Figure 2 ( A ) RCC1 . 24 ‐ NeoR and ‐ NucNeoR cells were incubated for 48 h with IFN ‐ γ and in addition with 7 . 5 mM 3 ‐ MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 ‐ 4 / A4 cells in a GM ‐ CSF release assay . Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing . ( B ) LCL1 . 11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise . Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors , indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing . ( C ) To exclude toxic side effects of the inhibitors , LCL1 . 11 cells were incubated for 20 h with 200 ng / mL recombinant NeoR protein in the presence or absence of the inhibitors . Afterwards , residual protein and inhibitors were removed by washing and the cells were co ‐ cultured with the NeoR ‐ specific CD4 + T cell clone 20 ‐ 4 / A4 . The presentation of exogenous NeoR on MHC class II was not affected by 3 ‐ MA , but was impaired when lysosomal processing was blocked with chloroquine or leupeptin . ( D ) LCL1 . 11 cells were transfected with siRNA specific for Atg12 or GFP , and Atg12 mRNA levels were determined 24 h later by qPCR . The relative amount of Atg12 mRNA was reduced approximately fourfold . ( E ) At 24 h after siRNA transfection , NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later . In cells transfected with Atg12 siRNA , but not in mock ‐ transfected cells or cells transfected with GFP siRNA , antigen presentation of both NeoR and NucNeoR was greatly reduced .

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

Figure 2(A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing.(B) LCL1.11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise. Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors, indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing.(C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin.(D) LCL1.11 cells were transfected with siRNA specific for Atg12 or GFP, and Atg12 mRNA levels were determined 24 h later by qPCR. The relative amount of Atg12 mRNA was reduced approximately fourfold.(E) At 24 h after siRNA transfection, NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with Atg12 siRNA, but not in mock‐transfected cells or cells transfected with GFP siRNA, antigen presentation of both NeoR and NucNeoR was greatly reduced.


Figure 3 ( A ) RCC1 . 24 cells were transfected with the NES ‐ GFP ‐ NeoR ‐ GFP ‐ NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence . As compared to untreated cells ( a ) , cells treated with 8 nM LMB for 20 h ( b ) showed a strong accumulation of the protein in the nucleus . ( a ' ) and ( b ' ) are the corresponding daylight photographs of the transfected cells . ( B ) NeoR ‐ and NucNeoR ‐ transfected LCL1 . 11 cells were either left untreated or incubated for 20 h with 8 nM LMB . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and probed with the NeoR ‐ specific T cells . LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II , indicating that CRM1 ‐ dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RCC1.24 cells were transfected with the NES‐GFP‐NeoR‐GFP‐NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence. As compared to untreated cells (a), cells treated with 8 nM LMB for 20 h (b) showed a strong accumulation of the protein in the nucleus. (a') and (b') are the corresponding daylight photographs of the transfected cells.(B) NeoR‐ and NucNeoR‐transfected LCL1.11 cells were either left untreated or incubated for 20 h with 8 nM LMB. Subsequently, the cells were fixed with 0.5% paraformaldehyde and probed with the NeoR‐specific T cells. LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II, indicating that CRM1‐dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II.


Figure 1A . qRT - PCR analysis of RNA samples from normal human skin ( NS ) and AK for activin βA ( INHBA ) , normalized to hypoxanthine phosphoribosyltransferase ( HPRT ) . Expression levels in one of the normal skin samples were set to 1 . Scatter plots are shown . NS : N = 4 ; AK : N = 21 . ns = non - significant ( Mann - Whitney test ) . B . qRT - PCR analysis of RNA samples from normal back skin of wild - type mice ( N = 4 , n = 4 ) , non - tumorigenic back skin from HPV8 / wt mice ( N = 7 , n = 9 ) and papillomas from HPV8 / wt transgenic mice ( N = 8 , n = 11 ) for Inhba , normalized to glyceraldehyde 3 - phosphate dehydrogenase ( Gapdh ) . Expression levels in one of the wild - type back skin samples were set to 1 . Statistical significance was determined using 1 - way ANOVA and Bonferroni ` s Multiple Comparisons Test of log - transformed data . N , number of mice ; n , number of biopsies . C - E . Sections from a tumor collected from a 64 week - old HPV8 / wt mouse ( C , D ) or from non - tumorigenic skin of 5 week - old HPV8 / wt mice ( E ) stained with an anti - activin A antibody and counterstained with hematoxylin . E : Epidermis , D : Dermis , HF : Hair follicle . ( D ) Shows a higher magnification of the boxed area in ( C ) . Scale bars : 200μm ( C ) and 100μm ( D , E ) . F . Kinetics of tumor incidence in HPV8 / wt and HPV8 / Act mice ; N = 20 mice per genotype . Statistical significance was determined using Log - rank ( Mantel - Cox ) test . G . Age of the mice when the first tumors appeared on certain body sites . Each dot represents one tumor . H . Representative macroscopic pictures of ear tumors ( left panel ) and H & amp ; amp ; E - stained sections of ear acanthopapillomas ( upper and middle panel ) or an ear acanthopapilloma with trichoepitheliomatous differentiation ( lower panel ) . Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel . E : epidermis ; D : dermis ; C : cartilage . Scale bars : 200μm ( left panel ) , 100μm ( middle panel ) or 50μm ( right panel ) .

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 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 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 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 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

Figure 1A. qRT-PCR analysis of RNA samples from normal human skin (NS) and AK for activin βA (INHBA), normalized to hypoxanthine phosphoribosyltransferase (HPRT). Expression levels in one of the normal skin samples were set to 1. Scatter plots are shown. NS: N=4; AK: N=21. ns = non-significant (Mann-Whitney test).B. qRT-PCR analysis of RNA samples from normal back skin of wild-type mice (N=4, n=4), non-tumorigenic back skin from HPV8/wt mice (N=7, n=9) and papillomas from HPV8/wt transgenic mice (N=8, n=11) for Inhba, normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Expression levels in one of the wild-type back skin samples were set to 1. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test of log-transformed data. N, number of mice; n, number of biopsies.C-E. Sections from a tumor collected from a 64 week-old HPV8/wt mouse (C,D) or from non-tumorigenic skin of 5 week-old HPV8/wt mice (E) stained with an anti-activin A antibody and counterstained with hematoxylin. E: Epidermis, D: Dermis, HF: Hair follicle. (D) Shows a higher magnification of the boxed area in (C). Scale bars: 200μm (C) and 100μm (D,E).F. Kinetics of tumor incidence in HPV8/wt and HPV8/Act mice; N=20 mice per genotype. Statistical significance was determined using Log-rank (Mantel-Cox) test.G. Age of the mice when the first tumors appeared on certain body sites. Each dot represents one tumor.H. Representative macroscopic pictures of ear tumors (left panel) and H&amp;E-stained sections of ear acanthopapillomas (upper and middle panel) or an ear acanthopapilloma with trichoepitheliomatous differentiation (lower panel). Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel. E: epidermis; D: dermis; C: cartilage. Scale bars: 200μm (left panel), 100μm (middle panel) or 50μm (right panel).


Figure 2A , B . Representative pictures of ear sections from 10 week - old mice stained for δTcR ( green ) ; counterstained with Hoechst ( blue ) ( A ) and quantification of δTcR + cells in the ear epidermis ( B ) . Dotted lines indicate the epidermal - dermal border . Scale bar : 20μm . N = 6 wt / wt mice , N = 7 HPV8 / wt mice , N = 6 wt / Act mice and ( N = 8 ) HPV8 / Act mice . C . Quantification of βTcR + cells in ear skin cryosections from 10 week - old wt / wt ( N = 5 ) ; HPV8 / wt ( N = 5 ) ; wt / Act ( N = 8 ) or HPV8 / Act ( N = 8 ) mice . D - F . Percentage of viable CD4 + βTcR + ( D ) , CD8 + βTcR + ( E ) or CD4 + CD25 + ( F ) cells among all epidermal cells analyzed by flow cytometry . Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown . Representative flow cytometry scatter plots are shown in Appendix Fig S1A - C . G . Representative pictures of ear cryosections from 10 week - old mice stained with antibodies against Foxp3 ( red ) and counterstained with Hoechst ( blue ) . Insert shows higher magnification of the Foxp3 - positive cells in wt / Act skin . Dotted lines indicate the epidermal - dermal border . Scale bar : 50μm . H . Kinetics of tumor incidence in HPV8 / Act / wt ( N = 29 ) , HPV8 / Act / CD4KO ( N = 10 ) , HPV8 / wt / wt ( N = 23 ) and HPV8 / wt / CD4KO ( N = 22 ) mice per genotype . Statistical significance was determined using the Log - rank ( Mantel - Cox ) test ; ns = non - significant .

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Figure 2A,B. Representative pictures of ear sections from 10 week-old mice stained for δTcR (green); counterstained with Hoechst (blue) (A) and quantification of δTcR+ cells in the ear epidermis (B). Dotted lines indicate the epidermal-dermal border. Scale bar: 20μm. N=6 wt/wt mice, N=7 HPV8/wt mice, N=6 wt/Act mice and (N=8) HPV8/Act mice.C. Quantification of βTcR+ cells in ear skin cryosections from 10 week-old wt/wt (N=5); HPV8/wt (N=5); wt/Act (N=8) or HPV8/Act (N=8) mice.D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C.G. Representative pictures of ear cryosections from 10 week-old mice stained with antibodies against Foxp3 (red) and counterstained with Hoechst (blue). Insert shows higher magnification of the Foxp3-positive cells in wt/Act skin. Dotted lines indicate the epidermal-dermal border. Scale bar: 50μm.H. Kinetics of tumor incidence in HPV8/Act/wt (N=29), HPV8/Act/CD4KO (N=10), HPV8/wt/wt (N=23) and HPV8/wt/CD4KO (N=22) mice per genotype. Statistical significance was determined using the Log-rank (Mantel-Cox) test; ns = non-significant.


Figure 3A , B . Representative pictures of ear skin cryosections from 10 - 12 week - old mice stained with anti - CD206 antibodies ( green ) ; counterstained with Hoechst ( blue ) ( A ) , and quantification of CD206 + area as a percentage of total dermal area ( B ) in wt / wt ( N = 3 ) , HPV8 / wt ( N = 7 ) , wt / Act ( N = 2 ) or HPV8 / Act ( N = 8 ) mice . The basement membrane is indicated with a white dotted line . C . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12 - 16 week - old mice . N = 5 - 6 . Representative flow cytometry scatter plots are shown in Appendix Fig S1D . D . Quantification of CD206 + area as a percentage of total dermal area in wt ( N = 7 ) and Act ( N = 6 ) mice in back skin treated 8x with TPA ( Antsiferova et al , 2011 ) . E . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of pre - tumorigenic ear skin from 12 week - old mice expressing the CCR2 - CFP - DTR transgene ( wt / CCR2 ) or double - transgenic animals ( Act / CCR2 ) , untreated or treated with diphtheria toxin ( + DT ) 3 times every 48h and analyzed 24h after the last injection . Percentage of CD45 + CD11b + MerTK + CD64 + macrophages among live cells is shown . N = 3 control wt / CCR2 and Act / CCR2 mice ; N = 4 DT - injected wt / CCR2 and Act / CCR2 mice . Representative flow cytometry scatter plots are shown in Appendix Fig S1E . F , G . Inflammatory thioglycollate - elicited peritoneal macrophages ( F ) or mouse peripheral blood monocytes ( G ) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay . Activin A 0 . 1ng / ml ( A0 . 1 ) , 1ng / ml ( A1 ) , or 10ng / ml ( A10 ) was added to the lower well . Alternatively , 10ng / ml was added to the lower well and the insert ( A10 / A10 ) . Random migration to medium ( ctrl ) was set to 1 for each mouse ; fold over control is shown . Log - transformed data were used for statistical analysis . Results in ( F ) are representative of 4 independent experiments performed with 8 mice in total ( results obtained in one experiment performed with 3 mice are shown ) . Results in ( G ) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice .

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

Figure 3A,B. Representative pictures of ear skin cryosections from 10-12 week-old mice stained with anti-CD206 antibodies (green); counterstained with Hoechst (blue) (A), and quantification of CD206+ area as a percentage of total dermal area (B) in wt/wt (N=3), HPV8/wt (N=7), wt/Act (N=2) or HPV8/Act (N=8) mice. The basement membrane is indicated with a white dotted line.C. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12-16 week-old mice. N = 5-6. Representative flow cytometry scatter plots are shown in Appendix Fig S1D.D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).E. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of pre-tumorigenic ear skin from 12 week-old mice expressing the CCR2-CFP-DTR transgene (wt/CCR2) or double-transgenic animals (Act/CCR2), untreated or treated with diphtheria toxin (+DT) 3 times every 48h and analyzed 24h after the last injection. Percentage of CD45+CD11b+MerTK+CD64+ macrophages among live cells is shown. N=3 control wt/CCR2 and Act/CCR2 mice; N=4 DT-injected wt/CCR2 and Act/CCR2 mice. Representative flow cytometry scatter plots are shown in Appendix Fig S1E.F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.


Figure 4B . Analysis of FACS data of sorted macrophages shows the relative percentage of CD45 + CD11b + F4 / 80 + cells in each genotype and the absolute number of sorted cells used for RNA sequencing . Representative flow cytometry gating strategy for sorting is shown in Fig EV3B . N = 5 - 9 . Statistical significance was determined using 1 - way ANOVA and Bonferroni ` s Multiple Comparisons Test . C . Differential expression of genes was analyzed using the edgeR software package . IPA was performed with filtered lists of up - and downregulated genes that exhibited an FDR - value of < 0 . 1 in different genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt ) . The number of differentially expressed genes used for analysis is indicated . Data are presented as pseudo - heatmap with magnitudes of Activation Z - scores color - coded as indicated in the legend ; all non - grey comparisons are - log10 ( p - value ) > 2 . 5 unless otherwise indicated . D . Gene Set Enrichment Analysis ( GSEA ) was performed to compare transcriptomes of genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt , [ all ] / Act vs [ all ] / wt ) to 3 published datasets of Tumor - Associated Macrophages ( TAM ) ( Franklin et al . 2014 ; Ojalvo et al , 2009 ; Galletti et al , 2016 ) . Gene sets are composed of top genes upregulated ( TAM UP ) or downregulated ( TAM DOWN ) in TAM vs normal tissue macrophages . Positive Normalized Enrichment Score ( NES ) corresponds to enrichment of gene set in activin - upregulated genes ; negative NES corresponds to enrichment in activin - downregulated genes . Data are presented as pseudo - heatmap with magnitudes of NES color - coded as indicated in the legend ; all non - grey comparisons are FDR < 0 . 10 . E . Differential GSEA was performed on combined genotype comparison [ all ] / Act vs [ all ] / wt and two datasets of human AK vs normal skin ( GSE2503 ; Nindl et al , 2006 ) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets ( top ) ; and gene sets derived from top upregulated genes in datasets of differential human macrophage activation ( IFN - γ , TNF - α , LPS , IL - 4 vs non - treated ) and selected human myeloid / lymphoid cell type - specific gene expression profiles ( macrophage vs monocyte , dendritic cell , and T - cell ) ( Xue et al , 2014 ; bottom ) . Activin target genes enriched in the AK transcriptomes are shown ( right ) . Data are presented as pseudo - heatmaps with magnitudes of Normalized Enrichment Scores ( NES ) and Log2 ( Fold Change ) color - coded as indicated in the respective legends ; all non - grey comparisons are FDR < 0 . 05 for NES and p < 0 . 10 for Fold Change . G . Heatmap of 28 differentially expressed genes ( FDR ≤ 0 . 05 , | log2Ratio | ≥ 1 ) overlapping in all 3 comparisons ( wt / Act vs wt / wt mice , HPV8 / Act vs HPV8 / wt and HPV8 / Act vs wt / wt ) . Genes with a published pro - tumorigenic function are highlighted in red .

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 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 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 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 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

Figure 4B. Analysis of FACS data of sorted macrophages shows the relative percentage of CD45+CD11b+F4/80+ cells in each genotype and the absolute number of sorted cells used for RNA sequencing. Representative flow cytometry gating strategy for sorting is shown in Fig EV3B. N=5-9. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test.C. Differential expression of genes was analyzed using the edgeR software package. IPA was performed with filtered lists of up- and downregulated genes that exhibited an FDR-value of <0.1 in different genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt). The number of differentially expressed genes used for analysis is indicated. Data are presented as pseudo-heatmap with magnitudes of Activation Z-scores color-coded as indicated in the legend; all non-grey comparisons are -log10(p-value)>2.5 unless otherwise indicated.D. Gene Set Enrichment Analysis (GSEA) was performed to compare transcriptomes of genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt, [all]/Act vs [all]/wt) to 3 published datasets of Tumor-Associated Macrophages (TAM) (Franklin et al. 2014; Ojalvo et al, 2009; Galletti et al, 2016). Gene sets are composed of top genes upregulated (TAM UP) or downregulated (TAM DOWN) in TAM vs normal tissue macrophages. Positive Normalized Enrichment Score (NES) corresponds to enrichment of gene set in activin-upregulated genes; negative NES corresponds to enrichment in activin-downregulated genes. Data are presented as pseudo-heatmap with magnitudes of NES color-coded as indicated in the legend; all non-grey comparisons are FDR<0.10.E. Differential GSEA was performed on combined genotype comparison [all]/Act vs [all]/wt and two datasets of human AK vs normal skin (GSE2503; Nindl et al, 2006) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets (top); and gene sets derived from top upregulated genes in datasets of differential human macrophage activation (IFN-γ, TNF-α, LPS, IL-4 vs non-treated) and selected human myeloid/lymphoid cell type-specific gene expression profiles (macrophage vs monocyte, dendritic cell, and T-cell) (Xue et al, 2014; bottom). Activin target genes enriched in the AK transcriptomes are shown (right). Data are presented as pseudo-heatmaps with magnitudes of Normalized Enrichment Scores (NES) and Log2(Fold Change) color-coded as indicated in the respective legends; all non-grey comparisons are FDR<0.05 for NES and p<0.10 for Fold Change.G. Heatmap of 28 differentially expressed genes (FDR≤0.05, |log2Ratio|≥1) overlapping in all 3 comparisons (wt/Act vs wt/wt mice, HPV8/Act vs HPV8/wt and HPV8/Act vs wt/wt). Genes with a published pro-tumorigenic function are highlighted in red.


Figure 5A , B , F . Expression of Cx3cr1 ( A ) , Spp1 ( B ) relative to Rps29 was analyzed by qRT - PCR in peritoneal macrophages , resident or inflammatory , treated in vitro with 10 ng / ml of activin A ( Act ) or untreated ( Ctrl , arbitrarily set to 1 ) . Results are combined from 2 - 5 independent experiments performed with cells pooled from at least 5 wt mice . C , D . Western blot analysis of total ear protein lysates of 12 - 14 week - old mice for osteopontin ( OPN ) and GAPDH ( C ) . Each lane represents an individual mouse ( n = 3 ) . ( D ) Band intensities were quantified and normalized to GAPDH . The expression level in one of the wt mice was arbitrarily set to 1 . E . Ear skin sections from 10 week - old mice stained with anti - CD68 ( red ) or anti - osteopontin antibody ( green ) ; counterstained with Hoechst ( blue ) . Osteopontin - positive macrophages in wt / Act and HPV8 / Act mice are indicated with arrows . Representative of N = 2 wt / wt and N = 3 HPV8 / wt , wt / Act and HPV8 / Act mice . Boxed areas in top panel are shown at higher magnification in bottom panel . Scale bar top : 100μm , bottom : 20μm . A , B , F . Expression of Arg1 ( F ) relative to Rps29 was analyzed by qRT - PCR in peritoneal macrophages , resident or inflammatory , treated in vitro with 10 ng / ml of activin A ( Act ) or untreated ( Ctrl , arbitrarily set to 1 ) . Results are combined from 2 - 5 independent experiments performed with cells pooled from at least 5 wt mice . G . In situ gelatin zymography ( top panel ) and methylene blue staining ( bottom panel ) of the ear skin from 10 week - old mice . Representative images of 2 - 3 mice per genotype are shown . Scale bar = 500μm . H , I . Gelatin zymography of ear skin lysates from 10 week - old mice . Inverted image of a representative gel with 2 ( wt / wt ) or 3 ( HPV8 / wt , wt / Act , HPV8 / Act ) mice per genotype is shown in ( H ) , quantification of the results obtained in 2 independent experiments is shown in ( I ) . N = 5 wt / wt , N = 6 HPV8 / wt , wt / Act and HPV8 / Act mice . J . Representative pictures of ear skin from 10 week - old mice stained for MECA32 ( red ) , LYVE1 ( green ) ; counterstained with Hoechst ( blue ) . Boxed areas in top panel are shown at higher magnification in bottom panel . Scale bar top : 100μm , bottom : 20μm . K , L . Quantification of MECA32 + area ( K ) and average distance between MECA32 + vessels and basement membrane ( BM ) ( L ) .

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 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 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 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 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 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

Figure 5A,B,F. Expression of Cx3cr1 (A), Spp1 (B) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.C,D. Western blot analysis of total ear protein lysates of 12-14 week-old mice for osteopontin (OPN) and GAPDH (C). Each lane represents an individual mouse (n=3). (D) Band intensities were quantified and normalized to GAPDH. The expression level in one of the wt mice was arbitrarily set to 1.E. Ear skin sections from 10 week-old mice stained with anti-CD68 (red) or anti-osteopontin antibody (green); counterstained with Hoechst (blue). Osteopontin-positive macrophages in wt/Act and HPV8/Act mice are indicated with arrows. Representative of N=2 wt/wt and N=3 HPV8/wt, wt/Act and HPV8/Act mice. Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.A,B,F. Expression of Arg1 (F) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.G. In situ gelatin zymography (top panel) and methylene blue staining (bottom panel) of the ear skin from 10 week-old mice. Representative images of 2-3 mice per genotype are shown. Scale bar = 500μm.H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice.J. Representative pictures of ear skin from 10 week-old mice stained for MECA32 (red), LYVE1 (green); counterstained with Hoechst (blue). Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.K,L. Quantification of MECA32+ area (K) and average distance between MECA32+ vessels and basement membrane (BM) (L).


Figure 6A . Kinetics of tumor incidence in HPV8 / Act mice treated for 5 weeks with 50mg / kg anti - CSF1R antibody ( AFS98 ) or with control rat IgG . N = 10 mice per treatment group . B . Representative pictures of anti - CD68 - or CD206 - stained cryosections of ear skin from 10 week - old mice treated for 5 weeks with anti - CSF1R antibody or control rat IgG . Scale bar = 100μm . C . Quantification of the CD68 + or CD206 + area relative to total dermis area . N = 10 mice per treatment group . D . Representative pictures of anti - BrdU ( green ) and anti - K14 ( red ) stained cryosections of ear skin from 10 week - old mice treated for 5 weeks with anti - CSF1R antibody or control rat IgG . Scale bar : 50μm . E . Quantification of BrdU + keratinocytes per mm of basement membrane . N = 8 anti - CSF1R - injected mice ; N = 4 rat IgG - injected mice . F . Expression of Mmp12 relative to Rps29 was analyzed by qRT - PCR in total ear skin from mice treated with anti - CSF1R antibody or with rat IgG . Expression in one of the IgG - injected mice was set to 1 . N = 10 per group . G . Representative pictures of the ear skin from 10 week - old mice treated for 5 weeks with anti - CSF1R or control rat IgG , stained for CD31 ( red ) , LYVE1 ( green ) , and counterstained with Hoechst ( blue ) . Boxed areas are shown at higher magnification in the top - right corner . Scale bar : 100μm . H , I . Quantification of CD31 + LYVE1 - area ( H ) and number of CD31 + LYVE1 - vessels ( I ) .

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 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 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 O O O O O O O O O

Figure 6A. Kinetics of tumor incidence in HPV8/Act mice treated for 5 weeks with 50mg/kg anti-CSF1R antibody (AFS98) or with control rat IgG. N=10 mice per treatment group.B. Representative pictures of anti-CD68- or CD206-stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar = 100μm.C. Quantification of the CD68+ or CD206+ area relative to total dermis area. N=10 mice per treatment group.D. Representative pictures of anti-BrdU (green) and anti-K14 (red) stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar: 50μm.E. Quantification of BrdU+ keratinocytes per mm of basement membrane. N=8 anti-CSF1R-injected mice; N=4 rat IgG-injected mice.F. Expression of Mmp12 relative to Rps29 was analyzed by qRT-PCR in total ear skin from mice treated with anti-CSF1R antibody or with rat IgG. Expression in one of the IgG-injected mice was set to 1. N=10 per group.G. Representative pictures of the ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R or control rat IgG, stained for CD31 (red), LYVE1 (green), and counterstained with Hoechst (blue). Boxed areas are shown at higher magnification in the top-right corner. Scale bar: 100μm.H,I. Quantification of CD31+LYVE1- area (H) and number of CD31+LYVE1-vessels (I).


Figure 1Growth curve of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or vehicle alone ( mock ) for the indicated time . The cell number at the indicated days relative to the number at the start of the treatment ( d0 ) is shown . The dotted line represents the cell number at d0 . NIH - 3T3 cells were treated with TGFβ or mock for 48h and incubated with O - propargyl - puromycin ( OPP ) for the last 60 min . The translation rate was determined by flow cytometry for OPP incorporation into proteins . A representative plot is shown on the left , and the quantification is shown on the right . Values are relative to mock - treated cells . Western blot of NIH - 3T3 cells treated with TGFβ or mock for 48h . Western blot of ECM from NIH - 3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH - 3T3 cells grown on a parallel plate . Note the absence of tubulin and the electromobility shift of collagens in ECM extracts . ECM was produced from NIH - 3T3 cells according to ( d ) , and collagen abundance was measured by picrosirius red staining , normalized to the packed cell volume before ECM extraction , and expressed relative to mock - treated cells . NIH - 3T3 cells were treated with TGFβ or mock for 48h . The medium was replaced in the last 12h of the treatment ( with the same treatments as before ) , and glucose consumption from the fresh media were measured using the YSI bioanalyzer . ATP production rate from glycolysis ( JATPgly ) and mitochondrial oxidative phosphorylation ( JATPox ) from NIH - 3T3 cells treated with TGFβ or mock for 48h , calculated using Seahorse data shown in ( k ) and EV Figure 1f . Pearson correlation of mitochondrial ATP production rate ( JATPox ) and OPP incorporation of NIH - 3T3 cells treated with TGFβ or mock for 2h , 4h , 8h , 24h and 48h , relative to mock - treated cells . Also see EV Figure 1k and l . Note that the datapoints of the 2h and 4h measurements overlap . NIH - 3T3 cells were treated with TGFβ or mock for 48h . The medium was replaced in the last 12h of the treatment ( with the same treatments as before ) Glutamine consumption measured as in ( g ) . NIH - 3T3 cells were treated with TGFβ or mock for 48h , and the oxygen consumption rate ( OCR ) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer . Oligo , Oligomycin ; Rot / Anti - A , Rotenone / Antimycin - A ; PCV , packed cell volume . NIH - 3T3 cells were treated with TGFβ or mock for 48h and reduced ( GSH ) and oxidized ( GSSG ) glutathione were measured by LC - MS / MS . Shown is the GSH / GSSG ratio . NIH - 3T3 cells were treated with TGFβ for 48h or mock and incubated with CM - H2DCFDA for the last 30 min . ROS were measured by flow cytometry for CM - H2DCFDA . Values are relative to mock - treated cells .

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 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 B-PANEL_START 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 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 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 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 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

Figure 1Growth curve of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or vehicle alone (mock) for the indicated time. The cell number at the indicated days relative to the number at the start of the treatment (d0) is shown. The dotted line represents the cell number at d0.NIH-3T3 cells were treated with TGFβ or mock for 48h and incubated with O-propargyl-puromycin (OPP) for the last 60 min. The translation rate was determined by flow cytometry for OPP incorporation into proteins. A representative plot is shown on the left, and the quantification is shown on the right. Values are relative to mock-treated cells.Western blot of NIH-3T3 cells treated with TGFβ or mock for 48h.Western blot of ECM from NIH-3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH-3T3 cells grown on a parallel plate. Note the absence of tubulin and the electromobility shift of collagens in ECM extracts.ECM was produced from NIH-3T3 cells according to (d), and collagen abundance was measured by picrosirius red staining, normalized to the packed cell volume before ECM extraction, and expressed relative to mock-treated cells.NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before), and glucose consumption from the fresh media were measured using the YSI bioanalyzer.ATP production rate from glycolysis (JATPgly) and mitochondrial oxidative phosphorylation (JATPox) from NIH-3T3 cells treated with TGFβ or mock for 48h, calculated using Seahorse data shown in (k) and EV Figure 1f.Pearson correlation of mitochondrial ATP production rate (JATPox) and OPP incorporation of NIH-3T3 cells treated with TGFβ or mock for 2h, 4h, 8h, 24h and 48h, relative to mock-treated cells. Also see EV Figure 1k and l. Note that the datapoints of the 2h and 4h measurements overlap.NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before) Glutamine consumption measured as in (g).NIH-3T3 cells were treated with TGFβ or mock for 48h, and the oxygen consumption rate (OCR) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer. Oligo, Oligomycin; Rot/Anti-A, Rotenone/Antimycin-A; PCV, packed cell volume.NIH-3T3 cells were treated with TGFβ or mock for 48h and reduced (GSH) and oxidized (GSSG) glutathione were measured by LC-MS/MS. Shown is the GSH/GSSG ratio.NIH-3T3 cells were treated with TGFβ for 48h or mock and incubated with CM-H2DCFDA for the last 30 min. ROS were measured by flow cytometry for CM-H2DCFDA. Values are relative to mock-treated cells.


Figure 2Comparison of the amino acid representation in all human proteins and all collagens . The x - axis denotes individual amino acids shown by single letter code . Glycine and proline representation in protein acid hydrolysates of untreated NIH - 3T3 cells and their secreted ECM , produced as in Figure 1d . Amino acids were measured by GC - MS . Tracing of [ U - 13C ] L - glutamine ( [ U - 13C ] Gln ) into indicated metabolites . NIH - 3T3 cells were treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 48h , and the medium was replaced ( including all treatments ) with DMEM lacking L - glutamine and supplemented with [ U - 13C ] Gln for the last 8h . Metabolic steady state was reached at this time point ( see kinetic labeling curves in Figure EV3b ) . Metabolites were measured by LC - MS . The left graph shows the total pool size normalized to the packed cell volume ; the right side shows the percent labeling . αKG , alpha - ketoglutarate ; AU , arbitrary units . Glutamine flux into glutamate , αKG and proline in NIH - 3T3 cells treated with TGFβ or mock for 48h , relative to mock - treated cells . Abundance of indicated metabolites in NIH - 3T3 cells treated with TGFβ or mock for 48h , relative to mock - treated cells . Metabolites were measured by GC - MS . Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ( [ 12C ] Gln , left ) and fully labeled glutamine ( [ U - 13C ] Gln , right ) . Peaks representing fully labeled ( m + 5 , m + 10 ) proline are highlighted in red . Peptides were measured by LC - MS . Relative abundance of the analytical peptide containing unlabeled ( m + 0 ) or one / two fully labelled prolines ( m + 5 / m + 10 ) from the CO1A1 peptide in ECM derived from NIH - 3T3 cells treated with TGFβ or mock in the presence of fully labelled L - glutamine ( [ U - 13C ] Gln ) . Peptides were measured by LC - MS . The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions .

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 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 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 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 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 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

Figure 2Comparison of the amino acid representation in all human proteins and all collagens. The x-axis denotes individual amino acids shown by single letter code.Glycine and proline representation in protein acid hydrolysates of untreated NIH-3T3 cells and their secreted ECM, produced as in Figure 1d. Amino acids were measured by GC-MS.Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units.Glutamine flux into glutamate, αKG and proline in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells.Abundance of indicated metabolites in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Metabolites were measured by GC-MS.Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ([12C] Gln, left) and fully labeled glutamine ([U-13C] Gln, right). Peaks representing fully labeled (m+5, m+10) proline are highlighted in red. Peptides were measured by LC-MS.Relative abundance of the analytical peptide containing unlabeled (m+0) or one/two fully labelled prolines (m+5/m+10) from the CO1A1 peptide in ECM derived from NIH-3T3 cells treated with TGFβ or mock in the presence of fully labelled L-glutamine ([U-13C] Gln). Peptides were measured by LC-MS. The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions.


Figure 3Western blot of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 48h in the presence of 0 . 5 % FBS . Left : NIH - 3T3 cells expressing sgCtrl , sgP5CS - 1 or sgP5CS - 2 were treated with TGFβ or mock for 48h , and proline abundance was measured by GC - MS . Values are relative to mock - treated sgCtrl expressing cells . Right : western blot of NIH - 3T3 cells expressing sgCtrl , sgP5CS - 1 or sgP5CS - 2 . Western blot of NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 and treated with TGFβ or mock for 48h in the presence or absence of 0 . 15 mM proline . Collagen abundance in ECM produced by NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 grown in the presence or absence of TGFβ and 0 . 15 mM proline , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing cells . Proline abundance in NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA , measured by GC - MS . Values are relative to mock - treated empty vector expressing cells . Western blot of NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA . Collagen abundance in ECM produced by NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treatedAnalysis of the indicated gene expression datasets for mRNA levels of P5CS : ( i ) lung tissue from mice with pulmonary fibrosis induced by bleomycin ( Bleo ) treatment compared to saline treatment ( GSE112827 ) ; two datasets ( GSE110147 , GSE32537 ) from lungs of patients with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . The number of patients per group is indicated . Pearson correlation of P5CS mRNA level and forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as percentage of what was predicted for each patient , from clinical data of GSE32537 . Tracing of [ U - 13C ] L - glutamine ( [ U - 13C ] Gln ) into indicated metabolites . NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 48h and the medium was replaced ( including all treatments ) with DMEM without L - glutamine and supplemented with [ U - 13C ] Gln for the last 8h . Metabolites were measured by GC - MS .

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 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 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 B-PANEL_START 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 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 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 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 3Western blot of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 48h in the presence of 0.5% FBS.Left: NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2 were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2.Western blot of NIH-3T3 cells expressing sgCtrl or sgP5CS-2 and treated with TGFβ or mock for 48h in the presence or absence of 0.15 mM proline.Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl or sgP5CS-2 grown in the presence or absence of TGFβ and 0.15 mM proline, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.Proline abundance in NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.Western blot of NIH-3T3 cells expressing empty vector or HA-P5CS cDNA.Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treatedAnalysis of the indicated gene expression datasets for mRNA levels of P5CS: (i) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.Pearson correlation of P5CS mRNA level and forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient, from clinical data of GSE32537.Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 48h and the medium was replaced (including all treatments) with DMEM without L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolites were measured by GC-MS.


Figure 4Mitochondrial basal respiration ( a ) and FCCP - induced maximal respiration ( b ) of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 2h , 4h , 8h , 24h or 48h , based on Seahorse OCR measurements . The relative values were calculated by normalizing the respective values of TGFβ - treated cells to those of mock - treated cells for each time point . Left : western blot of NIH - 3T3 cells expressing empty vector , mitoTPNOX or mitoLbNOX . MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG - tag . Right : NIH - 3T3 cells expressing empty vector , mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC - MS . Values are normalized to mock - treated empty vector expressing cells . NIH - 3T3 cells were treated with rotenone , antimycin , myxothiazol or vehicle control for 1h , and proline abundance was measured by GC - MS . Values are normalized to control - treated cells . NIH - 3T3 cells were treated with cobalt chloride ( CoCl2 ) or vehicle control for 6h , and proline abundance was measured by GC - MS . Values are normalized to control - treated cells . NIH - 3T3 cells were treated with TGFβ or mock for 48h , and FCCP or vehicle control was added for the last 6h of the treatment . Proline abundance was measured by GC - MS . Values are normalized to mock - and control - treated cells . NIH - 3T3 cells were treated with TGFβ or mock for 48h , and oligomycin or vehicle control was added for the last 1h or 6h of the treatment . Proline abundance was measured by GC - MS . Values are normalized to mock - and control - treated cells .

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 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 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 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 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

Figure 4Mitochondrial basal respiration (a) and FCCP-induced maximal respiration (b) of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 2h, 4h, 8h, 24h or 48h, based on Seahorse OCR measurements. The relative values were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.Left: western blot of NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX. MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG-tag. Right: NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are normalized to mock-treated empty vector expressing cells.NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.NIH-3T3 cells were treated with cobalt chloride (CoCl2) or vehicle control for 6h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.NIH-3T3 cells were treated with TGFβ or mock for 48h, and FCCP or vehicle control was added for the last 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.NIH-3T3 cells were treated with TGFβ or mock for 48h, and oligomycin or vehicle control was added for the last 1h or 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.


Figure 5NIH - 3T3 cells were treated with TGFβ or mock for 1h , 6h , 24h or 48h . ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment ( Red line ) . Proline abundance was measured by GC - MS ( Blue line ) . The relative levels were calculated by normalizing the respective values of TGFβ - treated cells to those of mock - treated cells for each time point . Western blot of NIH - 3T3 cells were treated with TGFβ or mock for 1h , 6h , 24h or 48h . NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are normalized to mock - treated sgCtrl expressing cells . Western blot of NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 and treated with TGFβ or mock for 6h in the presence of 0 . 5 % FBS . NIH - 3T3 cells expressing mito - Grx1 - roGFP2 or cyto - Grx1 - roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control . Emission was measured with a 520 / 10nm filter after excitation with 405nm and 488nm lasers using flow cytometry . Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm . Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest . NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 were treated with TGFβ or mock for 48h in the presence of 0 . 15 mM proline . ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment .

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

Figure 5NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment (Red line). Proline abundance was measured by GC-MS (Blue line). The relative levels were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.Western blot of NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h.NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are normalized to mock-treated sgCtrl expressing cells.Western blot of NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 and treated with TGFβ or mock for 6h in the presence of 0.5% FBS.NIH-3T3 cells expressing mito-Grx1-roGFP2 or cyto-Grx1-roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control. Emission was measured with a 520/10nm filter after excitation with 405nm and 488nm lasers using flow cytometry. Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm. Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest.NIH-3T3 cells expressing sgCtrl or sgP5CS-2 were treated with TGFβ or mock for 48h in the presence of 0.15 mM proline. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment.


Figure 6NIH - 3T3 cells were treated with vehicle control or CTPi at increasing concentrations ( 0 . 1 mM , 0 . 2 mM , 0 . 5 mM ) or TGFβ ( 2 ng / mL for all experiments ) for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to control - treated cells . P - values represent comparison of individual samples to the control . Left : NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to mock - treated sgCtrl expressing cells . Right : western blot of NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 . NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC - MS . Values are relative to mock - treated sgCtrl expressing cells . Western blot of NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 and treated with TGFβ or mock for 48h . Collagen abundance in ECM produced by NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 grown in the presence or absence of TGFβ , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing cells . NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to mock - treated empty vector expressing cells . NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h , and proline abundance was measured by GC - MS . Values are relative to mock - treated empty vector expressing cells . Western blot of NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h . Collagen abundance in ECM produced by NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated empty vector expressing cells . Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1 , as described in Figure 3 : ( j ) lung tissue from mice with pulmonary fibrosis induced by bleomycin ( Bleo ) treatment compared to saline treatment ( GSE112827 ) ; Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1 , as described in Figure 3 : two datasets ( GSE110147 , GSE32537 ) from lungs of patients with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . The number of patients per group is indicated . Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537 . IPF patients from GSE32537 were assigned into P5CSlow / SLC25A1high and P5CShigh / SLC25A1low groups based on the expression level of P5CS and SLC25A1 . " High " represents patients with P5CS or SLC25A1 expression values being above the 75 % percentile of the respective gene expression ; " low " represents patients with expression values being below the 25 % percentile of gene expression . The forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as percentage of what was predicted for each patient was compared between the groups .

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 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 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 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 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 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 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 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 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

Figure 6NIH-3T3 cells were treated with vehicle control or CTPi at increasing concentrations (0.1 mM, 0.2 mM, 0.5 mM) or TGFβ (2 ng/mL for all experiments) for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to control-treated cells. P-values represent comparison of individual samples to the control.Left: NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2.NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells.Western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 and treated with TGFβ or mock for 48h.Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated empty vector expressing cells.NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.Western blot of NIH-3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h.Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated empty vector expressing cells.Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: (j) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537. IPF patients from GSE32537 were assigned into P5CSlow/SLC25A1high and P5CShigh/SLC25A1low groups based on the expression level of P5CS and SLC25A1. "High" represents patients with P5CS or SLC25A1 expression values being above the 75% percentile of the respective gene expression; "low" represents patients with expression values being below the 25% percentile of gene expression. The forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient was compared between the groups.


Figure 1b , c Phospho - sites identified after TiO2 enrichment b and phospho - tyrosine immunoprecipitation ( pY - IP ) c . Left panel : pie chart presenting the proportion of identified phosphorylated serines ( pS ) , threonines ( pT ) and tyrosines ( pY ) in the two data sets . Right panel : cumulative number of unique identified phospho - sites across the biological replicates ( 3 and 4 independent experiments for the TiO2 and pY - IP , respectively ) . Phosphorylated serines , threonines and tyrosines are presented individually next to the total number of identified sites ( pSTY ) . The phospho - sites that were not previously reported in any species in the PhosphoSitePlus database ( www . phosphosite . org ) are reported in isolation in the graph on the right .

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

Figure 1b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.


Figure 2 a , b Output of the statistical analyses of the TiO2 - fractionated a and the pY - IP - fractionated b data sets . Left panel : number of phospho - sites significantly regulated upon TCR stimulation presented in bar plots for each time point , next to the total number of regulated sites across the entire time course ( total ) . The number of phosphorylated threonines ( pT ) and serines ( pS ) regulated in the TiO2 data set are indicated in yellow and green , respectively . Right panel : representative volcano plot presenting the statistical significance distribution against the log2 - transformed fold change between 300 seconds and the unstimulated control . For each condition , phospho - sites were considered significantly up - ( red ) or down - ( blue ) regulated when displaying a corrected P - value ≤ 0 . 05 ( Anova test ) and an absolute fold change ≥ 1 . 75 ( see Materials and Methods for more detailed information ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 a, b Output of the statistical analyses of the TiO2-fractionated a and the pY-IP-fractionated b data sets. Left panel: number of phospho-sites significantly regulated upon TCR stimulation presented in bar plots for each time point, next to the total number of regulated sites across the entire time course (total). The number of phosphorylated threonines (pT) and serines (pS) regulated in the TiO2 data set are indicated in yellow and green, respectively. Right panel: representative volcano plot presenting the statistical significance distribution against the log2-transformed fold change between 300 seconds and the unstimulated control. For each condition, phospho-sites were considered significantly up- (red) or down- (blue) regulated when displaying a corrected P-value ≤ 0.05 (Anova test) and an absolute fold change ≥ 1.75 (see Materials and Methods for more detailed information).


Figure 4 a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster . Black dots indicate clusters with significantly enriched kinase activity ( hypergeometric test P - value ≤ 0 . 05 , fold - change ≥ 2 , number of annotated sites ≥ 2 ; Grey squares : fold - change < 1 ) . d Immunoblot analysis of equal amounts of proteins from total lysates of CD4 + T cells treated with DMSO , U0126 ( 10 μM ) or AKTi VIII ( 10 μM ) and left unstimulated ( - ) or stimulated for 15 , 120 , or 300 sec with anti - CD3 and anti - CD4 antibodies , probed with the indicated phospho - specific antibodies . Anti - ERK1 / 2 immunoblot served as a loading control . e Equal amounts of proteins from total lysates of CD4 + T cells left unstimulated ( - ) or stimulated for 120 , 300 or 600 sec with anti - CD3 and anti - CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho - sites / 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4 a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1).  d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control.  e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.


Figure 5 b Cas9 - EGFP OT - I CD8 + T cells were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers ( 0 . 1 nM ) in presence or absence of soluble anti - CD28 antibody or with IL - 7 as control . Cell surface expression of CD69 and CD5 were analyzed by flow cytometry ( Z - score normalization of the Geometric Means of fluorescence MFI per experiment ) in four independent experiments . Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t - test . Boxplot elements : center line corresponds to median , box limits correspond to the first and third quartiles , whiskers indicate variability from Q1 - 1 . 5 . IQR to Q3 + 1 . 5 IQR . Outliers are shown as black dots . c Proliferation of OT - I Cas9 - EGFP CD8 + T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers ( 0 . 01 - 0 . 1 nM ) in the presence or absence of soluble anti - CD28 antibody or with PMA and ionomycin ( PMA / Iono ) or with IL - 7 . Data are presented as mean ± standard deviation ( SD ) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections . Data are representative of three independent experiments . d IFN - γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation . e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti - CD28 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5 b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots. c Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments.  d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation. e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.


Figure 6 a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP , sgITSN2 - 1 or sgITSN2 - 2 and left unstimulated ( - ) or stimulated for 2 or 5 min with N4 tetramers ( 20 nM ) . Upper panel : membrane probed with antibody to phosphorylated tyrosine ( Anti - p - Tyr ) or anti - LAT ( loading control ) . Lower panel : membrane probed with antibodies to anti - CBL - pY774 , anti - ZAP70 - pY319 / 352 , anti - AKT - pT308 , anti - ERK1 / 2 - pY204 / T202 or anti - ERK1 / 2 ( loading control ) . b Heat map showing normalized levels ( z - score ) of indicated surface markers and protein phosphorylations ( left margin ) from Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP or sgITSN2 - 1 left unstimulated ( - ) or stimulated for 1 , 3 or 10 min with N4 tetramers ( 1 or 10 nM ) and analyzed by mass cytometry . Z - scores were calculated from hyperbolic arcsine ( arcsinh ) transformed intensities . c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points ( left panel ) or stimulated with increased doses of N4 peptides for 4 h ( right panel ) , were analyzed by FACS for surface TCR expression . Data are presented as mean ± SEM and comparisons were performed using a paired t - test ( * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ) . A representative analysis of three independent experiments is shown . d Heatmap showing scaled expression levels ( z - score ) of indicated surface markers and protein phosphorylations ( left margin ) from Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP or sgITSN2 - 1 and stimulated for 6 h with increasing doses of N4 peptides or PMA / Ionomycin ( PI ) analyzed by single - cell mass cytometry ( left ) . Z - scores were calculated from hyperbolic arcsine ( arcsinh ) transformed intensities . Signal difference ( mean arcsinh difference ) between sgEGFP and sgITSN2 - 1 transfected cells is depicted on the right panel .

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

Figure 6 a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control).  b Heat map showing normalized levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 left unstimulated (-) or stimulated for 1, 3 or 10 min with N4 tetramers (1 or 10 nM) and analyzed by mass cytometry. Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities.  c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points (left panel) or stimulated with increased doses of N4 peptides for 4 h (right panel), were analyzed by FACS for surface TCR expression. Data are presented as mean ± SEM and comparisons were performed using a paired t-test (*, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001). A representative analysis of three independent experiments is shown.  d Heatmap showing scaled expression levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 and stimulated for 6 h with increasing doses of N4 peptides or PMA/Ionomycin (PI) analyzed by single-cell mass cytometry (left). Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Signal difference (mean arcsinh difference) between sgEGFP and sgITSN2-1 transfected cells is depicted on the right panel.


Figure 1Epi - ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes , calculated as described in Appendix Supplementary Methods , where 0 means a wild - type H3K79me level ( log2 scale ) . The gray dots indicate accessory subunits . UpTag and DownTag are barcode reporters in a promoter and terminator context , respectively . Data was obtained on all Rpd3L / Rpd3S subunits except Sds3 . Mass spectrometry analysis of H3K79 methylation in wild - type and mutant strains . Mean and individual data points of two biological replicates . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type . Heatmaps of H3K79me1 , H3K79me3 , H2Bub and H3 in wild - type cells , aligned on the TSS . Genes were sorted based on the average H3K79me3 / H3K79me1 ratio in the first 500 bp . Snapshot of depth - normalized ChIP - seq data tracks from wild - type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF , which is the top gene in the heatmap in panel ( F ) . All tracks have the same y axis ( 0 - 20 RPM ) . A snapshot of another top - regulated gene is shown in Fig EV1D . Heatmap of the H3K79me3 / H3 change in rpd3Δ versus wild - type cells , aligned on the TSS . Genes were sorted based on the average ratio in the first 500 bp .

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 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 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

Figure 1Epi-ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes, calculated as described in Appendix Supplementary Methods, where 0 means a wild-type H3K79me level (log2 scale). The gray dots indicate accessory subunits. UpTag and DownTag are barcode reporters in a promoter and terminator context, respectively. Data was obtained on all Rpd3L/Rpd3S subunits except Sds3.Mass spectrometry analysis of H3K79 methylation in wild-type and mutant strains. Mean and individual data points of two biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.Heatmaps of H3K79me1, H3K79me3, H2Bub and H3 in wild-type cells, aligned on the TSS. Genes were sorted based on the average H3K79me3/H3K79me1 ratio in the first 500 bp.Snapshot of depth-normalized ChIP-seq data tracks from wild-type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF, which is the top gene in the heatmap in panel (F). All tracks have the same y axis (0-20 RPM). A snapshot of another top-regulated gene is shown in Fig EV1D.Heatmap of the H3K79me3/H3 change in rpd3Δ versus wild-type cells, aligned on the TSS. Genes were sorted based on the average ratio in the first 500 bp.


Figure 2ChIP - seq and RNA - seq data for genes ranked on H3K79me3 / H3 in rpd3Δ / WT , smoothed using locally weighed regression . The gray band around the line shows the 95 % confidence interval . Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion . ChIP - seq data of H3K79me1 , H3K79me3 and H2Bub were generated in this study ( plotted is the average coverage in reads per genomic content , RPGC ) , Rpd3 binding , H4ac and WT gene expression data were from ( McKnight et al , 2015 ) , and the relative expression in rpd3Δ / WT from Kemmeren et al ( 2014 ) . H3K79me3 / H3 ChIP - qPCR efficiencies ( relative to a non - transcribed region , which was unaffected by RPD3 deletion ) in wild - type and in rpd3Δ cells harboring empty or RPD3 - encoding CEN plasmids . The H188A and H150A - H151A mutations have previously been shown to abrogate catalytic activity ( Kadosh & Struhl , 1998 ) . Error bars indicate standard deviation of three biological replicates . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type . Gene set enrichment analysis on genes ranked on H3K79me3 / H3 in rpd3Δ / WT ; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines . Meiotic ( C ) and Ume6 - bound ( D ) genes are enriched among the genes at which Rpd3 represses H3K79 methylation , and subtelomeric genes ( < 30 kb of telomere ) ( E ) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells .

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Figure 2ChIP-seq and RNA-seq data for genes ranked on H3K79me3/H3 in rpd3Δ/WT, smoothed using locally weighed regression. The gray band around the line shows the 95% confidence interval. Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion. ChIP-seq data of H3K79me1, H3K79me3 and H2Bub were generated in this study (plotted is the average coverage in reads per genomic content, RPGC), Rpd3 binding, H4ac and WT gene expression data were from (McKnight et al, 2015), and the relative expression in rpd3Δ/WT from Kemmeren et al (2014).H3K79me3/H3 ChIP-qPCR efficiencies (relative to a non-transcribed region, which was unaffected by RPD3 deletion) in wild-type and in rpd3Δ cells harboring empty or RPD3-encoding CEN plasmids. The H188A and H150A-H151A mutations have previously been shown to abrogate catalytic activity (Kadosh & Struhl, 1998). Error bars indicate standard deviation of three biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.Gene set enrichment analysis on genes ranked on H3K79me3/H3 in rpd3Δ/WT; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines. Meiotic (C) and Ume6-bound (D) genes are enriched among the genes at which Rpd3 represses H3K79 methylation, and subtelomeric genes (<30 kb of telomere) (E) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells.


Figure 3Mass spectrometry analysis of H3K79 methylation in thymuses from 3 - week - old mice , either wild - type ( Cre - ) or with deleted Hdac1 or Dot1L alleles , as indicated . Mean and individual data points of biological replicates ; H3K79me0 is the predominant state , the y axis is truncated at 70 % for readability . The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed ( yet ) . p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type . Representative H & E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes . A picture with lower magnification of independent samples is included in Fig EV3B . Kaplan - Meier curves of mice harboring thymocytes with indicated genotypes . An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma . Mice that died due to other causes or were still alive and event - free at the end of the experiment were censored . Mice for which the cause of death could not be determined were removed from the data . Wild - type mice were the Cre - littermates of the mice that were used for the other curves .

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Figure 3Mass spectrometry analysis of H3K79 methylation in thymuses from 3-week-old mice, either wild-type (Cre-) or with deleted Hdac1 or Dot1L alleles, as indicated. Mean and individual data points of biological replicates; H3K79me0 is the predominant state, the y axis is truncated at 70% for readability. The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed (yet). p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.Representative H&E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes. A picture with lower magnification of independent samples is included in Fig EV3B.Kaplan-Meier curves of mice harboring thymocytes with indicated genotypes. An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma. Mice that died due to other causes or were still alive and event-free at the end of the experiment were censored. Mice for which the cause of death could not be determined were removed from the data. Wild-type mice were the Cre- littermates of the mice that were used for the other curves.


Figure 4Immunoblots showing HDAC1 status and H3K79me / H3K9ac levels in nuclear lysates of Hdac1 - proficient ( p53 - null ) and Hdac1 - deficient thymic lymphoma cell lines . The top four and bottom two panels are from separate lysates of the same cell lines . Mass spectrometry analysis of H3K79 methylation in the cell lines from panel A . Mean and individual data points of two independent cell lines ; H3K79me0 is the predominant state , the y axis is truncated at 80 % for readability . p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA . Immunoblot showing H2BK120 ubiquitination levels in Hdac1 - proficient and - deficient cell lines ( two independent lines each ) . Growth curves of Hdac1 - proficient and - deficient cell lines that were left untreated or were infected with empty virus ( pLKO ) or shRNAs against Dot1L and selected with puromycin . Growth curves were determined by a series of resazurin assays , which measure metabolic activity , starting four days post - infection . Error bars indicate the range of two replicates from independent cell lines . Inhibitor dose - response curves of the two DOT1L inhibitors EPZ - 5676 ( Pinometostat ) and SGC - 0946 in Hdac1 - proficient and - deficient cell lines . Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO - treated cells . Two independent cell lines are plotted separately ; error bars indicate the range of two biological replicates .

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Figure 4Immunoblots showing HDAC1 status and H3K79me/H3K9ac levels in nuclear lysates of Hdac1-proficient (p53-null) and Hdac1-deficient thymic lymphoma cell lines. The top four and bottom two panels are from separate lysates of the same cell lines.Mass spectrometry analysis of H3K79 methylation in the cell lines from panel A. Mean and individual data points of two independent cell lines; H3K79me0 is the predominant state, the y axis is truncated at 80% for readability. p<0.01 (**) and p<0.001 (***) by two-way ANOVA.Immunoblot showing H2BK120 ubiquitination levels in Hdac1-proficient and -deficient cell lines (two independent lines each).Growth curves of Hdac1-proficient and -deficient cell lines that were left untreated or were infected with empty virus (pLKO) or shRNAs against Dot1L and selected with puromycin. Growth curves were determined by a series of resazurin assays, which measure metabolic activity, starting four days post-infection. Error bars indicate the range of two replicates from independent cell lines.Inhibitor dose-response curves of the two DOT1L inhibitors EPZ-5676 (Pinometostat) and SGC-0946 in Hdac1-proficient and -deficient cell lines. Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO-treated cells. Two independent cell lines are plotted separately; error bars indicate the range of two biological replicates.