( D - G ) Kaplan Meier analysis to address correlation of TNC expression ( D ) , total numbers of CD8 TIL ( E ) , combined high ( above median ) CD8 TIL and high or low ( above / below median ) TNC ( F ) and stromal CD8 cells ( G ) with metastasis - free survival ( MFS ) of breast cancer patients . Stromal CD8 - or CD8 + is defined as below or above median stromal CD8 TIL per area . Log - rank test .

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(D-G) Kaplan Meier analysis to address correlation of TNC expression (D), total numbers of CD8 TIL (E), combined high (above median) CD8 TIL and high or low (above/below median) TNC (F) and stromal CD8 cells (G) with metastasis-free survival (MFS) of breast cancer patients. Stromal CD8- or CD8+ is defined as below or above median stromal CD8 TIL per area. Log-rank test.


( H , I ) Correlation of TNC and CD8a levels with overall survival ( OS ) using survival analysis on breast cancer patient cohort GSE 19783 - GPL6580 . Hazard ratio ( HR ) and p values are indicated in Appendix Fig . S5N . Log - rank test .

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(H, I) Correlation of TNC and CD8a levels with overall survival (OS) using survival analysis on breast cancer patient cohort GSE 19783-GPL6580. Hazard ratio (HR) and p values are indicated in Appendix Fig. S5N. Log-rank test.


( A ) Representative IHC images of CXCL12 intensity and staining pattern in the TMA . N = 219 tumors . Upper panel , scale bar , 400 μm ; lower panel , scale bar , 100 μm . Arrows point at punctate staining in the stroma .

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(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA. N = 219 tumors. Upper panel, scale bar, 400 μm; lower panel, scale bar, 100 μm. Arrows point at punctate staining in the stroma.


( D ) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median , p = 0 . 002 . Mann - Whitney test . ( E ) Survival analysis of breast cancer patients in the GSE 19783 - GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12 . Log - rank test . HR and p values are indicated ,

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(D) Kaplan Meier survival analysis of breast cancer patients upon stratification into tumors with a CXCL12 Hscore below or above the median, p = 0.002. Mann-Whitney test. (E) Survival analysis of breast cancer patients in the GSE 19783-GPL6580 cohort upon stratification into tumors with high or low expression of TNC and CXCL12. Log-rank test. HR and p values are indicated,


A . Ribbon representation of SARS - CoV - 2 N - NTD molecules in one asymmetric unit . The four molecules are highlighted with different color

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A. Ribbon representation of SARS-CoV-2 N-NTD molecules in one asymmetric unit. The four molecules are highlighted with different color respectively


Naïve CD4 + T cells were isolated from the spleen and LNs of WT , Stim2fl / fl Cd4Cre ( S2 ) , Orai1fl / fl Cd4Cre ( O1 ) and Stim1fl / fl Cd4Cre ( S1 ) mice and differentiated into Th1 , Th17 and iTreg cells . ( A ) Fura - 2 loaded cells were stimulated with 0 . 3 μM ionomycin to induce SOCE . Representative Ca2 + traces ( left ) and peak Ca2 + levels ( right ) . Data are from the mean ± SEM of 4 mice per group .

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Naïve CD4+ T cells were isolated from the spleen and LNs of WT, Stim2fl/fl Cd4Cre (S2), Orai1fl/fl Cd4Cre (O1) and Stim1fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17 and iTreg cells. (A) Fura-2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca2+ traces (left) and peak Ca2+ levels (right). Data are from the mean ± SEM of 4 mice per group.


( B ) Induction of colitis in Rag1 - / - mice by adoptive transfer of naive CD4 + T cells from wildtype , Stim2fl / fl Cd4Cre , Orai1fl / fl Cd4Cre and Stim1fl / fl Cd4Cre mice . Weight curves of recipient Rag1 - / - mice following injection of naive CD4 + T cells . Data are from two independent experiments with 8 recipient mice per group .

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(B) Induction of colitis in Rag1-/- mice by adoptive transfer of naive CD4+ T cells from wildtype, Stim2fl/fl Cd4Cre, Orai1fl/fl Cd4Cre and Stim1fl/fl Cd4Cre mice. Weight curves of recipient Rag1-/- mice following injection of naive CD4+ T cells. Data are from two independent experiments with 8 recipient mice per group.


( C ) H & E stains of colon tissues and histological inflammation scores of Rag1 - / - mice 8 weeks after T cell transfer . Data are from one representative experiment with 5 mice per group .

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(C) H&E stains of colon tissues and histological inflammation scores of Rag1-/- mice 8 weeks after T cell transfer. Data are from one representative experiment with 5 mice per group.


( D - E ) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ , IL - 17A , TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 - / - mice . Data are from 2 independent experiments with 3 - 8 mice per group .

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(D-E) Representative flow cytometry plots and frequencies of donor CD4+ T cells producing IFNγ, IL-17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1-/- mice. Data are from 2 independent experiments with 3-8 mice per group.


( A , B ) Ca2 + influx in human lamina propria CD4 + and CD8 + T cells from 3 IBD patient samples analyzed by flow cytometry . ( A ) Experimental design . ( B ) Ca2 + influx rates in T cells pre - incubated for 4 h in the presence of 15 - 1000 nM BTP2 , which was present until data acquisition . SOCE was induced by stimulation with the sarco - endoplasmic reticulum Ca2 + ATPase ( SERCA ) inhibitor thapsigargin ( TG ) in Ca2 + free buffer followed by the addition of 2 mM Ca2 + . Bar graphs show the mean ± SEM of Ca2 + influx rates after Ca2 + readdition from 1 experiment ( n = 3 IBD patients ) .

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(A,B) Ca2+ influx in human lamina propria CD4+ and CD8+ T cells from 3 IBD patient samples analyzed by flow cytometry. (A) Experimental design. (B) Ca2+ influx rates in T cells pre-incubated for 4 h in the presence of 15-1000 nM BTP2, which was present until data acquisition. SOCE was induced by stimulation with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG) in Ca2+ free buffer followed by the addition of 2 mM Ca2+. Bar graphs show the mean ± SEM of Ca2+ influx rates after Ca2+ readdition from 1 experiment (n=3 IBD patients).


( D ) viSNE plots of CD45 + CD3 + LPMCs stimulated with 20 ng / ml PMA and 1 µg / ml ionomycin for 4 hours in the presence of 15 - 1000 nM BTP2 . viSNE plots are colored according to the expression level of IL - 2 ( blue : low , red : high ) and are representative of one CD patient .

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(D) viSNE plots of CD45+CD3+ LPMCs stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 hours in the presence of 15-1000 nM BTP2. viSNE plots are colored according to the expression level of IL-2 (blue: low, red: high) and are representative of one CD patient.


( E ) Dose - response curves of the frequencies of cytokine producing CD4 + and CD8 + T cells after PMA / ionomycin stimulation for 4h in the presence of increasing doses of BTP2 . The frequencies of cytokine producing cells were normalized to control samples treated with PMA / ionomycin alone . Bar graphs show the mean ± SEM of 5 CD patient samples .

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(E) Dose-response curves of the frequencies of cytokine producing CD4+ and CD8+ T cells after PMA/ionomycin stimulation for 4h in the presence of increasing doses of BTP2. The frequencies of cytokine producing cells were normalized to control samples treated with PMA/ionomycin alone. Bar graphs show the mean ± SEM of 5 CD patient samples.


( F ) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45 + CD3 + LPMCs stimulated for 4h with PMA / ionomycin in the presence of BTP2 ; data are normalized to PMA / ionomycin treatment alone .

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(F) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45+CD3+ LPMCs stimulated for 4h with PMA/ionomycin in the presence of BTP2; data are normalized to PMA/ionomycin treatment alone.


( G ) Cytokine production by CD19 + HLA - DR + B cells and CD11c + dendritic cells in the presence of increasing doses ( 15 - 1 , 000 nM ) of BTP2 . Left : Dose - response curves showing frequencies of cytokine - producing cells after 4h stimulation with PMA / ionomycin ex vivo . Data are normalized to control samples stimulated with PMA / ionomycin in the absence of BTP2 . Error bars represent SEM obtained from 1 experiment ( n = 5 CD patients ) . Right : heatmaps showing the median fold change of cytokines and surface markers in CD19 + HLA - DR + B cells and CD11c + dendritic cells treated as described for the left panel .

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(G) Cytokine production by CD19+HLA-DR+ B cells and CD11c+ dendritic cells in the presence of increasing doses (15-1,000 nM) of BTP2. Left: Dose-response curves showing frequencies of cytokine-producing cells after 4h stimulation with PMA/ionomycin ex vivo. Data are normalized to control samples stimulated with PMA/ionomycin in the absence of BTP2. Error bars represent SEM obtained from 1 experiment (n=5 CD patients). Right: heatmaps showing the median fold change of cytokines and surface markers in CD19+HLA-DR+ B cells and CD11c+ dendritic cells treated as described for the left panel.


( A ) Experimental setup for mass cytometric assays . Non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 . ( B ) FlowSOM plot of merged FCS files from LPMCs of non - inflamed controls , UC or CD patients after stimulation with PMA / ionomycin ± 1 μM BTP2 for 4h ex vivo . Colors of the t - SNE plot represent 20 clusters of distinct CD45 + LPMC lineages . The heatmap shows the expression levels of 21 markers used for defining cell clusters .

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(A) Experimental setup for mass cytometric assays. Non-inflamed: n = 4, CD: n =6, UC: n =6. (B) FlowSOM plot of merged FCS files from LPMCs of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± 1 μM BTP2 for 4h ex vivo. Colors of the t-SNE plot represent 20 clusters of distinct CD45+ LPMC lineages. The heatmap shows the expression levels of 21 markers used for defining cell clusters.


( C ) FlowSOM map of merged FCS files from CD45 + cells of non - inflamed controls , UC and CD patients that were left unstimulated or treated with PMA / ionomycin ± 1 μM BTP2 . Colors indicate altered cell clusters in CD patients vs . controls , UC patients vs . controls and CD vs . UC patients . ( D ) viSNE plots of one exemplary non - inflamed control , UC and CD patient colored by marker expression levels ( blue : low , red : high ) .

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(C) FlowSOM map of merged FCS files from CD45+ cells of non-inflamed controls, UC and CD patients that were left unstimulated or treated with PMA/ionomycin ± 1 μM BTP2. Colors indicate altered cell clusters in CD patients vs. controls, UC patients vs. controls and CD vs. UC patients. (D) viSNE plots of one exemplary non-inflamed control, UC and CD patient colored by marker expression levels (blue: low, red: high).


( E ) Quantified frequencies ( % ) of each cell subset defined by the cluster analysis . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure ( * padj < 0 . 05 , * * padj < 0 . 01 , * * * padj < 0 . 001 ) . Boxes extend from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median from 1 experiment ( non - inflamed n = 4 , CD : n = 6 , UC : n = 6 patients ) .

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(E) Quantified frequencies (%) of each cell subset defined by the cluster analysis. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure (*padj< 0.05, **padj < 0.01, ***padj< 0.001). Boxes extend from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median from 1 experiment (non-inflamed n = 4, CD: n =6, UC: n =6 patients).


( F ) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45 + LPMCs that were isolated from non - inflamed , UC and CD samples and activated for 4h with PMA / ionomycin ex vivo . Statistical significance was calculated using an unpaired t test with FDR adjustment to 1 % using the Benjamini , Krieger and Yekutieli procedure ; *

(F) Heatmaps representing the median fold change of cytokine and surface marker expression in CD45+ LPMCs that were isolated from non-inflamed, UC and CD samples and activated for 4h with PMA/ionomycin ex vivo. Statistical significance was calculated using an unpaired t test with FDR adjustment to 1% using the Benjamini, Krieger and Yekutieli procedure; *


( A ) FlowSOM plot of merged FCS files from samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 + LPMCs . Heatmaps show the expression levels of 24 markers used for cluster determination . The table shows the mean ± SEM of frequencies ( % ) for each cell subset defined in non - inflamed controls , UC and CD patients after 4h stimulation with PMA / ionomycin in vitro . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg procedure .

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

(A) FlowSOM plot of merged FCS files from samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3+ LPMCs. Heatmaps show the expression levels of 24 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-inflamed controls, UC and CD patients after 4h stimulation with PMA/ionomycin in vitro. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure.


( B ) Box plots showing frequencies ( % ) of each cell subset defined by the cluster analysis in non - inflamed , UC and CD samples following stimulation with PMA / ionomycin ± BTP2 for 4h in vitro . Statistical significances were calculated using a one - tailed paired Wilcoxon matched - pairs signed - rank test , * p < 0 . 05 . Boxes range from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 patients ) .

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(B) Box plots showing frequencies (%) of each cell subset defined by the cluster analysis in non-inflamed, UC and CD samples following stimulation with PMA/ionomycin ± BTP2 for 4h in vitro. Statistical significances were calculated using a one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).


( C ) viSNE plots of one exemplary CD patient . Identification of cell subsets ( left ) and expression levels of cytokines after PMA / ionomycin stimulation in the presence or absence of BTP2 ( blue ; low , red : high ) .

(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cytokines after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).


( D ) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45 + CD3 + LPMCs stimulated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

(D) Heatmaps indicating the median fold change expression of cytokines and surface molecules in CD45+CD3+ LPMCs stimulated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


( A ) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA / ionomycin ± 1 µM BTP2 ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 ) . Colors of FlowSOM plot indicate 20 distinct clusters of CD45 + CD3 - LPMCs . Heatmap clusters showing the expression levels of 34 markers used for cluster determination . The table shows the mean ± SEM of frequencies ( % ) for each cell subset defined in non - Inflamed , UC and CD samples after stimulation with PMA / ionomycin for 4h . Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10 % using the Benjiamini - Hochberg

(A) FlowSOM plot of merged FCS files from unstimulated samples or samples treated with PMA/ionomycin ± 1 µM BTP2 (non-inflamed: n = 4, CD: n =6, UC: n =6). Colors of FlowSOM plot indicate 20 distinct clusters of CD45+CD3- LPMCs. Heatmap clusters showing the expression levels of 34 markers used for cluster determination. The table shows the mean ± SEM of frequencies (%) for each cell subset defined in non-Inflamed, UC and CD samples after stimulation with PMA/ionomycin for 4h. Statistical significance was calculated using the edgeR statistical framework with negative binomial GLM and a false discovery rate adjusted to 10% using the Benjiamini-Hochberg procedure


( B ) Box plots show frequencies ( % ) of each cell subset defined by the cluster analysis in samples of non - inflamed controls , UC or CD patients after stimulation with PMA / ionomycin ± BTP2 for 4h ex vivo . Statistical significances were calculated by one - tailed paired Wilcoxon matched - pairs signed - rank test , * p < 0 . 05 . Boxes range from the 25th to 75th percentiles . Whisker plots show the min ( smallest ) and max ( largest ) values . The line in the box denotes the median obtained from 1 experiment ( non - inflamed : n = 4 , CD : n = 6 , UC : n = 6 patients ) .

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(B) Box plots show frequencies (%) of each cell subset defined by the cluster analysis in samples of non-inflamed controls, UC or CD patients after stimulation with PMA/ionomycin ± BTP2 for 4h ex vivo. Statistical significances were calculated by one-tailed paired Wilcoxon matched-pairs signed-rank test, *p < 0.05. Boxes range from the 25th to 75th percentiles. Whisker plots show the min (smallest) and max (largest) values. The line in the box denotes the median obtained from 1 experiment (non-inflamed: n = 4, CD: n =6, UC: n =6 patients).


( C ) viSNE plots of one exemplary CD patient . Identification of cell subsets ( left ) and expression levels of cell markers after PMA / ionomycin stimulation in the presence or absence of BTP2 ( blue ; low , red : high ) .

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(C) viSNE plots of one exemplary CD patient. Identification of cell subsets (left) and expression levels of cell markers after PMA/ionomycin stimulation in the presence or absence of BTP2 (blue; low, red: high).


( D ) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45 + CD3 - LPMCs activated with PMA / ionomycin ± BTP2 for 4h in vitro . Values are normalized to stimulated , non - BTP2 treated samples .

(D) Heatmaps indicating the median fold change expression of cytokines and surface markers in CD45+CD3- LPMCs activated with PMA/ionomycin ± BTP2 for 4h in vitro. Values are normalized to stimulated, non-BTP2 treated samples.


( A - B ) Adult stem cell - derived intestinal epithelial spheroids of wild - type C57BL / 6 mice were cultured for 4 days in Wnt - free medium to induce differentiation . Initial viability was determined on day 0 . DMSO control ( vehicle ) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy . Scale bar depicts 100 µm . ( C ) Transepithelial electrical resistance ( TEER ) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2 .

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(A-B) Adult stem cell-derived intestinal epithelial spheroids of wild-type C57BL/6 mice were cultured for 4 days in Wnt-free medium to induce differentiation. Initial viability was determined on day 0. DMSO control (vehicle) or 1 μM BTP2 were added to the culture medium and viability was measured every 24h by resazurin viability assay and microscopy. Scale bar depicts 100 µm. (C) Transepithelial electrical resistance (TEER) was measured on differentiated murine colon monolayers in the presence or absence of 1 µM BTP2.


( D ) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt - free medium to induce differentiation into colonic organoids . Initial viability was determined on day 0 . DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2 , 4 and 6 .

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(D) Human colonic spheroids were generated from primary human epithelial crypts obtained from colon resectates of 2 CD patients and cultured for 6 days in Wnt-free medium to induce differentiation into colonic organoids. Initial viability was determined on day 0. DMSO control or 1 μM BTP2 were added to the culture medium and viability was measured on days 2, 4 and 6.


( E ) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2 .

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(E) TEER of differentiated human colon monolayers was analyzed in the presence or absence of 1 µM BTP2.


( F ) Relative mRNA expression of markers for enterocytes ( ALPI ) , goblet cells ( MUC2 ) , and entero - endocrine cells ( CHGA ) was quantified by RT - PCR in differentiated human colon monolayers after addition of 1 µM BTP2 .

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(F) Relative mRNA expression of markers for enterocytes (ALPI), goblet cells (MUC2), and entero-endocrine cells (CHGA) was quantified by RT-PCR in differentiated human colon monolayers after addition of 1 µM BTP2.


( A - C ) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents ( Icrac ) . ( A ) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1 . ( B ) Current - voltage ( I - V ) relationship of ICRAC before and after CM4620 treatment . I - Vs were obtained at the time points indicated by the arrowheads in ( A ) . ( C ) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration ( arrowheads ) using the formula 1 - ( ICM4620 / ICtrl ) . Data are from 8 cells per conditions and shown as the mean ± SEM .

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(A-C) Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current-voltage (I-V) relationship of ICRAC before and after CM4620 treatment. I-Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1- (ICM4620 / ICtrl). Data are from 8 cells per conditions and shown as the mean ± SEM.


( E ) Relative weight loss of mice treated with vehicle or CM4620 ; the start of treatment is indicated by the white arrow . Data are the mean ± SEM of 9 mice per cohort , statistical analysis by unpaired student ' s t - test , * p < 0 . 05 .

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(E) Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of 9 mice per cohort, statistical analysis by unpaired student's t-test , *p<0.05.


( F ) Concentrations of CM4620 in the serum of mice at day 49 , data are the mean ± SEM . Statistical analysis by unpaired student ' s t - test , * * * p < 0 . 001 .

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(F) Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired student's t-test, ***p<0.001.


( G ) Representative H & E staining of the distal colon of Rag1 - / - mice treated with vehicle or CM4620 . Colitis scores of 9 mice per cohort . Each symbol represents one mouse .

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(G) Representative H&E staining of the distal colon of Rag1-/- mice treated with vehicle or CM4620. Colitis scores of 9 mice per cohort. Each symbol represents one mouse.


( H ) Number of CD11b + Gr - 1 + neutrophils and CD4 + T cells ( normalized to grams of tissue ) in the colon lamina propria ( CLP ) of mice on day 49 quantified by flow cytometry . Each dot represents one mouse . Data are the mean ± SEM of 9 mice per cohort .

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(H) Number of CD11b+Gr-1+ neutrophils and CD4+ T cells (normalized to grams of tissue) in the colon lamina propria (CLP) of mice on day 49 quantified by flow cytometry. Each dot represents one mouse. Data are the mean ± SEM of 9 mice per cohort.


( I ) Frequencies of RORγt + Th17 cells and Foxp3 + Treg cells in the CLP of mice treated with CM4620 or vehicle . Each dot represents one mouse . Data are the mean ± SEM of 6 control and 5 CM4620 treated mice .

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(I) Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


( J ) Frequencies of IFN - γ , TNF - α and IL - 2 producing CD4 + T cells isolated from the CLP , restimulated ex vivo with PMA and ionomycin for 4 h ( without addition of CM4620 during stimulation in vitro ) and analyzed by flow cytometry . Bar graphs represent the percentages of IFN - γ + , TNF - α + , IL - 2 + cells in CD4 + cells ( top row ) and mean fluorescent intensity ( MFI ) of IFN - γ , TNF - α , IL - 2 on CD4 + cells ( bottom row ) . Each dot represents one mouse . Data are the mean ± SEM of 6 control and 5 CM4620 treated mice .

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(J) Frequencies of IFN-γ, TNF-α and IL-2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN-γ+, TNF-α+, IL-2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN-γ, TNF-α, IL-2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of 6 control and 5 CM4620 treated mice.


A Scheme of the lentiviral vector with inducible FlagOmomyc ( top ) and immunoblotting ( bottom ) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0 - 48 hours .

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A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FOcells for 0-48 hours.


B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0 - 96 h ( n = 3 ; mean ± SD ) . Viable cells were counted using a haemocytometer .

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B Proliferation curves of BT168FO and BT168GFPcells upon Dox treatment for 0-96 h (n = 3; mean ± SD). Viable cells were counted using a haemocytometer.


C - D Self - renewal assay upon Dox treatment . C : histograms showing the percentage of cells capable of re - forming a neurosphere seven days after dissociation ( n = 3 ; mean ± SD ) . D : representative micrographs of BT168FO cell neurospheres .

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C-D Self-renewal assay upon Dox treatment. C: histograms showing the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). D: representative micrographs of BT168FO cell neurospheres.


E qRT - PCR of proliferation , stem cell and differentiation markers ( CCND1 , PTEN , SOX2 , NOTCH1 , NESTIN , MYC ) in BT168FO cells after 48 h Dox treatment , compared to uninduced cells ( n = 3 ; mean ± SD ) . Expression levels in non - induced cells were set as 1 .

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E qRT-PCR of proliferation, stem cell and differentiation markers (CCND1, PTEN, SOX2, NOTCH1, NESTIN, MYC) in BT168FO cells after 48 h Dox treatment, compared to uninduced cells (n = 3; mean ± SD). Expression levels in non-induced cells were set as 1.


F . Transwell migration assay of BT168FO cells after 3 days with or without Dox ( n = 3 ; mean ± SD ) . 10 fields were counted per assay .

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F. Transwell migration assay of BT168FO cells after 3 days with or without Dox (n = 3; mean ± SD). 10 fields were counted per assay.


G . Immunofluorescence analyses of GSC differentiation . To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days . The upper panel displays immunofluorescence images of NESTIN , GFAP , ß - tubulin III , SOX2 , and FlagOmomyc expression . FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels , while inducing the onset of ß - tubulin III . The lower panel shows the percentage of positive cells for each cell marker evaluated ( n = 4 ; mean ± SD ) . 16 fields for each assay were examined ; scale bar = 100 μm .

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G. Immunofluorescence analyses of GSC differentiation. To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days. The upper panel displays immunofluorescence images of NESTIN, GFAP, ß-tubulin III, SOX2, and FlagOmomyc expression. FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels, while inducing the onset of ß-tubulin III. The lower panel shows the percentage of positive cells for each cell marker evaluated (n = 4; mean ± SD). 16 fields for each assay were examined; scale bar = 100 μm.


A - H Immunostaining of representative xenografts derived from naïve ( control ) and Omomyc expressing BT168FO cells . A - D ) Flag , Ki67 , CD31 , and DCX ( doublecortin ) immunostaining of adjacent sections in control and Omomycxenografts . E - G ) Differentiation phenotype investigated by GFAP , ß - tubulin III , and OLIG2 staining . H ) Immunostaining for anti - human nuclei . Representative images are shown . Scale bar = 100μm .

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A-H Immunostaining of representative xenografts derived from naïve (control) and Omomyc expressing BT168FO cells. A-D) Flag, Ki67, CD31, and DCX (doublecortin) immunostaining of adjacent sections in control and Omomycxenografts. E-G) Differentiation phenotype investigated by GFAP, ß-tubulin III, and OLIG2 staining. H) Immunostaining for anti-human nuclei. Representative images are shown. Scale bar = 100μm.


A - B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter - target genes in BT168FO ( A ) cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows , in cells treated or not with Dox .

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A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in BT168FO (A) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.


A - B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter - target genes in U87FO ( B ) cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in units of tags per 50bp . The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows , in cells treated or not with Dox .

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A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in U87FO (B) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.


A Heatmap of RNA Pol II levels around TSSs of MYC promoter - target genes in BT168FO cells , treated or not with Dox for 24 h . TSS regions are ranked by decreasing MYC occupancy in untreated cells . Each row shows the ± 5 kb region centred on TSSs . Colour scaled intensities are in tags / 50bp .

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A Heatmap of RNA Pol II levels around TSSs of MYC promoter-target genes in BT168FO cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in tags/50bp.


B Tracks of Pol II binding signals ( ChIP - seq ) at NCL , miR - 17 - 92 , OLIG2 , HDAC1 , and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h . The y - axis shows Pol II binding signals as tags / 500bp / million reads . The x - axis displays genomic positions ; introns as lines , exons as boxes . Arrowheads denote direction of transcription . Blue and green bars denote the presence of MYC or Omomyc peaks . Grey boxes are TSS regions .

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B Tracks of Pol II binding signals (ChIP-seq) at NCL, miR-17-92, OLIG2, HDAC1, and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h. The y-axis shows Pol II binding signals as tags/500bp/million reads. The x-axis displays genomic positions; introns as lines, exons as boxes. Arrowheads denote direction of transcription. Blue and green bars denote the presence of MYC or Omomyc peaks. Grey boxes are TSS regions.


C Transcript levels ( FPKM from RNA - seq data ) in BT168FO cells of the genes shown in B . Data are means of 3 independent biological replicates .

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C Transcript levels (FPKM from RNA-seq data) in BT168FO cells of the genes shown in B. Data are means of 3 independent biological replicates.


D Ratio between fold enrichments ( specific antibody / IgG signal ) derived from qChIPs of Pol II - Ser2p and Pol II - Ser5p ( n = 3 ; mean ± SD ) .

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D Ratio between fold enrichments (specific antibody / IgG signal) derived from qChIPs of Pol II-Ser2p and Pol II-Ser5p (n = 3; mean ± SD).


E RNA Polymerase II traveling ratio distribution from the Pol II ChIP - seqs , in the presence or absence of Dox .

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E RNA Polymerase II traveling ratio distribution from the Pol II ChIP-seqs, in the presence or absence of Dox.


A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline ( black line ) . In doxycycline treated cells promoter occupancy by MYC is lower , and no longer increases together with transcript levels . The scatter plot displays MYCChIP - seq reads at promoters ( - 1000 , + 100 regions with respect to TSS ) in BT168FO cells - untreated ( black ) or upon 48 h Dox treatment ( blue ) - versus transcript levels ( FPKM from RNA - seq data ) .

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A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline (black line). In doxycycline treated cells promoter occupancy by MYC is lower, and no longer increases together with transcript levels. The scatter plot displays MYCChIP-seq reads at promoters (-1000, +100 regions with respect to TSS) in BT168FO cells - untreated (black) or upon 48 h Dox treatment (blue) - versus transcript levels (FPKM from RNA-seq data).


B Distribution of transcript level differences ( Δ FPKM ) of single genes classified as promoter - targets ( green ) , intragenic - targets ( blue ) , intergenic - targets ( red ) in Dox treated ( 24 h ) versus untreated cells , plotted against expression in untreated cells ( n = 3 ) . Δ FPKM = FPKM _ 24h Dox - FPKM _ no Dox .

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B Distribution of transcript level differences (Δ FPKM) of single genes classified as promoter-targets (green), intragenic-targets (blue), intergenic-targets (red) in Dox treated (24 h) versus untreated cells, plotted against expression in untreated cells (n=3). Δ FPKM = FPKM_24h Dox - FPKM_no Dox.


C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment , subdivided in promoter - , intragenic - , intergenic - targets ( ± 50 kbp from TSS ) , and the rest . The following rows depict - for each gene class - mean ( average ) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change ( Fc ) . Data are from 3 independent biological replicates .

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C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment, subdivided in promoter-, intragenic-, intergenic-targets (± 50 kbp from TSS), and the rest. The following rows depict - for each gene class - mean (average) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change (Fc). Data are from 3 independent biological replicates.


D Transcript level distribution of MYC promoter - targets significantly down ( red dots ) or up ( green dots ) modulated in cells treated with Dox for 24 h vs . untreated cells . Transcript levels , expressed as FPKM , represent averages of three independent experiments .

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D Transcript level distribution of MYC promoter-targets significantly down (red dots) or up (green dots) modulated in cells treated with Dox for 24 h vs. untreated cells. Transcript levels, expressed as FPKM, represent averages of three independent experiments.


A RNA - seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction ( n = 3 independent biological replicates ) . The block on the left represents DUSP ( dual specificity phosphatase ) family genes . The middle block contains transcription factors ( TFs ) : the upper thirteen ( ASCL1 - SALL1 ) are GSC - specific and the remaining ones are oncogenic . The right block contains genes , including well - known MYC targets , with functions in proliferation , neurodifferentiation , and gliomagenesis . The first column of each block represents the average expression ( log2 FPKM ) in untreated cells ( 0 h ) in the colour scale illustrated by the lower bar : violet indicates low and blue high expression . The other columns depict relative expression versus untreated cells ( average log2 FC ) at different times ( 8 - 48h ) of Dox treatment , according to the scale shown by the upper bar : red indicating low and green high expression . The table in the upper left summarizes the GSEA score of the set 19 GSC - specific transcription factors [ 40 ] , showing a significant downregulation by Omomyc .

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A RNA-seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction (n = 3 independent biological replicates). The block on the left represents DUSP (dual specificity phosphatase) family genes. The middle block contains transcription factors (TFs): the upper thirteen (ASCL1-SALL1) are GSC-specific and the remaining ones are oncogenic. The right block contains genes, including well-known MYC targets, with functions in proliferation, neurodifferentiation, and gliomagenesis. The first column of each block represents the average expression (log2 FPKM) in untreated cells (0 h) in the colour scale illustrated by the lower bar: violet indicates low and blue high expression. The other columns depict relative expression versus untreated cells (average log2 FC) at different times (8-48h) of Dox treatment, according to the scale shown by the upper bar: red indicating low and green high expression. The table in the upper left summarizes the GSEA score of the set 19 GSC-specific transcription factors [40], showing a significant downregulation by Omomyc.


B Enrichment plots obtained by GSEA ( Gene Set Enrichment Analysis ) of RNA - seq data from BT168FO cells . Left : genes downregulated by Omomyc in BT168FO identify the set of genes targeted by the GSC core TFs [ 40 ] . Middle : Omomyc downregulated genes identify a gene set upregulated by MYC in cancer cells [ 66 ] . Right : genes upregulated by Omomyc identify genes downregulated in small cell lung cancers carrying MYC amplification [ 2 ] . .

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B Enrichment plots obtained by GSEA (Gene Set Enrichment Analysis) of RNA-seq data from BT168FO cells. Left: genes downregulated by Omomyc in BT168FO identify the set of genes targeted by the GSC core TFs [40]. Middle: Omomyc downregulated genes identify a gene set upregulated by MYC in cancer cells [66]. Right: genes upregulated by Omomyc identify genes downregulated in small cell lung cancers carrying MYC amplification [2]. .


B Immunoblots of EGFR , ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with ( + ) or without ( - ) Dox . Representative images are shown .

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B Immunoblots of EGFR, ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with (+) or without (-) Dox. Representative images are shown.


C RT - qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence ( + ) or absence ( - ) of Dox ( n = 3 ; mean ± SD ) . Expression levels were normalized using U6RNA as control . Expression levels in non - induced cells were set as 1 .

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C RT-qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence (+) or absence (-) of Dox (n = 3; mean ± SD). Expression levels were normalized using U6RNA as control. Expression levels in non-induced cells were set as 1.


D EGFR , ZEB1 , and GAPDHimmunoblots in the same cells as in panel C . Representative images are shown .

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D EGFR, ZEB1, and GAPDHimmunoblots in the same cells as in panel C. Representative images are shown.


E EGFR , ZEB1 , and GAPDHimmunoblots in uninduced U87FO cells ( - ) and in cells transfected with LNA oligos targeting miR - 200a - 200b - 429 ( LNA 200 ) and miR - 146b ( LNA 146b ) , or with a non - targeting LNA ( C ) , and induced with Dox for 48 h ( + Dox ) . Representative images are shown .

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E EGFR, ZEB1, and GAPDHimmunoblots in uninduced U87FO cells (-) and in cells transfected with LNA oligos targeting miR-200a-200b-429 (LNA 200) and miR-146b (LNA 146b), or with a non-targeting LNA (C), and induced with Dox for 48 h (+Dox). Representative images are shown.


F MTS cell proliferation assay of uninduced or Dox induced U87FO cells , transfected with either a non - targeting LNA ( LNA C ) , or LNA oligos targeting the miR - 200a - 200b - 429 cluster ( LNA 200 ) , miR - 146b ( LNA 146b ) , or both ( LNAmix ) and analysed at 24 , 48 and 72h ( n = 3 ; mean ± SD ) .

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F MTS cell proliferation assay of uninduced or Dox induced U87FO cells, transfected with either a non-targeting LNA (LNA C), or LNA oligos targeting the miR-200a-200b-429 cluster (LNA 200), miR-146b (LNA 146b), or both (LNAmix) and analysed at 24, 48 and 72h (n = 3; mean ± SD).


G Migration assay of U87FO cells transfected with non - targeting control LNA oligos ( LNA C ) or with LNA oligos targeting miR - 200a - 200b - 429 ( LNA 200 ) ; cells were either untreated ( - ) or treated with Dox for 24 h ( + Dox ) ; ( n = 3 ; mean ± SD ) .

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G Migration assay of U87FO cells transfected with non-targeting control LNA oligos (LNA C) or with LNA oligos targeting miR-200a-200b-429 (LNA 200); cells were either untreated (-) or treated with Dox for 24 h (+Dox); (n = 3; mean ± SD).


A . Relative antiviral activity of IFNα ( circles ) or IFNλ ( triangles ) . AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations ( ng / ml ) and induction of indicated ISGs was assessed by qPCR ( data shown is representative of four independent experiments , n = 3 - 4 ) .

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A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).


B , C . Mice were pretreated with equivalent doses of IFNα ( 1 . 45μg / 50μl ) or IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( squares , 50μl PBS ) 24hrs prior to infection with PR8 ; weight loss and survival was assessed throughout infection ( B ) , and viral load ( C ) assessed at 4dpi ( data shown is representative of two independent experiments , n = 8 - 10 ( B ) , n = 3 ( C ) ) .

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B, C. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)).


D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; survival and weight loss was monitored ( D , data pooled from 4 independent experiments , n = 12 - 29 ) ( data representative of two independent experiments , n = 3 - 5 ) .

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D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) (data representative of two independent experiments, n=3-5).


D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; viral load assessed at 4dpi ( E ) ( data representative of two independent experiments , n = 3 - 5 ) .

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D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).


A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay ( A ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) (data shown is representative of two independent experiments, n=2-6).


A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed ( B ) ( data shown is representative of two independent experiments , n = 2 - 6 ) .

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A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6).


C , D . Lung sections from control and infected mice treated as indicated , were stained by TUNEL for apoptotic cells at 6dpi . Quantification of TUNEL + cells in whole lung slides by Icy Spot Detector ( ICY - R3M2Y2 ) ( C ) ( data shown is pooled from three independent experiments , n = 3 - 8 ) . Red arrowheads indicate TUNEL signal ( D ) . Scale bar , 200 μM ( data shown is representative of two independent experiments , n = 3 - 4 ) .

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C, D. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).


A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants ( A ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) or LPS ( AEC only ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).


A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in Macrophage , pDC and cDC culture supernatants ( B ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) ( data shown is representative of two independent experiments , n = 3 - 6 ) .

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A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) (data shown is representative of two independent experiments, n=3-6).


C . BAL samples taken from mice treated with IFNα , IFNλ or Veh Ctrl at specified time points ( data shown is representative of two independent experiments , n = 5 - 6 ) .

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C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).


Mice were treated with IFNα ( 1 . 45μg / 50μl ) , IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( 50μl PBS ) , and whole lungs were taken at 18hrs post treatment for global analysis by Illumina . SingleColor . Mouse WG - 6 _ V2 _ 0 _ R0 _ 1127 microarrays . Samples ( n = 5 ) were normalized to the median of the vehicle control group and filtered for a fold change of 1 . 5 , yielding 553 genes differently regulated between treatments ( One way ANOVA , P < 0 . 01 , Benjamini - Hochberg multiple test correction ) , of which 429 genes are upregulated . K means clustering revealed six gene clusters , one of which encompassed genes primarily induced by IFNα4 ( A ) , while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 ( B ) . The two clusters of genes were analysed by Ingenuity Pathway Analysis ( IPA ) ( C , D ) .

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Mice were treated with IFNα (1.45μg/50μl), IFNλ (2.6μg/50μl) or Veh Ctrl (50μl PBS), and whole lungs were taken at 18hrs post treatment for global analysis by Illumina.SingleColor.Mouse WG-6_V2_0_R0_1127 microarrays. Samples (n=5) were normalized to the median of the vehicle control group and filtered for a fold change of 1.5, yielding 553 genes differently regulated between treatments (One way ANOVA, P<0.01, Benjamini-Hochberg multiple test correction), of which 429 genes are upregulated. K means clustering revealed six gene clusters, one of which encompassed genes primarily induced by IFNα4 (A), while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 (B). The two clusters of genes were analysed by Ingenuity Pathway Analysis (IPA) (C,D).


A . HumanAEC cultures were stimulated for 4hrs with IFNα ( circles ) or IFNλ ( triangles ) at specified concentrations then assessed for stated ISG induction by qPCR ( data is representative of 2 independent experiments , n = 3 ) .

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A. HumanAEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3).


B , C . ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα ( 21 U / ml ) or IFNλ ( 1 . 2 ng / ml ) stimulation ( B ) ( data shown is pooled from 6 independent donors ) .

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B, C. ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B) (data shown is pooled from 6 independent donors).


B , C . PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ ( C ) ( data shown is pooled from 6 independent donors ) .

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B, C. PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).


( A ) Laser injured DRG axons showing growth cone regeneration ( upper panels ) and regenerative failure ( lower panels ) . Arrows as indicated .

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(A) Laser injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated.


( B ) Upper graph shows percentage of regenerating axons of DIV 1 - 2 adult DRG neurons in the presence of p110 inhibitors , 2 h after injury . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , and maintained throughout : LY294002 , 20 μM . A66 5 μM . XL - 147 5 μM . TGX221 500 nM . IC - 87114 10μM . Idelalisib 500 nM . Upper graph data are shown as the mean + / - SEM . P values indicate statistical significance analysed by Fishers exact ( upper graph ) or Kruskal - Wallis test ( lower graph ) .

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(B) Upper graph shows percentage of regenerating axons of DIV 1-2 adult DRG neurons in the presence of p110 inhibitors, 2 h after injury. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: LY294002, 20 μM. A66 5 μM. XL-147 5 μM. TGX221 500 nM. IC-87114 10μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance analysed by Fishers exact (upper graph) or Kruskal-Wallis test (lower graph).


( C ) Examples of static vs . growing uninjured axons treated with p110α or δ inhibitors as indicated . Both arrows in bottom right image indicate growing axons .

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(C) Examples of static vs. growing uninjured axons treated with p110α or δ inhibitors as indicated. Both arrows in bottom right image indicate growing axons.


( D ) Percentage of uninjured , growing axons in the presence of p110 inhibitors . Inhibitors were added as for ( B ) . Lower graph shows length grown in 2 h . Data are shown as the mean + / - SEM . P values indicate significance measured by Fishers exact test ( upper graph ) or ANOVA with Tukey ' s post - hoc analysis ( lower graph ) .

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(D) Percentage of uninjured, growing axons in the presence of p110 inhibitors. Inhibitors were added as for (B). Lower graph shows length grown in 2 h. Data are shown as the mean +/- SEM. P values indicate significance measured by Fishers exact test (upper graph) or ANOVA with Tukey's post-hoc analysis (lower graph).


( E ) Adult DRG neurons in microfluidic compartmental chambers . Cell bodies on the left side , axons extending through microchannels on the right . Lower panels are schematics .

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(E) Adult DRG neurons in microfluidic compartmental chambers. Cell bodies on the left side, axons extending through microchannels on the right. Lower panels are schematics.


( F ) Upper graph shows percentage of regenerating axons in microfluidic chambers . p110α or δ inhibitors were applied to either soma or axons . Lower graph shows time taken to regenerate a new growth cone . Inhibitors were added 1hr before axons were injured , and maintained throughout : A66 5 μM . Idelalisib 500 nM . Upper graph data are shown as the mean + / - SEM . P values indicate statistical significance as measured by Fishers exact test ( upper graph ) or by Kruskal - Wallis test ( lower graph ) .

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(F) Upper graph shows percentage of regenerating axons in microfluidic chambers. p110α or δ inhibitors were applied to either soma or axons. Lower graph shows time taken to regenerate a new growth cone. Inhibitors were added 1hr before axons were injured, and maintained throughout: A66 5 μM. Idelalisib 500 nM. Upper graph data are shown as the mean +/- SEM. P values indicate statistical significance as measured by Fishers exact test (upper graph) or by Kruskal-Wallis test (lower graph).


( A ) DIV 3 cortical neurons immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( B ) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( C ) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3 . Spectrum heatmap shows fluorescence intensity . ( D ) Somatic PIP3 quantification in the soma at increasing days in vitro . Data are shown as the mean + / - SEM . P values are significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 neurons . ( E ) Growth cone PIP3 quantification at increasing days in vitro . Data are shown as the mean + / - SEM . P values indicate significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 growth cones .

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(A) DIV 3 cortical neurons immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (B) DIV 8 cortical neuron transfected with GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (C) DIV 16 cortical neuron transfected GFP and immunolabelled for PIP3. Spectrum heatmap shows fluorescence intensity. (D) Somatic PIP3 quantification in the soma at increasing days in vitro. Data are shown as the mean +/- SEM. P values are significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (E) Growth cone PIP3 quantification at increasing days in vitro. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


( A ) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP . Spectrum heatmap shows fluorescence intensity . ( B ) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP . Spectrum heatmap shows fluorescence intensity . ( C ) Quantification of PIP3 immunofluorescence in the soma . Data are shown as the mean + / - SEM . P values indicate significance as measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 neurons . ( D ) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone . Data are shown as the mean + / - SEM . P values indicate significance measured by ANOVA with Tukey ' s post - hoc analysis . N = 3 experiments , 60 growth cones .

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(A) PIP3 immunofluorescence in the soma of DIV 16 cortical neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (B) PIP3 immunofluorescence at the axonal growth cone of DIV 16 neurons expressing p110 isoforms and GFP. Spectrum heatmap shows fluorescence intensity. (C) Quantification of PIP3 immunofluorescence in the soma. Data are shown as the mean +/- SEM. P values indicate significance as measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 neurons. (D) Graph showing PIP3 quantification of immunofluorescence at the axon growth cone. Data are shown as the mean +/- SEM. P values indicate significance measured by ANOVA with Tukey's post-hoc analysis. N=3 experiments, 60 growth cones.


( A ) DIV 4 cortical neurons expressing p110 isoforms and GFP . Arrow marks the axon tip . ( B ) Quantification of axon length . n = 3 experiments , 45 neurons per group . ( C ) Quantification of dendrite length . n = 3 experiments , 45 neurons per group . ( D ) Quantification of the axon : dendrite length ratio . n = 3 experiments , 45 neurons per group .

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(A) DIV 4 cortical neurons expressing p110 isoforms and GFP. Arrow marks the axon tip. (B) Quantification of axon length. n = 3 experiments, 45 neurons per group. (C) Quantification of dendrite length. n = 3 experiments, 45 neurons per group. (D) Quantification of the axon:dendrite length ratio. n = 3 experiments, 45 neurons per group.


( E ) DIV 14 cortical neurons expressing p110 isoforms and GFP , immunolabelled for pS6 . ( F ) Sholl analysis of branches . n = 60 neurons per group . Data are shown as the mean + / - SEM . P values indicate significance measured by repeated measure ANOVA with Bonferonni ' s post - hoc test . ( G ) Quantification of the total neurite length . n = 3 experiments , 60 neurons per group . ( H ) Quantification of soma area . n = 3 experiments , 60 neurons per group . ( I ) Quantification of the pS6 immunofluorescence . n = 3 experiments , 60 neurons per group .

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(E) DIV 14 cortical neurons expressing p110 isoforms and GFP, immunolabelled for pS6. (F) Sholl analysis of branches. n = 60 neurons per group. Data are shown as the mean +/- SEM. P values indicate significance measured by repeated measure ANOVA with Bonferonni's post-hoc test. (G) Quantification of the total neurite length. n = 3 experiments, 60 neurons per group. (H) Quantification of soma area. n = 3 experiments, 60 neurons per group. (I) Quantification of the pS6 immunofluorescence. n = 3 experiments, 60 neurons per group.


( A ) Axotomised DIV 14 - 16 rat cortical neurons expressing p110δ and GFP or GFP alone . Axons were cut > 1000 μm from the soma imaged for 14 h . Black arrows mark the cut site , white arrows marks the axon tip . See also Movies EV7 and EV8 . ( B ) Percentage of regenerating axons 14 h after laser axotomy . Numbers on bars are injured axons per group , from 4 experiments . ( C ) Quantification of axon regeneration length 14 h post - axotomy . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R and 27 for p110δ from 4 experiments . ( D ) Quantification of time to start of regeneration . n = 9 axons for GFP , 9 for p110α , 25 for p110αH1047R and 27 for p110δ from 4 experiments .

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(A) Axotomised DIV 14-16 rat cortical neurons expressing p110δ and GFP or GFP alone. Axons were cut >1000 μm from the soma imaged for 14 h. Black arrows mark the cut site, white arrows marks the axon tip. See also Movies EV7 and EV8. (B) Percentage of regenerating axons 14 h after laser axotomy. Numbers on bars are injured axons per group, from 4 experiments. (C) Quantification of axon regeneration length 14 h post-axotomy. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments. (D) Quantification of time to start of regeneration. n=9 axons for GFP, 9 for p110α, 25 for p110αH1047R and 27 for p110δ from 4 experiments.


( E ) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone . Black arrow marks the cut site , white arrows mark the axon tip . ( F ) Percentage of regenerating axons of human hESC neurons , Numbers on bars are injured axons per group , from 5 experiments . ( G ) Quantification of axon regeneration length . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as the mean + / - SEM . Data were analysed by two tailed Student ' s T - test . ( H ) Quantification of time to start of regeneration . n = 22 axons for GFP , 44 for p110δ , from 5 experiments . Data are shown as the mean + / - SEM . Data were analysed by two tailed Student ' s T - test .

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(E) Axotomised human embryonic stem cell neurons p110δ and GFP or GFP alone. Black arrow marks the cut site, white arrows mark the axon tip. (F) Percentage of regenerating axons of human hESC neurons, Numbers on bars are injured axons per group, from 5 experiments. (G) Quantification of axon regeneration length. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test. (H) Quantification of time to start of regeneration. n=22 axons for GFP, 44 for p110δ, from 5 experiments. Data are shown as the mean +/- SEM. Data were analysed by two tailed Student's T-test.


( A ) Percentage of regenerating axons after laser axotomy of DIV 14 - 17 rat cortical neurons expressing either GFP ( control ) or GFP and p110δ in the presence of the indicated inhibitors , 14h after laser injury . Numbers on bars are injured axons per group .

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(A) Percentage of regenerating axons after laser axotomy of DIV 14-17 rat cortical neurons expressing either GFP (control) or GFP and p110δ in the presence of the indicated inhibitors, 14h after laser injury. Numbers on bars are injured axons per group.


( B ) Single confocal section through a DIV 16 neuron expressing p110δ and GFP ( upper panels ) . Green colour is GFP , red is overexpressed p110δ , detected with anti - p110δ . Lower panels show p110δ and GFP in a distal axon section , and at the growth cone . Arrow indicates the axon .

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(B) Single confocal section through a DIV 16 neuron expressing p110δ and GFP (upper panels). Green colour is GFP, red is overexpressed p110δ, detected with anti-p110δ. Lower panels show p110δ and GFP in a distal axon section, and at the growth cone. Arrow indicates the axon.


( C ) Active ARF and total ARF6 ( red ) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ , as indicated . Arrows indicate axons . ( D ) Quantification of mean axonal ARF activity and total ( mean ) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n = 60 axons for each condition .

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(C) Active ARF and total ARF6 (red) in axons of DIV 16 rat cortical neurons expressing GFP or GFP and p110δ, as indicated. Arrows indicate axons. (D) Quantification of mean axonal ARF activity and total (mean) ARF6 in DIV 16 neurons expressing GFP or GFP and p110δ n=60 axons for each condition.


( E ) Kymographs showing dynamics of α9 integrin - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( F ) Quantification of α9 integrin - GFP dynamics in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( G ) Quantification of total α9 integrin - GFP number in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 47 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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(E) Kymographs showing dynamics of α9 integrin-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (F) Quantification of α9 integrin-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (G) Quantification of total α9 integrin-GFP number in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 47 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.


( H ) Kymographs showing dynamics of Rab11 - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . ( I ) Quantification of Rab11 - GFP dynamics in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n numbers are axon sections analysed for each condition . ( J ) Quantification of total Rab11 - GFP in the distal axons of DIV 14 - 16 neurons , control or co - transfected with p110δ . n = 43 for control , 53 for co - transfected with p110δ . n numbers are axon sections analysed for each condition .

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(H) Kymographs showing dynamics of Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. (I) Quantification of Rab11-GFP dynamics in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n numbers are axon sections analysed for each condition. (J) Quantification of total Rab11-GFP in the distal axons of DIV 14-16 neurons, control or co-transfected with p110δ. n=43 for control, 53 for co-transfected with p110δ. n numbers are axon sections analysed for each condition.


C ) Retinal sections from AAV - injected mice , immunolabelled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset images show individual colours at the same scale as the full image .

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C) Retinal sections from AAV-injected mice, immunolabelled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image.


( D ) Percentage of phospho - S6 positive cells in the RGC layer 2 weeks after delivery of AAV - Cre - GFP or AAV - GFP .

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(D) Percentage of phospho-S6 positive cells in the RGC layer 2 weeks after delivery of AAV-Cre-GFP or AAV-GFP.


( E ) Retinal whole mounts of AAV - injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival .

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(E) Retinal whole mounts of AAV-injected mice immunolabelled for the RGC marker Brn3A to indicate cell survival.


( F ) Analysis of RGC survival 28 days after optic nerve crush ( counts of Brn3A positive RGC neurons ) .

(F) Analysis of RGC survival 28 days after optic nerve crush (counts of Brn3A positive RGC neurons).


G ) Representative images of CTB - labelled RGC axons 4 weeks after optic nerve crush in wild type ( c57bl / 6 ) , Rosa26 - p110αH1047R , and Rosa26 - p110δ transgenic mice injected with AAV2 . Cre . GFP . Dashed line indicates crush site .

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G) Representative images of CTB-labelled RGC axons 4 weeks after optic nerve crush in wild type (c57bl/6), Rosa26-p110αH1047R, and Rosa26-p110δ transgenic mice injected with AAV2.Cre.GFP. Dashed line indicates crush site.


( H ) Regenerating axons at different distances distal to lesion sites .

(H) Regenerating axons at different distances distal to lesion sites.


( A ) Retinal sections from mice injected with AAV2 viruses as indicated , immunolabeled for phospho - S6 . Blue colour is DAPI to indicate nuclei . Inset images show individual colours at the same scale as the full image . ( B ) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2 . shScramble . GFP or AAV2 . shPTEN . GFP . ( C ) Number of pS6 - postive RGCs of mice injected with AAV2 . GFP or AAV2 . p110δ .

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(A) Retinal sections from mice injected with AAV2 viruses as indicated, immunolabeled for phospho-S6. Blue colour is DAPI to indicate nuclei. Inset images show individual colours at the same scale as the full image. (B) Percentage of phospho S6 positive cells in the retinal ganglion layer of mice injected with AAV2.shScramble.GFP or AAV2.shPTEN.GFP. (C) Number of pS6-postive RGCs of mice injected with AAV2.GFP or AAV2.p110δ.


( D ) Representative images of retinal whole mounts from AAV - injected mice stained for the RGC marker Brn3A to indicate RGC survival . ( E ) RGC survival 28 days after optic nerve crush .

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(D) Representative images of retinal whole mounts from AAV-injected mice stained for the RGC marker Brn3A to indicate RGC survival. (E) RGC survival 28 days after optic nerve crush.


( F ) CTB - labelled RGC axons 4 weeks after optic nerve crush . Boxes i . to iv . show regenerating axons . Dashed line indicates crush site . White arrows mark the end of axons . Graph shows quantification of regenerating axons at different distances distal to the lesion sites .

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(F) CTB-labelled RGC axons 4 weeks after optic nerve crush. Boxes i. to iv. show regenerating axons. Dashed line indicates crush site. White arrows mark the end of axons. Graph shows quantification of regenerating axons at different distances distal to the lesion sites.


Giemsa staining of a femoral section from 8 - day - old mice showing endochondral ossification defects . Scale bar : 100 μm .

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Giemsa staining of a femoral section from 8-day-old mice showing endochondral ossification defects. Scale bar: 100 μm.


Representative femur sections from 8 - day - old wild - type and Dnmt2 − / − mice . The staining of perivascular cells ( GLS , green ) is strongly reduced in Dnmt2 − / − bone marrow . Nuclei are counterstained with DAPI . Scale bar : 100 μm .

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Representative femur sections from 8-day-old wild-type and Dnmt2−/− mice. The staining of perivascular cells (GLS, green) is strongly reduced in Dnmt2−/− bone marrow. Nuclei are counterstained with DAPI. Scale bar: 100 μm.


Quantitative analysis of capillary numbers . GLS - positive capillaries were counted in wild - type ( wt ) and Dnmt2 − / − ( D2 - ) mice , in three independent high - power fields ( HPF ) per mouse ( n = 7 ) .

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Quantitative analysis of capillary numbers. GLS-positive capillaries were counted in wild-type (wt) and Dnmt2−/− (D2-) mice, in three independent high-power fields (HPF) per mouse (n = 7).


Quantification of cell numbers in haematopoietic tissues of 8 - day - old mice ( n = 8 ) .

Quantification of cell numbers in haematopoietic tissues of 8-day-old mice (n = 8).


Representative FACS plots of wild - type and Dnmt2 − / − BM stained for Lin − Sca1 + and cKit + cells .

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Representative FACS plots of wild-type and Dnmt2−/− BM stained for Lin− Sca1+ and cKit+ cells.


Quantification of bone marrow LSK cells in wild - type ( three independent experiments , each with pooled BM samples from 4 to 6 mice ) and Dnmt2 − / − bone marrow ( three independent experiments , each with pooled BM samples of 5 - 9 mice ) .

Quantification of bone marrow LSK cells in wild-type (three independent experiments, each with pooled BM samples from 4 to 6 mice) and Dnmt2−/−bone marrow (three independent experiments, each with pooled BM samples of 5-9 mice).


Restoration of the LSK cell population in adult Dnmt2 − / − mice . Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild - type littermates at the indicated age ( 2 - 3 independent experiments , each with pooled bone marrow samples from 3 to 9 mice ) .

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Restoration of the LSK cell population in adult Dnmt2−/− mice. Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild-type littermates at the indicated age (2-3 independent experiments, each with pooled bone marrow samples from 3 to 9 mice).


Increased LSK CD34 − and reduced LSK CD34 + cell populations in 8 - day - and 1 - year - old Dnmt2 − / − mice compared to wild - type ( two independent experiments , each with pooled bone marrow samples from 3 - 9 mice ) .

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Increased LSK CD34− and reduced LSK CD34+cell populations in 8-day- and 1-year-old Dnmt2−/− mice compared to wild-type (two independent experiments, each with pooled bone marrow samples from 3-9 mice).


qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues , normalized to GAPDH .

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qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues, normalized to GAPDH.


Western blot analysis of Dnmt2 protein expression in haematopoietic tissues .

Western blot analysis of Dnmt2 protein expression in haematopoietic tissues.


Proliferation analysis of primary bone marrow suspension cells . Primary bone marrow suspension cells isolated from the femurs of 8 - day - old mice were grown together with the attached stromal cells . Dnmt2 − / − population doubling levels of three biological replicates , each plated three times , were calculated relative to wild - type culture .

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Proliferation analysis of primary bone marrow suspension cells. Primary bone marrow suspension cells isolated from the femurs of 8-day-old mice were grown together with the attached stromal cells. Dnmt2−/− population doubling levels of three biological replicates, each plated three times, were calculated relative to wild-type culture.


Reduced replating abilities of primary Dnmt2 − / − bone marrow cells in a myeloid colony formation assay . For primary ( 1 ° ) colony formation , 1 . 25 × 104 total bone marrow cells were plated in semisolid medium allowing myeloid differentiation . For secondary ( 2 ° ) colony formation , cells were harvested and 0 . 1 % of primary colonies were replated into semisolid medium . Results are from two independent experiments analyzing 9 wild - type and 13 Dnmt2 − / − mice in total .

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Reduced replating abilities of primary Dnmt2−/−bone marrow cells in a myeloid colony formation assay. For primary (1°) colony formation, 1.25 × 104 total bone marrow cells were plated in semisolid medium allowing myeloid differentiation. For secondary (2°) colony formation, cells were harvested and 0.1% of primary colonies were replated into semisolid medium. Results are from two independent experiments analyzing 9 wild-type and 13 Dnmt2−/−mice in total.


Reduced secondary CFU - preB colony formation of Dnmt2 − / − cells . Numbers of CFU - preB - cell colonies are indicated per 5 × 104 wild - type or Dnmt2 − / − total bone marrow cells . 10 % of primary colonies were replated into secondary CFU - preB assays .

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Reduced secondary CFU-preB colony formation of Dnmt2−/− cells. Numbers of CFU-preB-cell colonies are indicated per 5 × 104 wild-type or Dnmt2−/− total bone marrow cells. 10% of primary colonies were replated into secondary CFU-preB assays.


Stable engraftment ( upper panel ) and multi - lineage reconstitution ( lower panel ) of wild - type and Dnmt2 − / − bone marrow transplants . CD11b : macrophages ; Ly6G : granulocytes ; CD45R : B cells ; CD3 : T cells .

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Stable engraftment (upper panel) and multi-lineage reconstitution (lower panel) of wild-type and Dnmt2−/− bone marrow transplants. CD11b: macrophages; Ly6G: granulocytes; CD45R: B cells; CD3: T cells.


Flow cytometry analysis of bone marrow primary mesenchymal cells .

Flow cytometry analysis of bone marrow primary mesenchymal cells.


Quantification of fibroblast colony - forming units ( CFU - F ) .

Quantification of fibroblast colony-forming units (CFU-F).


Quantification of alkaline phosphatase colony - forming units ( CFU - ALP ) .

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Quantification of alkaline phosphatase colony-forming units (CFU-ALP).


Dnmt2 expression after 3 days of mesenchymal cell differentiation .

Dnmt2 expression after 3 days of mesenchymal cell differentiation.


Alizarin Red staining , indicating enhanced osteoblast differentiation of primary stromal cells from Dnmt2 − / − mice . Scale bar : 50 μm . Quantification was performed with three biological replicates .

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Alizarin Red staining, indicating enhanced osteoblast differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.


qPCR analysis of the osteoblast markers Osterix and Osteocalcin .

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qPCR analysis of the osteoblast markers Osterix and Osteocalcin.


Oil Red O / hematoxylin staining , showing reduced adipocyte differentiation of primary stromal cells from Dnmt2 − / − mice . Scale bar : 50 μm . Quantification was performed with three biological replicates .

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Oil Red O/hematoxylin staining, showing reduced adipocyte differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.


qPCR analysis of the adipocyte markers CEBp alpha and Glut4 .

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qPCR analysis of the adipocyte markers CEBp alpha and Glut4.


Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines ( boxes ) within tRNAs from bone marrow ( BM ) , mesenchymal stromal cells ( MSC ) , and in vitro differentiated adipocytes and osteoblasts . Methylation levels are indicated on a color scale from green ( unmethylated ) to red ( completely methylated ) .

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Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines (boxes) within tRNAs from bone marrow (BM), mesenchymal stromal cells (MSC), and in vitro differentiated adipocytes and osteoblasts. Methylation levels are indicated on a color scale from green (unmethylated) to red (completely methylated).