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f3a-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).


f4a , 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 ) . 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 .

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f4a, 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).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.


Figure 1G . 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 .

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Figure 1G. 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.


Figure 2A . 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 . 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 . 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 . 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 ) . K . Quantification of data in ( J ) for S phase ( EdU - positive ) cells ( red bars , mean ) .

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Figure 2A. 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.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.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.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).K. Quantification of data in (J) for S phase (EdU-positive) cells (red bars, mean).


Figure 3A . 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 . 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 . 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 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 ) . 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 .

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Figure 3A. 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.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.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 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). 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.


Figure 4B . 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 . 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 . 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 . 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 . 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 . 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 ) .

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Figure 4B. 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.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.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.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.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.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).


Figure 5B . 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 . 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 . 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 . 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 . 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 .

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Figure 5B. 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.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.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.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.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.


Figure 1A , 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 ) . 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 ) .

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Figure 1A, 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). 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).


Figure 2A , 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, 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.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.


Figure 3B , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B, 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).


Figure 4B , 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 ) . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B, 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).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).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).


Figure 5C , 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C, 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.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.


Figure 6B , 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B, 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).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).


Figure 1 ( 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 ) . 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 MSOKinetic 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).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 MSOKinetic 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).


Figure 2Evaluation 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 ) . 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 ) . ( 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 ) . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Evaluation 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).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).(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).(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).(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.


Figure 3Quantification 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 ) . 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 ) . 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 ) . 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 ) .

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Figure 3Quantification 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).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).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).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).


Figure 4Quantification 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 ) . ( 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 ) . 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 ) . ( I ) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate - treated mice ( n = 6 ) .

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Figure 4Quantification 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).(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).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).(I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).


Figure 5 ( 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 ) . 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 ) .

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Figure 5(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).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).


Figure 6Intradermal 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 ) . ( 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 ) . ( 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 ) . 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 . 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 ) . ( 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 ) .

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Figure 6Intradermal 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).(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).(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).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.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).(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).


Figure 7Quantification 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 . ( 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 ) .

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Figure 7Quantification 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.(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).


Figure 8Evaluation 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 ) . 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 . 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 . 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 ) . 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 .

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Figure 8Evaluation 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).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.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.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).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.


Figure 9 ( 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 ) . 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 ) . 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 ) . 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 ) .

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Figure 9(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).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).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).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).


Figure 1Release 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 , DRelease 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 mic ( 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 ( HLeukocyte 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 - tesA . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Release 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, DRelease 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 mic(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 (HLeukocyte 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-tesA. 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


Figure 2TNF 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 - testTNF 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 - 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 - 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 - 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 ) ( 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 ( 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 -

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Figure 2TNF 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-testTNF 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-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-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-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)  (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  (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


Figure 3 ( 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 su ( D ) IRAK4 S186 and T345 phospho - site containing IRAK4 peptides quantified in proteome - wide phospho - screen in THP - 1 cells ( n = 1 ) Data informatio I one representative of ' n ' independent experiments is shown ( mean + SD ) for details on data acquisition and analysis for panel see Expanded View Information ( F ) Microarray intensities ( excerpt ) of chitin - specific signature genes ( n = 5 ) Data information represent data ( mean + SEM ) from ' n ' technical or biological replicates ( donors ) for details on data acquisition and analysis for panels ( D - F ) see Expanded View Information ( G ) Relative cytokine mRNAs in whole blood stimulation ( n = 5 - 13 ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (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 su (D) IRAK4 S186 and T345 phospho-site containing IRAK4 peptides quantified in proteome-wide phospho-screen in THP-1 cells (n=1) Data informatio I one representative of 'n' independent experiments is shown (mean+SD) for details on data acquisition and analysis for panel see Expanded View Information (F) Microarray intensities (excerpt) of chitin-specific signature genes (n=5) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) for details on data acquisition and analysis for panels (D-F) see Expanded View Information (G) Relative cytokine mRNAs in whole blood stimulation (n=5-13) Data information represent data (mean+SEM) from 'n' technical or biological replicates (donors) * p<0.05 according to Wilcoxon signed rank su


Figure 4Flow cytometric quantificatio of Alexa647 - labeled C10 - 15 interaction with mTLR2 - Fc Protein ( n = 4 each ) Data information : Quantification sub - panel represent data ( mean + SE combined from ' n ' biological replicate mic I one representative of ' n ' independent experiments is show p < 0 . 05 according to Mann - Whitney U ( Amicroscale thermophoresis ( MST ) analysis ( B ) of Alexa647 - labeled C10 - 15 interaction with mTLR2 - Fc Protein ( n = 4 each ) I one representative of ' n ' independent experiments is show ( C ) Flow cytometr of Candida albicans stained with control IgG or mTLR2 - Fc anti - Fc - Alexa594 , together with ConA - Alexa488 and CFW ( n = 4 each ) . Scale bar = 5 μm Data information : Quantification sub - panel represent data ( mean + SE combined from ' n ' biological replicate mic I one representative of ' n ' independent experiments is show one - sample t - test ( C ( D ) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2 - Fc anti - Fc - Alexa594 , together with ConA - Alexa488 and CFW ( n = 4 each ) . Scale bar = 5 μm I one representative of ' n ' independent experiments is show ( g ) MST analysis of Alexa647 - labeled C10 - 15 and mTLR2 - Fc protein with SSL3 titration ( n = 2 ) Data information I one representative of ' n ' independent experiments is show ( H ) NF - κB activation in TLR2 - transfected HEK293T cells ( n = 3 Data information I one representative of ' n ' independent experiments is shown ( for asmean + SD ) ) , Student ' s t - tesIL - 8 release from primary PMNs ( n = 2 ) without or with SSL3 Data information I one representative of ' n ' independent experiments is show asmean + SD ) Student ' s t - tes ( J ) BALF TNF in C57BL / 6 mice ( n = 5 / group ) upon C10 - 15 administration without or with SSL3 Data information : Quantificatio - panel represent data ( mean + SE combined from ' n ' biological replicate mic one - way ANOVA with Dunnett ' s multiple comparison ( J ) ( K ) ROS production between 0 and 45 min post chitin application in A . thaliana leaf pieces ( n = 6 ) Data information : Quantificatio panels ( represent dat mean ± SD in K combined from ' n ' biological replicate plant leave ( L ) MST analysis of Alexa647 - labeled C10 - 15 and mTLR2 - Fc Protein with C5 titration ( n = 2 ) Data informatio I one representative of ' n ' independent experiments is

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Flow cytometric quantificatio of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show p<0.05 according to Mann-Whitney U (Amicroscale thermophoresis (MST) analysis (B) of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each) I one representative of 'n' independent experiments is show(C) Flow cytometr of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm Data information: Quantification sub-panel represent data (mean+SE combined from 'n' biological replicate mic I one representative of 'n' independent experiments is show one-sample t-test (C(D) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm I one representative of 'n' independent experiments is show (g) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc protein with SSL3 titration (n=2) Data information I one representative of 'n' independent experiments is show(H) NF-κB activation in TLR2-transfected HEK293T cells (n=3 Data information I one representative of 'n' independent experiments is shown (for asmean+SD) ), Student's t-tesIL-8 release from primary PMNs (n=2) without or with SSL3 Data information I one representative of 'n' independent experiments is show asmean+SD) Student's t-tes (J) BALF TNF in C57BL/6 mice (n=5/group) upon C10-15 administration without or with SSL3 Data information: Quantificatio -panel represent data (mean+SE combined from 'n' biological replicate mic one-way ANOVA with Dunnett's multiple comparison (J) (K) ROS production between 0 and 45 min post chitin application in A. thaliana leaf pieces (n=6) Data information: Quantificatio panels ( represent dat mean±SD in K combined from 'n' biological replicate plant leave (L) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc Protein with C5 titration (n=2) Data informatio I one representative of 'n' independent experiments is show


f1 ( b , c ) Effects of miR - 212 and miR - 132 precursors and inhibitors ( anti ) on cardiomyocyte cell size as compared with the effects of scrambled ( Scr ) controls ( n = 5 - 13 ) . Representative images used for quantification of cardiomyocyte cell size are shown in c . ( d ) Effects of various pro - hypertrophic stimuli on miR - 212 and miR - 132 expression in neonatal cardiomyocytes ( n = 6 - 10 ) . ( e ) miR - 212 and miR - 132 expression levels during pressure - induced left ventricular hypertrophy 3 and 21 days after transaortic constriction ( TAC ) surgery of mice ( n = 4 ) . ( f ) Cardiomyocyte diameters after Sham operation or 3 , 14 and 35 days after TAC ( n = 4 per group ) . ( g , h ) miR - 212 and miR - 132 expression ( normalized to sno - 202 levels ) in fractionated cardiomyocytes and cardiac fibroblasts derived from adult mice after 3 and 35 days of TAC . All values represent mean ± s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 005 . Scale bar in c represents 50 μm . ACTN2 , α - cardiac actinin ; Ang II , angiotensin - 2 ; a . u . , arbitrary unit ; d , day ; DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; FC , fold change ; NS , no significant difference ; P / I , phenylephrine / isoprenaline .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(b,c) Effects of miR-212 and miR-132 precursors and inhibitors (anti) on cardiomyocyte cell size as compared with the effects of scrambled (Scr) controls (n=5-13). Representative images used for quantification of cardiomyocyte cell size are shown in c.(d) Effects of various pro-hypertrophic stimuli on miR-212 and miR-132 expression in neonatal cardiomyocytes (n=6-10).(e) miR-212 and miR-132 expression levels during pressure-induced left ventricular hypertrophy 3 and 21 days after transaortic constriction (TAC) surgery of mice (n=4).(f) Cardiomyocyte diameters after Sham operation or 3, 14 and 35 days after TAC (n=4 per group).(g,h) miR-212 and miR-132 expression (normalized to sno-202 levels) in fractionated cardiomyocytes and cardiac fibroblasts derived from adult mice after 3 and 35 days of TAC. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bar in c represents 50 μm. ACTN2, α-cardiac actinin; Ang II, angiotensin-2; a.u., arbitrary unit; d, day; DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; NS, no significant difference; P/I, phenylephrine/isoprenaline.