula versus compacted morula/blastocyst.


F , Representative images of embryoid bodies ( EBs ) derived from wild - type iPSCs or ESCs , or from miiPSC and miESCs at day 30 of differentiation . Scale bars , 500 μm . G , Quantification of the percentage of EBs from panel ( F ) presenting internal large cavities and EBs beating during the indicated time course . Data are represented as mean ± s . e . m . ( n = 3 independent experiments ) . * P < 0 . 05 ; * * * P < 0 . 001 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of embryoid bodies (EBs) derived from wild-type iPSCs or ESCs, or from miiPSC and miESCs at day 30 of differentiation. Scale bars, 500 μm. G, Quantification of the percentage of EBs from panel (F) presenting internal large cavities and EBs beating during the indicated time course. Data are represented as mean ± s.e.m. (n=3 independent experiments). *P<0.05; ***P<0.001 (Student´s t-test).


H , Representative images of EBs derived from human iPSCs transiently transfected with either control ( left ) or miR - 203 mimics ( right ) , at different time points during the differentiation process . Scale bars , 500 μm . I , Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR - 203 mimics as in panel ( H ) at different time points during the differentiation process . The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel . Data are mean ± s . e . m . ( n = 3 independent experiments ) . * * * P < 0 . 001 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of EBs derived from human iPSCs transiently transfected with either control (left) or miR-203 mimics (right), at different time points during the differentiation process. Scale bars, 500 μm. I, Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR-203 mimics as in panel (H) at different time points during the differentiation process. The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel. Data are mean ± s.e.m. (n=3 independent experiments). ***P<0.001 (Student´s t-test).


A , Teratoma volume ( mm3 ) 20 - 25 days after subcutaneous injection of wild - type iPSCs or miiPSCs expressing GFP . Data are represented as mean ± s . e . m . ( n = 8 tumors per genotype ; 292 , 8 ± 66 , 67 versus 1216 ± 140 , 1 ; difference between means : 923 , 5 ± 155 , 2 ; 95 % confidence interval : 1256 to - 590 , 7 ) ; * * * P < 0 . 001 ( 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 O O O O O O O O O O O O O O O O O O O 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, Teratoma volume (mm3) 20-25 days after subcutaneous injection of wild-type iPSCs or miiPSCs expressing GFP. Data are represented as mean ± s.e.m. (n=8 tumors per genotype; 292,8 ± 66,67 versus 1216 ±140,1; difference between means: 923,5 ± 155,2; 95% confidence interval: 1256 to -590,7); ***P<0.001 (Student´s t-test).


B , Incidence and representative images of specific highly differentiated tissues in teratomas . The number of tumors included in the analysis is indicated in the panel . * * * P < 0 . 001 ( Student ´ s t - test ) . 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

B, Incidence and representative images of specific highly differentiated tissues in teratomas. The number of tumors included in the analysis is indicated in the panel. ***P<0.001 (Student´s t-test). Scale bars, 50 μm.


D , Representative example of E - Ls generated after i . p . injection of GFP - expressing miiPSCs . H / E , hematoxylin and eosin . The following antigens were detected by immunohistochemistry : Sox2 ( ectoderm ) , Cd34 ( mesoderm ) , Gata4 ( endoderm ) , AFP and CK8 ( visceral endoderm of the yolk sac ) and Ter119 ( nucleated erythroid cells ) . Scale bars , 500 μm and 100 μm for higher magnifications .

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

D, Representative example of E-Ls generated after i.p. injection of GFP-expressing miiPSCs. H/E, hematoxylin and eosin. The following antigens were detected by immunohistochemistry: Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP and CK8 (visceral endoderm of the yolk sac) and Ter119 (nucleated erythroid cells). Scale bars, 500 μm and 100 μm for higher magnifications.


B , Representative images showing the contribution of human control iPSCs or miiPSCs cells to mouse blastocysts . These structures were co - immunostained with ( human ) anti - OCT4 and anti - HuNu antibodies . Td - Tomato , red ; 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

B, Representative images showing the contribution of human control iPSCs or miiPSCs cells to mouse blastocysts. These structures were co-immunostained with (human) anti-OCT4 and anti-HuNu antibodies. Td-Tomato, red; Scale bars, 50 μm.


C , Interspecies chimera assays in which the 8C - stage mouse embryos indicated in ( A , B ) , injected with one single human iPSCs and cultured in vitro for 48 h to reach the blastocyst stage , were then transfected to 2 . 5 days post coitum pseudo - pregnant females . The post - implantation mouse conceptuses were dissected at the E9 . 5 developmental stage and analyzed by immunofluorescence . Representative images showing the integration of control human iPSCs ( top ) or miiPSCs ( bottom ) into mouse E9 . 5 embryos . Anti - human HuNu antibody was co - stained with Td - Tomato direct fluorescence to detect human cells in E9 . 5 mouse embryos . Scale bars , 20 μm . D , E , Quantification of the Td - Tomato - ( D ) or HuNu - ( E ) positive area in 40 - 50 different cryosections of these embryos randomly and blindly selected for IF analysis . Data are mean ± s . e . m . * * P < 0 . 01 ; * * * P < 0 . 001 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Interspecies chimera assays in which the 8C-stage mouse embryos indicated in (A, B), injected with one single human iPSCs and cultured in vitro for 48 h to reach the blastocyst stage, were then transfected to 2.5 days post coitum pseudo-pregnant females. The post-implantation mouse conceptuses were dissected at the E9.5 developmental stage and analyzed by immunofluorescence. Representative images showing the integration of control human iPSCs (top) or miiPSCs (bottom) into mouse E9.5 embryos. Anti-human HuNu antibody was co-stained with Td-Tomato direct fluorescence to detect human cells in E9.5 mouse embryos. Scale bars, 20 μm. D, E, Quantification of the Td-Tomato-(D) or HuNu-(E) positive area in 40-50 different cryosections of these embryos randomly and blindly selected for IF analysis. Data are mean ± s.e.m. **P<0.01; ***P< 0.001 (Student´s t-test).


B , C , Relative Luciferase Units ( RLU ; normalized to Renilla luciferase and relative to DNA amount ) in 293T cells transfected with DNA constructs carrying the wild - type 3 ´ UTRs from the indicated transcripts ( B ) or the mutated versions of Dnmt3a and Dnmt3b 3 ´ UTRs , downstream of the luciferase reporter ( C ) . Cells were co - transfected with Renilla luciferase as a control of transfection , and a plasmid expressing GFP or miR - 203 - GFP . Data are represented as mean ± s . d . ( n = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C, Relative Luciferase Units (RLU; normalized to Renilla luciferase and relative to DNA amount) in 293T cells transfected with DNA constructs carrying the wild-type 3´UTRs from the indicated transcripts (B) or the mutated versions of Dnmt3a and Dnmt3b 3´UTRs, downstream of the luciferase reporter (C). Cells were co-transfected with Renilla luciferase as a control of transfection, and a plasmid expressing GFP or miR-203-GFP. Data are represented as mean ± s.d. (n=3 independent experiments).


E , Representative images of embryoid bodies ( EBs ) derived from wild - type iPSCs in which the expression of Dnmt3a and Dnmt3b was transiently repressed by siRNAs . Scale bars , 500 μm . Plots show the quantification of the size of EBs and the percentage of EBs with large cavities or beating at different time points during the differentiation process . Data are represented as mean ± s . e . m . ( n = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs in which the expression of Dnmt3a and Dnmt3b was transiently repressed by siRNAs. Scale bars, 500 μm. Plots show the quantification of the size of EBs and the percentage of EBs with large cavities or beating at different time points during the differentiation process. Data are represented as mean ± s.e.m. (n=3 independent experiments).


F , Expression levels of Dnmt3a or Dnmt3b transcripts after transfection of wild - type iPSCs with specific siRNAs either against Dnmt3a , Dnmt3b or a combination of both ( Dnmt3a / b ) ( as indicated in E ) . RNA expression was measured 24 hours after the transfection protocols and was normalized by GAPDH mRNA levels . Data are represented as mean ± s . e . m . ( n = 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 O O O O O O O O O O O O O O O O O O O O O O O O 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, Expression levels of Dnmt3a or Dnmt3b transcripts after transfection of wild-type iPSCs with specific siRNAs either against Dnmt3a, Dnmt3b or a combination of both (Dnmt3a/b) (as indicated in E). RNA expression was measured 24 hours after the transfection protocols and was normalized by GAPDH mRNA levels. Data are represented as mean ± s.e.m. (n=3 independent experiments).


G , Representative images of EBs derived from miiPSCs that were transiently and simultaneously transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors , and simultaneously treated with Dox to induce miR - 203 expression . Scale bars , 500 μm . Plots show the quantification of EB size , percentage of EBs with large cavities , and beating EBs at different time points during differentiation . Data are represented as mean ± s . e . m . ( n = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of EBs derived from miiPSCs that were transiently and simultaneously transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors, and simultaneously treated with Dox to induce miR-203 expression. Scale bars, 500 μm. Plots show the quantification of EB size, percentage of EBs with large cavities, and beating EBs at different time points during differentiation. Data are represented as mean ± s.e.m. (n=3 independent experiments).


H , Expression levels of miR - 203 , Dnmt3a or Dnmt3b transcripts in iPSCs after induction of miR - 203 ( miiPSCs ) and transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors ( as indicated in G ) . RNA expression was measured 24 hours after the transfection protocols and was normalized by a control miRNA ( miR - 142 ) or GAPDH mRNA , respectively . Data are represented as mean ± s . e . m . ( n = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Expression levels of miR-203, Dnmt3a or Dnmt3b transcripts in iPSCs after induction of miR-203 (miiPSCs) and transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors (as indicated in G). RNA expression was measured 24 hours after the transfection protocols and was normalized by a control miRNA (miR-142) or GAPDH mRNA, respectively. Data are represented as mean ± s.e.m. (n=3 independent experiments).


C , DNA methylation distribution of the indicated samples , smoothed over 100 kb - blocks .

C, DNA methylation distribution of the indicated samples, smoothed over 100 kb-blocks.


H , The specific differentially methylated regions ( DMR ) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite - modified DNA . Ten independent clones were sequenced per condition . The quantification of methylated ( filled circles ) vs . unmethylated ( empty circles ) CpGs is shown in the histogram in the indicated conditions .

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 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

H, The specific differentially methylated regions (DMR) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite-modified DNA. Ten independent clones were sequenced per condition. The quantification of methylated (filled circles) vs. unmethylated (empty circles) CpGs is shown in the histogram in the indicated conditions.


I , Heatmaps representing the methylation levels at the top Differentially Methylated Regions ( DMRs ; n = 100 ; upper panel ) or the Imprinting Control Regions ( ICRs ; n = 103 different imprinting loci ; lower panel ) in the indicated samples ( referred to panels A , B ) . Color code and scale of DNA methylation , from 0 . 0 ( red ) to 1 . 0 ( green ) is applicable to both heatmaps .

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

I, Heatmaps representing the methylation levels at the top Differentially Methylated Regions (DMRs; n=100; upper panel) or the Imprinting Control Regions (ICRs; n=103 different imprinting loci; lower panel) in the indicated samples (referred to panels A,B). Color code and scale of DNA methylation, from 0.0 (red) to 1.0 (green) is applicable to both heatmaps.


B , Representative immunofluorescences showing cardiac Troponin T ( cTnT , green ) and nuclei ( Hoechst , blue ) staining of in vitro - generated cardiomyocytes derived from wild - type iPSCs transiently transfected with either control mimics , miR - 203 mimics or miR - 203 mimics + Dnmt3a / b cDNAs . Pictures were taken at day 15 of differentiation . Scale bars , 68 μm . The cTnT - positive area in these cardiomyocytes is shown in the right histogram . Data are represented as mean ± s . d . ( n = 2 independent experiments with 6 replicates each ) .

B, Representative immunofluorescences showing cardiac Troponin T (cTnT, green) and nuclei (Hoechst, blue) staining of in vitro-generated cardiomyocytes derived from wild-type iPSCs transiently transfected with either control mimics, miR-203 mimics or miR-203 mimics + Dnmt3a/b cDNAs. Pictures were taken at day 15 of differentiation. Scale bars, 68 μm. The cTnT-positive area in these cardiomyocytes is shown in the right histogram. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).


C , Beating frequency ( measured as number of beats per 5 seconds ) of these cardiomyocytes at day 15 of differentiation . Data are represented as mean ± s . d . ( n = 8 independent replicates ) .

C, Beating frequency (measured as number of beats per 5 seconds) of these cardiomyocytes at day 15 of differentiation. Data are represented as mean ± s.d. (n=8 independent replicates).


D , miRNA or mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples . Data are represented as mean ± s . d . ( n = 2 independent experiments with 6 replicates each ) .

D, miRNA or mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).


( A ) Expression in E . coli and purification of human GS ( hGS ) . Protein was separated by SDS - PAGE and stained with Coomassie Blue dye . M : marker Precision Plus Protein Dual Color Standard ( Biorad ) . From right Lanes 1 - 4 : E . coli Bl21 ( DE3 ) cells containing the expression vector without ( lanes 1 and 2 ) and with ( lanes 3 and 4 ) the coding sequence of hGS . Samples were taken immediately before ( lanes 1 and 3 ) and 3 , 5 h later ( lanes 2 and 4 ) the induction of expression with isopropil - β - D - 1 - tiogalattopiranoside ( IPTG ) 0 . 7mM . The same number of bacteria was analyzed in each sample . Lane 5 : isolated and purified inclusion bodies ( 4 μg ) . Adiacent boxed lane : Western Blotting analysis of GS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression in E. coli and purification of human GS (hGS). Protein was separated by SDS-PAGE and stained with Coomassie Blue dye. M: marker Precision Plus Protein Dual Color Standard (Biorad). From right Lanes 1-4: E. coli Bl21(DE3) cells containing the expression vector without (lanes 1 and 2) and with (lanes 3 and 4) the coding sequence of hGS. Samples were taken immediately before (lanes 1 and 3) and 3,5 h later (lanes 2 and 4) the induction of expression with isopropil-β-D-1-tiogalattopiranoside (IPTG) 0.7mM. The same number of bacteria was analyzed in each sample. Lane 5: isolated and purified inclusion bodies (4 μg). Adiacent boxed lane: Western Blotting analysis of GS.


Kinetic study of the reaction catalyzed by recombinant hGS . The reaction , started by adding ATP , was linear for at least 10 min at 25 ° C ( B ) .

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

Kinetic study of the reaction catalyzed by recombinant hGS. The reaction, started by adding ATP, was linear for at least 10 min at 25°C (B).


Kinetic study of the reaction catalyzed by recombinant hGS . Lineweaver - Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence ( ) , or in the presence of glufosinate ( C ) Symbols - ( ) : 0 . 025 glufosinate ; or 0 . 050 mM of glufosinate ; or 0 . 065 mM of glufosinate ; 0 . 075 mM of glufosinate .

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

Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of glufosinate (C) Symbols- (): 0.025 glufosinate; or 0.050 mM of glufosinate; or 0.065 mM of glufosinate; 0.075 mM of glufosinate.


Kinetic study of the reaction catalyzed by recombinant hGS . Lineweaver - Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence ( ) , or in the presence of MSO Symbols - ( ) : 2 mM MSO ; ( ) 3 mM of MSO ; ( ) 4 mM of MSO ; ( ) 5 mM of

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

Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of MSO Symbols- (): 2 mM MSO ; () 3 mM of MSO ; () 4 mM of MSO ; () 5 mM of MSO


Kinetic study of the reaction catalyzed by recombinant hGS . The insets represent the secondary plot of the slopes of Lineweaver - Burk plot obtained at the indicated concentrations of glufosinate ( C ) or MSO ( D ) used for determining the inhibitor constant Ki . GS inhibition ( % ) in presence of increasing concentrations of glufosinate ( ) or MSO ( ) results from the average of at least three independent experiments ( E ) . The control value for uninhibited hGS activity is 0 . 51 ± 0 . 08 μmol / ( min x mg protein ) .

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

Kinetic study of the reaction catalyzed by recombinant hGS. The insets represent the secondary plot of the slopes of Lineweaver-Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ±0.08 μmol/(min x mg protein).


Evaluation of M1 markers in macrophages by real - time PCR . Fold change of TNFA , CD80 , CXCL9 , and CXCL10 mRNA in IL10 , MSO - and glufosinate ( 10 and 20µM ) - stimulated IL10 macrophages ( n = 3 ) . Evaluation of M2 markers in macrophages by real - time PCR . Fold change of MRC1 , MSR1 , mRNA in IL10 , MSO - and glufosinate ( 10 and 20µM ) - stimulated IL10 macrophages ( n = 3 ) .

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

Evaluation of M1 markers in macrophages by real-time PCR. Fold change of TNFA, CD80, CXCL9, and CXCL10 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3). Evaluation of M2 markers in macrophages by real-time PCR. Fold change of MRC1, MSR1, mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).


Evaluation of M2 markers in macrophages by real - time PCR . Fold change of CCL17 and CCL18 mRNA in IL10 , MSO - and glufosinate ( 10 and 20µM ) - stimulated IL10 macrophages ( n = 3 ) .

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

Evaluation of M2 markers in macrophages by real-time PCR. Fold change of CCL17 and CCL18 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).


Evaluation of M1 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of TNFA , CXCL10 , CD86 mRNA in IL10 alone or glufosinate ( 10 and 20µM ) - and acriflavine / glufosinate ( 10 and 20µM ) - IL10 macrophages ( n = 3 ) .

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

Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of TNFA, CXCL10, CD86 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).


Evaluation of M1 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of CD80 and CXCL9 mRNA in IL10 alone or glufosinate ( 10 and 20µM ) - and acriflavine / glufosinate ( 10 and 20µM ) - IL10 macrophages ( n = 3 ) . Evaluation of M2 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of MRC1 , MSR1 , and CCL18 mRNA in IL10 , glufosinate ( 10 and 20µM ) - and acriflavine / glufosinate ( 10 and 20µM ) - IL10 macrophages ( n = 3 ) .

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

Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of CD80 and CXCL9 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3). Evaluation of M2 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of MRC1, MSR1, and CCL18 mRNA in IL10, glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).


( Q ) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10 , MSO / IL10 and glufosinate ( 10 and 20µM ) - IL10 treated macrophages after 24 h of incubation ( n = 6 ) .

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

(Q) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10, MSO/IL10 and glufosinate (10 and 20µM)- IL10 treated macrophages after 24 h of incubation (n=6).


( R ) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO / IL10 , and glufosinate ( 10 and 20µM ) / IL10 after 4 h of incubation with HUVEC cells ( n = 6 ) .

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

(R) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 after 4 h of incubation with HUVEC cells (n=6).


( S ) CD8 + T cell suppression by macrophages treated with IL10 or MSO / IL10 , and glufosinate ( 10 and 20µM ) / IL10 for 24 h ( n = 4 ) . Proliferation was evaluated by reading radioactivity as cpm ( counts per minute ) , after incubation with 1 μCi / well tritiated thymidine . The proliferation of T cells cultured without macrophages was used as control .

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

(S) CD8+ T cell suppression by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.


( T ) CD8 + T cell recruitment in a transwell system by macrophages treated with IL10 or MSO / IL10 , and glufosinate ( 10 and 20µM ) / IL10 for 24 h versus macrophages treated with LPS / IFNγ ; the migration of T cells cultured without macrophages ( Mφ - ) in presence of CXCL10 was used as positive control ( n = 4 ) .

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

(T) CD8+ T cell recruitment in a transwell system by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h versus macrophages treated with LPS/IFNγ; the migration of T cells cultured without macrophages (Mφ-) in presence of CXCL10 was used as positive control (n=4).


( U ) Representative image of Western blotting analysis of HIF1α , REDD1 , 4E - BP1 , S6 and P70S6K ( in their phosphorylated and unphosphorilated form ) to test mTOR activation in normoxic ( NRX ) and hypoxic ( HYP ) IL10 macrophages treated with glufosinate ( 20µM ) , rapamycin ( 20nM ) and a combination of both ( n = 3 ) .

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

(U) Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6 and P70S6K (in their phosphorylated and unphosphorilated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20µM), rapamycin (20nM) and a combination of both (n=3).


( V ) CD8 + T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate ( 20µM ) , rapamycin and a combination of both for 24 h ( n = 4 ) . Proliferation was evaluated by reading radioactivity as cpm ( counts per minute ) , after incubation with 1 μCi / well tritiated thymidine . The proliferation of T cells cultured without macrophages was used as control .

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

(V) CD8+ T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate (20µM), rapamycin and a combination of both for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.


Quantification of glufosinate ( B ) levels in wet tumor tissues of vehicle and glufosinate - treated mice ( n = 8 ) , by liquid chromatography - mass spectrometry ( LC - MS ) .

O O B-MEASURED_VAR 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

Quantification of glufosinate (B) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).


Quantification of glutamine ( GLN ) ( C ) levels in wet tumor tissues of vehicle and glufosinate - treated mice ( n = 8 ) , by liquid chromatography - mass spectrometry ( LC - MS ) .

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

Quantification of glutamine (GLN) (C) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).


Subcutaneous LLC tumor growth over the time ( D ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

Subcutaneous LLC tumor growth over the time (D) (pool of 2 independent experiments; 10 mice per condition in total).


number of lung metastatic nodules in vehicle and glufosinate ( 10 and 20mg / kg ) treated mice ( E ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

number of lung metastatic nodules in vehicle and glufosinate (10 and 20mg/kg) treated mice (E) (pool of 2 independent experiments; 10 mice per condition in total).


Quantification and representative images of F4 / 80 + CD11c + cells in the lung metastasis ( F ) , F4 / 80 + CD206 + ( G ) cells infiltration in tumors of vehicle and glufosinate - treated mice ( n = 6 ) .

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

Quantification and representative images of F4/80+ CD11c+ cells in the lung metastasis (F), F4/80+ CD206+ (G) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).


Quantification and representative images of F4 / 80 CD11c + ( H ) cells infiltration in tumors of vehicle and glufosinate - treated mice ( n = 6 ) .

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

Quantification and representative images of F4/80 CD11c+ (H) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).


RT - PCR quantification of M2 ( Ccl22 , Arg1 and Ccl17 ) ( I ) markers in vehicle and glufosinate treated mice ( n = 4 ) .

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

RT-PCR quantification of M2 (Ccl22, Arg1 and Ccl17) (I) markers in vehicle and glufosinate treated mice (n=4).


RT - PCR quantification of M1 ( Tnfa , Cxcl9 , Nos2 , Cd86 and Cd80 ) ( J ) markers in vehicle and glufosinate treated mice ( n = 4 ) .

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

RT-PCR quantification of M1 (Tnfa, Cxcl9, Nos2, Cd86 and Cd80) (J) markers in vehicle and glufosinate treated mice (n=4).


Quantification of total length of vessels ( A ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

Quantification of total length of vessels (A) in vehicle and glufosinate (20mg/kg) treated mice (n=6).


Quantification of CD31 + tumor vessel area ( B ) and of αSMA + pericyte - covered vessels over the total number of CD31 + vessels ( with representative images ) ( C ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

Quantification of CD31+ tumor vessel area (B) and of αSMA+ pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( D ) Quantification and representative images of Lectin + tumor vessel area over the total number of CD31 + vessels in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(D) Quantification and representative images of Lectin+ tumor vessel area over the total number of CD31+ vessels in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( E ) Quantification and representative images of PIMO + tumor hypoxic areas in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(E) Quantification and representative images of PIMO+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS quantification of TCRbeta + CD8 + cytotoxic T cells in tumors of vehicle and glufosinate - treated mice ( F ) .

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

FACS quantification of TCRbeta+ CD8+ cytotoxic T cells in tumors of vehicle and glufosinate-treated mice (F).


Evaluation of CD69 ( G ) and CD25 ( H ) proteins , expressed as Mean Fluorescence Intensity ( MFI ) , was determined by flow cytometry on the surface of responders CD8 + T cells ( n = 4 ) .

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

Evaluation of CD69 (G) and CD25 (H) proteins, expressed as Mean Fluorescence Intensity (MFI), was determined by flow cytometry on the surface of responders CD8+ T cells (n=4).


( I ) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate - treated mice ( n = 6 ) .

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

(I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).


( B ) Evaluation of body weight in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 8 ) . ( C ) Representative images of metastasis at the beginning and end of therapy . Evaluation of the metastatic burden following glufosinate treatment by H & E staining ( n = 8 ) .

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

(B) Evaluation of body weight in vehicle and glufosinate (20mg/kg) treated mice (n=8). (C) Representative images of metastasis at the beginning and end of therapy. Evaluation of the metastatic burden following glufosinate treatment by H&E staining (n=8).


( D ) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H & E staining in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 8 ) .

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

(D) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H&E staining in vehicle and glufosinate (20mg/kg) treated mice (n=8).


Quantification ( F ) of total metastatic area in LLC - tumor bearing mice preconditioned with IgG or anti - CSF1R antibody and treated with vehicle or glufosinate ( 20mg / Kg ) .

O O O 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

Quantification (F) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).


representative images ( G ) of total metastatic area in LLC - tumor bearing mice preconditioned with IgG or anti - CSF1R antibody and treated with vehicle or glufosinate ( 20mg / Kg ) .

O O O O 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

representative images (G) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).


Intradermal YUMM 1 . 7 ( CD90 . 1 + ) tumor growth over the time ( B ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

Intradermal YUMM 1.7 (CD90.1+) tumor growth over the time (B) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


number of metastatic lesions per lung ( C ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

number of metastatic lesions per lung (C) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


( D ) Representative images of metastatic lesions checked by DAB - H stain for CD90 . 1 positivity in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per lung were analysed . Scale bar : 2mm . Zoomed area scale bar : 50µm .

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

(D) Representative images of metastatic lesions checked by DAB-H stain for CD90.1 positivity in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per lung were analysed. Scale bar: 2mm. Zoomed area scale bar: 50µm.


( E , F ) FACS quantification of M1 - like MHC class IIhigh ( E ) and MHC class IIlow ( F ) CD11c + CD206 - TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(E, F) FACS quantification of M1-like MHC class IIhigh (E) and MHC class IIlow (F) CD11c+ CD206- TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( G ) Quantification and representative images of CD11c + cells over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . 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 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

(G) Quantification and representative images of CD11c+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.


FACS quantification of M2 - like MHC class IIhigh CD11c - CD206 + TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

O O O 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

FACS quantification of M2-like MHC class IIhigh CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS quantification of M2 - like MHC class IIlow ( I ) CD11c - CD206 + TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

O O O O O O 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

FACS quantification of M2-like MHC class IIlow (I) CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( J ) Quantification and representative images of CD206 + cells over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bars : 50 µm .

(J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.


FACS quantification of M1 - like MHC class IIhigh ( K ) TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

O O 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

FACS quantification of M1-like MHC class IIhigh (K) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS quantification of M1 - like M2 - like MHC class IIlow ( L ) TAMs over the total number of F4 / 80 + cells in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

O O O O O 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

FACS quantification of M1-like M2-like MHC class IIlow (L) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( M ) Quantification of MHC - II protein , expressed as Mean Fluorescence Intensity ( MFI ) , on F4 / 80 + cells , was obtained by flow cytometry ( n = 6 ) .

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

(M) Quantification of MHC-II protein, expressed as Mean Fluorescence Intensity (MFI), on F4/80+ cells, was obtained by flow cytometry (n=6).


( N ) RT - PCR quantification of M2 ( Arg1 and Cxcr4 ) and M1 ( Cxcl9 and Nos2 ) markers in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

O 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

(N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).


Quantification of total length of vessels ( A ) Six images per tumor were analysed . Scale bar : 50µm .

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

Quantification of total length of vessels (A) Six images per tumor were analysed. Scale bar: 50µm.


CD31 + tumor vessel area ( B ) and of αSMA + pericyte - covered vessels over the total number of CD31 + vessels ( with representative images ) ( C ) in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bar : 50µm .

O O O O O O O O O O O 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

CD31+ tumor vessel area (B) and of αSMA+pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.


( D ) Quantification and representative images of CA9 + tumor hypoxic areas in vehicle and glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six images per tumor were analysed . Scale bar : 50µm .

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

(D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.


( E , F ) FACS quantification of CD8 + cytotoxic T cells ( E ) and CD69 + CD8 + T cells ( F ) over the total number of CD45 + in vehicle or glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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

(E, F) FACS quantification of CD8+ cytotoxic T cells (E) and CD69+ CD8+ T cells (F) over the total number of CD45+ in vehicle or glufosinate (20mg/kg) treated mice (n=6).


Evaluation of 4T1 tumor growth over the time ( B ) , in vehicle and glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

Evaluation of 4T1 tumor growth over the time (B) , in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


mice body weight ( C ) in vehicle and glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

mice body weight (C) in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


number of metastatic nodules ( D ) , in vehicle and glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

number of metastatic nodules (D), in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


Total metastatic area ( E ) in vehicle and glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . Six images per lung were analysed . Scale bar : 2mm .

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

Total metastatic area (E) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.


number of lung metastatic lesions per size by H & E staining ( F ) with representative images ( G ) in vehicle and glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . Six images per lung were analysed . Scale bar : 2mm .

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

number of lung metastatic lesions per size by H&E staining (F) with representative images (G) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.


( H , I ) Quantification of F4 / 80 + macrophage density ( H ) , of F4 / 80 + CD206 + cells infiltration in the lung metastasis and representative images ( I ) , in vehicle and glufosinate - treated mice ( n = 6 ) . Six images per lung were analysed . Scale bar : 20µm .

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

(H, I) Quantification of F4/80+ macrophage density (H), of F4/80+ CD206+ cells infiltration in the lung metastasis and representative images (I), in vehicle and glufosinate-treated mice (n=6). Six images per lung were analysed. Scale bar: 20µm.


GFP + CTCs amount in blood , expressed as GFP expression levels , was determined by qPCR ( n = 8 ) ( K ) .

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

GFP+ CTCs amount in blood, expressed as GFP expression levels, was determined by qPCR (n=8) (K).


Blood was drawn from treated and untreated tumor - bearing mice at the time of tumor resection and plated in culture dishes . The formation of tumor cell colonies was traced over time . Graph shows the number of GFP + tumor cell colonies ( n = 8 ) ( L ) .

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

Blood was drawn from treated and untreated tumor-bearing mice at the time of tumor resection and plated in culture dishes. The formation of tumor cell colonies was traced over time. Graph shows the number of GFP+ tumor cell colonies (n=8) (L).


After treatment and tumor resection overall survival studies were performed until the natural death of the post - surgical mice ( n = 11 ) ( M ) .

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

After treatment and tumor resection overall survival studies were performed until the natural death of the post-surgical mice (n=11) (M).


( O ) GFP + cancer cells amount in lungs , expressed as GFP expression levels , was determined by qPCR in vehicle and glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . The results were normalized to the vehicle 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 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) GFP+ cancer cells amount in lungs, expressed as GFP expression levels, was determined by qPCR in vehicle and glufosinate (10mg/kg) treated mice (n=8). The results were normalized to the vehicle group.


( A ) Time course evaluation of murine body weight during treatment in vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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

(A) Time course evaluation of murine body weight during treatment in vehicle and glufosinate (10 and 20 mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


( B ) Blood counts of WBC and RBC in vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( n = 8 ) .

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

(B) Blood counts of WBC and RBC in vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of liver toxicity following glufosinate treatment . Quantification of liver glufosinate ( C ) in serum of vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( n = 8 ) .

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

Evaluation of liver toxicity following glufosinate treatment. Quantification of liver glufosinate (C) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of liver toxicity following glufosinate treatment . hepatotoxicity markers ALT ( Alanine Aminotransferase ) and AST ( Aspartate aminotransferase ) ( D ) in serum of vehicle and glufosinate ( 10 and 20 mg / kg ) treated mice ( n = 8 ) .

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

Evaluation of liver toxicity following glufosinate treatment. hepatotoxicity markers ALT (Alanine Aminotransferase) and AST (Aspartate aminotransferase) (D) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of neuronal toxicity following glufosinate treatment . Quantification of brain glufosinate ( E )

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

Evaluation of neuronal toxicity following glufosinate treatment. Quantification of brain glufosinate (E)


Evaluation of neuronal toxicity following glufosinate treatment . grip strength for front paws ( triangular bar ) ( F ) , as an indicator of the development of tactile allodynia in vehicle and glufosinate - treated mice ( n = 4 ) , before the treatment ( baseline ) , after 1 day ( acute Toxicity ) and after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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 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

Evaluation of neuronal toxicity following glufosinate treatment. grip strength for front paws (triangular bar) (F), as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).


Evaluation of neuronal toxicity following glufosinate treatment . grip strength for all paws ( rectangle grid ) ( G ) , of the latency to fall off an accelerating rotarod ( H ) , and of the mean paw withdrawal force that caused animals ' response in the von Frey test ( left hindpaw , I ; right hindpaw , J ) as an indicator of the development of tactile allodynia in vehicle and glufosinate - treated mice ( n = 4 ) , before the treatment ( baseline ) , after 1 day ( acute Toxicity ) and after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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 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

Evaluation of neuronal toxicity following glufosinate treatment. grip strength for all paws (rectangle grid) (G), of the latency to fall off an accelerating rotarod (H), and of the mean paw withdrawal force that caused animals' response in the von Frey test (left hindpaw, I; right hindpaw, J) as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).


Release of IL - 6 and / or TNF release ( ELISA ) from ( C ) human MoMacs ( n = 6 - 8 ) Data information represent data ( mean + SEM ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicates ( human donor * p < 0 . 05 according to Wilcoxon signed rank sum (

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

Release of IL-6 and/or TNF release (ELISA) from (C) human MoMacs (n=6-8) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicates (human donor * p<0.05 according to Wilcoxon signed rank sum (


Release of IL - 6 and / or TNF release ( ELISA ) fro ( D ) murine BMDMs ( n = 6 Data information represent data ( mean + SEM ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate mic * p < 0 . 05 according to Wilcoxon signed rank sum ( C ,

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

Release of IL-6 and/or TNF release (ELISA) fro (D) murine BMDMs (n=6 Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic * p<0.05 according to Wilcoxon signed rank sum (C, D


Release of IL - ( ELISA ) fro THP - 1 cells ( n = 3 ) Data information I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

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

Release of IL- (ELISA) fro THP-1 cells (n=3) Data information I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


( f ) GFP in vivo fluorescence post intradermal injection of chitin oligomers into LysMEGFP / + C57BL / 6 mice ( n = 3 / group ) Data information represent data ( mean + SEM ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate

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

(f) GFP in vivo fluorescence post intradermal injection of chitin oligomers into LysMEGFP/+ C57BL/6 mice (n=3/group) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic


( G ) Representative in vivo imaging results I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( 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

(G) Representative in vivo imaging results I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD)


Leukocyte infiltration in vivo after intratracheal administration assessed by ( H ) BALF flow cytometry ( n = 3 - 4 / group Data information represent data ( mean + SEM ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate mic ) , one - way ANOVA (

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

Leukocyte infiltration in vivo after intratracheal administration assessed by (H) BALF flow cytometry (n=3-4/group Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate mic ), one-way ANOVA (H


Leukocyte infiltration in vivo after intratracheal administration assessed b ( I ) histology analysis ( n = 2 ) , scale bar = 200 μm , arrows show cellular infiltrate I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( 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

Leukocyte infiltration in vivo after intratracheal administration assessed b (I) histology analysis (n=2), scale bar = 200 μm, arrows show cellular infiltrate I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD)


A . thaliana seedling ( J ) target gene mRNA induction ( n = 3 I one representative of ' n ' ( given in brackets for each panel ) independent experiments is shown ( mean + SD ) Student ' s t -

O O O O O O O O O O O O O O O O O O O O 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. thaliana seedling (J) target gene mRNA induction (n=3 I one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD) Student's t-tes


A . thalian leaf piece ( K ) ROS production ( 0 - 45 min , n = 6 ) Data information represent data ( mean + SEM ) combined from ' n ' ( given in brackets for each panel ) technical or biological replicate plant

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

A. thalian leaf piece (K) ROS production (0-45 min, n=6) Data information represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicate plant leave


TNF released from WT or Myd88 ( A KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Data information : I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate mic Student ' t -

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

TNF released from WT or Myd88 (A KO BMDM (total n=6-9/group combined from 3 experiments) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate mic Student' t-test


TNF released from W or Tlr KO BMDM ( total n = 6 - 9 / group combined from 3 experiments ) Student ' s t -

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

TNF released from W or Tlr KO BMDM (total n=6-9/group combined from 3 experiments) Student's t-tes


( C ) IL - 6 released from primary MoMacs ( n = 5 ) treated with non - targeting ( NT ) , TLR2 - or MyD88 - specific siRNA relative to NT condition one - sample t -

O O O O O O O O O O O O O O O O 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) IL-6 released from primary MoMacs (n=5) treated with non-targeting (NT), TLR2- or MyD88-specific siRNA relative to NT condition one-sample t-tes


( D ) CD62L shedding from human PMNs ( n = 4 - 8 ) with or without isotype control or anti - TLR2 blocking antibody Data information : I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD62L shedding from human PMNs (n=4-8) with or without isotype control or anti-TLR2 blocking antibody Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( E ) IL - 6 protein released from mock or TLR2 Cas9 - CRISPR - edited THP - 1 cells ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD ) ) , Student ' s t -

(E) IL-6 protein released from mock or TLR2 Cas9-CRISPR-edited THP-1 cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD) ), Student's t-tes


( F ) NF - κB activity in empty vector ( EV ) or TLR2 - transfected HEK293T cells ( n = 3 ) I one representative of ' n ' independent experiments is shown ( mean + SD )

(F) NF-κB activity in empty vector (EV) or TLR2-transfected HEK293T cells (n=3) I one representative of 'n' independent experiments is shown (mean+SD)


( G ) MPO release from intradermally injected WT or Tlr2 KO mice ( n = 5 - 15 biopsies each ) Data information I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate mic ) , Student ' s t -

(G) MPO release from intradermally injected WT or Tlr2 KO mice (n=5-15 biopsies each) Data information I represent data (mean+SEM) combined from 'n' technical or biological replicate mic ), Student's t-tes


( H ) BALF IL - 6 and TNF from intratracheally treated WT or Tlr2 KO mice ( n = 4 - 5 mice each ) one - way ANOVA with Dunnett ' s multiple compariso

O O O O O O O O O O O O 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) BALF IL-6 and TNF from intratracheally treated WT or Tlr2 KO mice (n=4-5 mice each) one-way ANOVA with Dunnett's multiple compariso


( I ) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers ( n = 5 each ) Data information : I represent data ( mean + SEM ) combined from ' n ' technical or biological replicate ( human donor * p < 0 . 05 according to Mann -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers (n=5 each) Data information: I represent data (mean+SEM) combined from 'n' technical or biological replicate (human donor * p<0.05 according to Mann-Whitney


( A ) NF - κB activity in TLR2 - transfected HEK293T cells ( n = 3 ) Data information I one representative of ' n ' independent experiments is shown ( mean + SD ) Student ' s t -

O O O O O O O O O O O O O O O O O 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) NF-κB activity in TLR2-transfected HEK293T cells (n=3) Data information I one representative of 'n' independent experiments is shown (mean+SD) Student's t-tes


( B ) IL - 8 release and ROS production from primary PMNs ( n = 7 ) Data information represent data ( mean + SEM ) from ' n ' technical or biological replicates ( donors ) * p < 0 . 05 according to Wilcoxon signed rank

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

(B) IL-8 release and ROS production from primary PMNs (n=7) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su