Figure 1A Range of example RAD51 nuclear expression score ( RAD51NES ) values with respective unmixed monochrome fluorescent IHC staining images . EpCAM is used as a tumour marker and an internal sample quality control . Scale bar is 50μm . C Distribution of RAD51 nuclear expression score ( RAD51NES ) in the BCC cohort . The cohort is divided into RAD51 - Low expressing cases ( first quartile , Q1 - blue ) , intermediate cases ( interquartile range , IQR - grey ) and RAD51 - High expressing cases ( fourth quartile , Q4 - brown ) . Dashed line denotes the median RAD51NES in this cohort . D Survival analysis of the BCC cohort . Kaplan - Meier plots for progression - free survival ( PFS ) ( left ) and overall survival ( OS ) ( right ) stratified according to fourth quartile ( Q4 ) and first quartile ( Q1 ) of RAD51NES . Log - rank test , shading denotes 95 % confidence intervals . E Number of cases with progression at 12 and 24 months . Chi - square test .

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Figure 1A Range of example RAD51 nuclear expression score (RAD51NES) values with respective unmixed monochrome fluorescent IHC staining images. EpCAM is used as a tumour marker and an internal sample quality control. Scale bar is 50μm.C Distribution of RAD51 nuclear expression score (RAD51NES) in the BCC cohort. The cohort is divided into RAD51-Low expressing cases (first quartile, Q1 - blue), intermediate cases (interquartile range, IQR - grey) and RAD51-High expressing cases (fourth quartile, Q4 - brown). Dashed line denotes the median RAD51NES in this cohort.D Survival analysis of the BCC cohort. Kaplan-Meier plots for progression-free survival (PFS) (left) and overall survival (OS) (right) stratified according to fourth quartile (Q4) and first quartile (Q1) of RAD51NES. Log-rank test, shading denotes 95% confidence intervals.E Number of cases with progression at 12 and 24 months. Chi-square test.


Figure 2A Distribution of RAD51NES in the SCOTROC4 cohort . Dashed line denotes the median RAD51NES in this cohort . B Survival analysis of the SCOTROC4 cohort . Kaplan - Meier plots for PFS ( left ) and OS ( right ) stratified according to quartiles of RAD51NES . Log - rank test , shading denotes 95 % confidence intervals . C Number of cases with progression at 12 and 24 months . Chi - square test . D Survival analysis of HRD positive patients according to quartile of RAD51NES . Log - rank test , shading denotes 95 % confidence intervals . E , Survival analysis of HRD negative patients . Log - rank test , shading denotes 95 % confidence intervals .

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Figure 2A Distribution of RAD51NES in the SCOTROC4 cohort. Dashed line denotes the median RAD51NES in this cohort.B Survival analysis of the SCOTROC4 cohort. Kaplan-Meier plots for PFS (left) and OS (right) stratified according to quartiles of RAD51NES. Log-rank test, shading denotes 95% confidence intervals.C Number of cases with progression at 12 and 24 months. Chi-square test.D Survival analysis of HRD positive patients according to quartile of RAD51NES. Log-rank test, shading denotes 95% confidence intervals. E, Survival analysis of HRD negative patients. Log-rank test, shading denotes 95% confidence intervals.


Figure 3A In - vitro cell survival assay of HGSOC cell lines upon stable RAD51 overexpression . Mean with standard deviation is shown of at least three biological replicates per point . Extra - sum - of - squares F test . B In - vitro colony forming assays comparing RAD51 - overexpressing and control HGSOC cell line , Caov - 3 , after treatment with increasing doses of carboplatin . Mean and SD of four biological replicates ( left ) a representative experiment ( right ) . P - value for a comparison between cell lines for each drug treatment condition is indicated above the bars . T - test . E Volcano plot for fold changes of immune genes enriched or depleted in RAD51 - High tumours vs RAD51 - Low tumours from TCGA . T - test ; dashed line denoted threshold of significance , Bonferroni corrected for multiple testing . F Immune genes enriched in RAD51 - High and RAD51 - Low tumours across four EOC mRNA cohorts ( TCGA , AOCS , MGH , Duke ) . G Example unmixed fluorescent IHC images demonstrating the presence of CD8 + T cells in the Tumour ( T ) and Stroma ( S ) in a RAD51 - High and a RAD51 - Low tumour from the BCC cohort . Scale bar is 50μm . H CD8 + cytotoxic T - cell ( Tcyt ) infiltration analysis in the BCC cohort . Absolute tumour cytotoxic T - cell infiltration numbers and tumour / stroma ( T / S ) cytotoxic T - cell number ratio in RAD51 - High and - Low cases ( left ) . Bar is median . Mann - Whitney test .

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Figure 3A In-vitro cell survival assay of HGSOC cell lines upon stable RAD51 overexpression. Mean with standard deviation is shown of at least three biological replicates per point. Extra-sum-of-squares F test.B In-vitro colony forming assays comparing RAD51-overexpressing and control HGSOC cell line, Caov-3, after treatment with increasing doses of carboplatin. Mean and SD of four biological replicates (left) a representative experiment (right). P-value for a comparison between cell lines for each drug treatment condition is indicated above the bars. T-test.E Volcano plot for fold changes of immune genes enriched or depleted in RAD51-High tumours vs RAD51-Low tumours from TCGA. T-test; dashed line denoted threshold of significance, Bonferroni corrected for multiple testing.F Immune genes enriched in RAD51- High and RAD51-Low tumours across four EOC mRNA cohorts (TCGA, AOCS, MGH, Duke).G Example unmixed fluorescent IHC images demonstrating the presence of CD8+ T cells in the Tumour (T) and Stroma (S) in a RAD51-High and a RAD51-Low tumour from the BCC cohort. Scale bar is 50μm.H CD8+ cytotoxic T-cell (Tcyt) infiltration analysis in the BCC cohort. Absolute tumour cytotoxic T-cell infiltration numbers and tumour/stroma (T/S) cytotoxic T-cell number ratio in RAD51-High and -Low cases (left). Bar is median. Mann-Whitney test.


Figure 1A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( A ) Supernatants were harvested after 18 h , and concentrations of IL - 6 and IL - 12 were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Mean ± SD of triplicate determinations from > 3 independent experiments are shown . A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( B ) mRNA was harvested after 2 h , and expression of IL - 6 and IL - 12 was measured by real - time PCR and normalized to G6pdx . Unpaired two - tailed t - test . Data ( mean ± SD ) are representative of three independent experiments . C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC . Periods of oxidation were 24 h for CuSO4 ( 10 μM ) , 48 h for FeSO4 ( 10 μM ) , and 72 h for air . BMDCs were treated for 60 min with lipids prior stimulation with R837 ( 5 μg / ml ) for 18 h . Cytokine concentrations in supernatants were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Data represent mean ± SEM of triplicate determinations . D PAPC ( 40 μg / ml ) oxidized with CuSO4 ( 10 μM ) for the indicated times was used for treatment of BMDCs prior to R837 ( 5 μg / ml ) stimulation and measurement of IL - 12 secretion . Mean ± SEM of triplicate determinations from three different oxidation series are shown . E , F Treatment of BMDCs with the indicated concentrations of CuSO4 - oxidized PAPC suppressed IL - 6 ( E ) and IL - 12 ( F ) secretion triggered by a variety of different TLR agonists . After OxPAPC treatment for 60 min , BMDCs were stimulated for 18 h with LTA ( 500 ng / ml ) , Poly I : C ( 50 μg / ml ) , LPS ( 10 ng / ml ) , R837 ( 5 μg / ml ) , and CpG ( 100 nM ) . Cytokine concentrations in the supernatant were quantified by ELISA . Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test . G - I Splenicdendritic cells were treated with OxPAPC or DPPC ( 40 μg / ml ) before co - culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61 . ( G ) After 4 days of cell culture , T - cell polarization was assessed by intracellular staining for the cytokines IL - 4 ( Th2 ) and IFN - γ ( Th1 ) . ( H ) Bar graphs represent the frequencies of IL - 4 - and IFN - γ - producing T cells after 4 days of co - culture with OxPAPC - treated and DPPC - treated splenicdendritic cells . Bars represent mean ± SD of duplicate experiments . * P ≤ 0 . 05 by unpaired two - tailed t - test . ( I ) IFN - γ production in supernatants of SMARTA CD4T cells ( stimulated with 1 , 000 nM gp61 ) and co - cultured with OxPAPC - and DPPC - treated splenicdendritic cells for 4 days . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Bars represent mean ± SD .

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

Figure 1A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.G-I Splenicdendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenicdendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenicdendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.


Figure 2Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4 - catalyzed oxidation of PAPC for 2 and 24 h . Mean ± SEM of duplicate determinations are shown . Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL - 12 secretion of thioglycollate - elicited macrophages . Dots represent data of individual OxPAPC preparations .

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Figure 2Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.


Figure 3Selected candidate lipids were tested for their inhibitory activity on R837 - induced ( 5 μg / ml ; 18 h ) cytokine secretion in BMDCs . Concentrations of indicated lipids : PECPC ( 10 μM ) , PEIPC ( 10 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , and KOdiAPC ( 40 μM ) . Representative data ( mean ± SD of triplicate determinations ) from one of three independent experiments are shown . * * * * P < 0 . 0001 ; ns , not significant ; as determined by one - way ANOVA adjusted by Sidak ' s multiple comparisons test . Dose - response curves of IL - 12 secretion in BMDCs pulsed for 60 min with the indicated lipids , followed by stimulation with R837 ( 5 μg / ml ) for 18 h . Toxic concentrations of lipids were excluded from analysis . Mean ± SEM of triplicate determinations are shown . EC50 values of anti - inflammatory lipid products normalized to the capacity of 15d - PGJ2 to suppress IL - 12 production in BMDCs ( left panel ) and their respective bioactivities ( right panel ) depicted as the fold increase relative to 15d - PGJ2 . Mean ± SEM of triplicate determinations are shown . IL - 12 production of BMDC stimulated via TLR 4 ( LPS ; 100 ng / ml ) , TLR 9 ( CpG ; 100 nM ) , and TLR 7 ( R837 ; 5 μg / ml ) after pretreatment with the indicated free and esterified versions of EC and 15d - PGJ2 . Lipids were used at 1 μM ( EC ) , 10 μM ( PECPC ) , or 20 μM ( 15d - PGJ2 and 15d - PGJ2PC ) . Data ( mean ± SEM ) are representative of 3 independent experiments . * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; determined by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Analysis of the capacity of various OxPL - derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset . Concentrations of indicated lipids : EC ( 1 μM ) , PECPC ( 10 μM ) , 15d - PGJ2 ( 20 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , KOdiAPC ( 40 μM ) .

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

Figure 3Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.Dose-response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.IL-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.Analysis of the capacity of various OxPL-derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).


Figure 4A IL - 12 production of BMDC from wild - type , Nrf2 − / − , Pparg − / − , and Pparg litter mate control mice normalized to medium control ( open bars ) . Cells were treated with the indicated lipids ( filled bars ) for 60 min prior TLR 7 ligation with R837 ( 5 μg / ml ) for 18 h . Lipids were used at starting concentrations of 40 μM ( POVPC , PGPC , and KOdiAPC ) , 40 μg / ml ( OxPAPC ) , 20 μM ( 15d - PGJ2 ) , and 1 . 25 μM ( EC ) , depicted as black bars , and 2 - fold serial dilutions thereof ( gray bars ) . Data represent mean ± SEM of triplicates from one of three independent experiments . B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild - type and Nrf2 − / − BMDM stimulated with EC ( 2 μM ) for 60 min . Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx . Data ( mean ± SEM ) are representative of two independent experiments . C , D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 ( C ) and of the pro - inflammatory cytokines IL - 6 and IL - 12 ( D ) in wild - type , PPAR - γ - deficient , and Nrf2 - deficient BMDM after treatment with EC or 15d - PGJ2 for 60 min followed by LPS treatment for 3 h . Expression levels are normalized to G6pdx . Data represent mean ± SEM of triplicate cultures from one of two independent experiments . E mRNA expression of the indicated chemokines as determined by qPCR . Wild - type BMDCs were treated with EC ( 1 μM ) or 15d - PGJ2 ( 20 μM ) for 60 min followed by R837 stimulation ( 5 μg / ml ) for 3 h . Expression levels are shown normalized to G6pdx . Data ( mean ± SEM , n = 2 ) are representative of three independent experiments .

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Figure 4A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/−BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.


Figure 5A C57BL / 6mice were treated ( i . v . ) with 500 μg EC or DPPC control 2 h prior to i . p injection of 150 ng / g LPS together with 800 μg / g D - galactosamine . 4 h after LPS application , lungs were perfused with PBS and embedded in paraffin . Tissue sections were hematoxylin - stained to visualize adherent cells . Bars represent 100 µm . B , C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in ( B ) and as averages of single mice in ( C ) . Pooled data of two independent experiments are shown ( n = 10 for EC , n = 14 for DPPC ) . Unpaired two - tailed t - test . D , E C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) and 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS and 800 μg / g D - galactosamine . Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils ( D ) . Unpaired two - tailed t - test . Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL cells of EC - / LPS - treated and DPPC - / LPS - treated mice . F , G The concentrations of IL - 6 ( F ) and IL - 12 ( G ) in the BAL of mice treated as in ( D ) were quantified by ELISA . Data ( mean ± SEM ) are representative of two independent experiments with 3 mice per group . * * * * P < 0 . 0001 ; unpaired two - tailed t - test . C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) and 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS and 800 μg / g D - galactosamine . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL cells of EC - / LPS - treated and DPPC - / LPS - treated mice .

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Figure 5A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test.C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


Figure 6Dose - response curves showing the modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h . Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in ( A ) , presented relative to EC .

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Figure 6Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.


Figure 7Quantification of IL - 6 and IL - 12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 ( 5 μg / ml ; 18 h ) . Bars represent mean ± SEM from one of three independent experiments . * * P < 0 . 01 ; * * * P < 0 . 001 ; one - way ANOVA adjusted by Dunnett ' s multiple comparisons test . mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression . BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed t - test . ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in ( E ) . Dose - response curves showing the modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h . mRNA quantification of the indicated chemokines relative to G6pdx expression . BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed t - test . Quantification and characterization of cellular infiltrates in BAL . Groups of six C57BL / 6mice were pretreated i . t . with 50 μg cEC , EC , or BisRed 24 and 2 h before challenge with 150 ng / g LPS in the presence of 800 μg / g D - galactosamine . BAL was harvested 4 h after LPS injection , and inflammatory cells were characterized by FACS analysis . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; one - way ANOVA with Sidak ' s multiple comparisons test . Comparison of the capacity of cEC , EC , and PECPC ( 1 μM ) to license splenicdendritic cells to polarize naïve CD4T cells into IFN - γ - producing ( Th1 ) and IL - 4 - producing ( Th2 ) effector cells . Data ( mean ± SD ) are representative of two independent experiments . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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Figure 7Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenicdendritic cells to polarize naïve CD4T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


Figure 1A . Immunostaining of TUJ1 , DESMIN and GATA6 after six days of mESC differentiation towards ectoderm , mesoderm and endoderm . Bar : 50 μm . C . Venn diagram of genes with differential expression during mESC differentiation towards the three germ layers . D - F . Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm ( E ) , mesoderm ( F ) and endoderm ( G ) .

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Figure 1A. Immunostaining of TUJ1, DESMIN and GATA6 after six days of mESC differentiation towards ectoderm, mesoderm and endoderm. Bar: 50 μm.C. Venn diagram of genes with differential expression during mESC differentiation towards the three germ layers.D-F. Hierarchical clustering of mRNA expression of mESCs differentiated towards ectoderm (E), mesoderm (F) and endoderm (G).


Figure 2A . Hierarchical clustering of gene expression profiles of mESCs differentiated towards the three germ layers and differentiated mouse tissues .

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Figure 2A. Hierarchical clustering of gene expression profiles of mESCs differentiated towards the three germ layers and differentiated mouse tissues.


Figure 3C . Hierarchical clustering of mRNA expression during differentiation of mESCs towards endoderm ( yellow ) or EpiSCs ( green ) .

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Figure 3C. Hierarchical clustering of mRNA expression during differentiation of mESCs towards endoderm (yellow) or EpiSCs (green).


Figure 5B . Ectoderm differentiation of the 3KI mESC line ( marked by the expression of Sox1 : : GFP ) . C . Mesendoderm differentiation of the 3KI mESC line ( marked by the expression of T : : 3xTagBFP ) . D . Endoderm differentiation of the 3KI mESC line ( marked by the expression of Eomes : : mCherry ) . E , F . Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers . Bar : 100 μm .

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Figure 5B. Ectoderm differentiation of the 3KI mESC line (marked by the expression of Sox1::GFP). C. Mesendoderm differentiation of the 3KI mESC line (marked by the expression of T::3xTagBFP).D. Endoderm differentiation of the 3KI mESC line (marked by the expression of Eomes::mCherry).E, F. Spontaneous differentiation of the 3KI mESC line leading to the coexistence of cells belonging to the three germ layers. Bar: 100 μm.


Figure 6B . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - and Zfp354c - / - mESCs in the 3KI background to ectoderm . C . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - and Zfp354c - / - mESCs in the 3KI background to mesendoderm . D . Differentiation of Atf1 - / - , Fos - / - Jun - / - , Foxj2 - / - , Meis3 - / - , Nr5a2 - / - , Sp1 + / - and Zff354c - / - mESCs in the 3KI background to definitive endoderm . F . Differentiation of wild type ( WT ) , Sp1 + / - , Nr5a2 - / - and Fos - / - Jun - / - mESCs in the 3KI background to definitive endoderm .

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Figure 6B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm. C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm. D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm.F. Differentiation of wild type (WT), Sp1+/-, Nr5a2-/- and Fos-/-Jun-/- mESCs in the 3KI background to definitive endoderm.


Figure 1 ( B , C ) HCT116 , PKcs - / - , and Lig4 - / - cells were subjected to Agilent Seahorse XFe24 analyzer to measure oxidative phosphorylation ( B ) via the oxygen consumption rate ( OCR ) and glycolysis ( C ) via the extracellular acidification rate ( ECAR ) . ( J , K ) M059K / J cells were also subjected to the OCR and ECAR analyses .

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Figure 1(B, C) HCT116, PKcs-/-, and Lig4-/- cells were subjected to Agilent Seahorse XFe24 analyzer to measure oxidative phosphorylation (B) via the oxygen consumption rate (OCR) and glycolysis (C) via the extracellular acidification rate (ECAR).(J, K) M059K/J cells were also subjected to the OCR and ECAR analyses.


Figure 2 ( B ) HeLa S3 total cell lysate , nuclear and cytosolic and mitochondrial fractions were analyzed by western blot to show sub - cellular localization of DNA - PKcs and ATM . ( C , D ) 100 μg of purified HeLa S3 mitochondria were subjected to limited protease K ( at the indicated amount ) digestion in a final 50 μl reaction volume at 37C for 30 minutes ( C ) or sodium carbonate ( 100 mM ) verse sodium chloride ( 133 mM ) extraction followed by western blot ( D ) .

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Figure 2(B) HeLa S3 total cell lysate, nuclear and cytosolic and mitochondrial fractions were analyzed by western blot to show sub-cellular localization of DNA-PKcs and ATM.(C, D) 100 μg of purified HeLa S3 mitochondria were subjected to limited protease K (at the indicated amount) digestion in a final 50 μl reaction volume at 37C for 30 minutes (C) or sodium carbonate (100 mM) verse sodium chloride (133 mM) extraction followed by western blot (D).


Figure 3 ( A ) HeLa cells expressing Flag - tagged ANT1 - 3 were subjected to Co - IP using anti - Flag antibody , then analyzed by western blot as indicated . ( E ) HeLa cells expressing Flag - tagged VDAC1 - 3 were subjected to Co - IP using anti - Flag antibody and analyzed by western blot as indicated . ( F ) PLA analysis show that the interaction between DNA - PKcs and Flag - ANT2 was disrupted by knockdown of VDAC2 , but not VDAC1 or VDAC3 . Quantification shown on the right . * * * , P < 0 . 001 . Data information : The bar graphs were generated from three independent biological replicates analyses ; data are represented as mean ± SD . Statistical significance was established using a two - way analysis of variance ( ANOVA , * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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Figure 3(A) HeLa cells expressing Flag-tagged ANT1-3 were subjected to Co-IP using anti-Flag antibody, then analyzed by western blot as indicated.(E) HeLa cells expressing Flag-tagged VDAC1-3 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated.(F) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by knockdown of VDAC2, but not VDAC1 or VDAC3. Quantification shown on the right. ***, P < 0.001. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).


Figure 4 ( A , B ) HCT116 , DNA - PKcs - / - , and Ligase 4 - / - cells were subjected to ADP - ATP exchange assay using MgGreen 5K + fluorescent dye . The exchange was stimulated by 5mM ADP and halted by ANTs inhibitor carboxyatractyloside ( cATR ) . Normalized MgGreen 5K + fluorescence was first measured to indicate changes in free magnesium concentration ( A ) . The decreased free magnesium concentration was converted into efflux of ATP ( [ ATP ] e , mM ) after the addition of ADP ( B ) . Bar graph on the right shows the normalized MgGreen 5K + fluorescence and efflux of ATP concentration after ADP until the reaction was stopped by cATR treatment . ( F ) shGFP and shATM pair of HeLa ( Right ) or MCF7 ( Left ) cells were subjected to an ADP - ATP exchange assay using MgGreen 5K + fluorescent dye .

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Figure 4(A, B) HCT116, DNA-PKcs-/-, and Ligase 4-/- cells were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The exchange was stimulated by 5mM ADP and halted by ANTs inhibitor carboxyatractyloside (cATR). Normalized MgGreen 5K+ fluorescence was first measured to indicate changes in free magnesium concentration (A). The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP (B). Bar graph on the right shows the normalized MgGreen 5K+ fluorescence and efflux of ATP concentration after ADP until the reaction was stopped by cATR treatment.(F) shGFP and shATM pair of HeLa (Right) or MCF7 (Left) cells were subjected to an ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye.


Figure 5 ( A ) Western blot results show attenuation of ANT2 protein level in DNA - PKcs - deficient and depleted cells . ( L ) Treatment with VDAC2 siRNA in Hela cells attenuated ADP - ATP exchange . Bar graph on the right shows the efflux of ATP concentration after adding ADP .

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Figure 5(A) Western blot results show attenuation of ANT2 protein level in DNA-PKcs-deficient and depleted cells.(L) Treatment with VDAC2 siRNA in Hela cells attenuated ADP-ATP exchange. Bar graph on the right shows the efflux of ATP concentration after adding ADP.


Figure 6 ( B ) PLA analysis show that the interaction between DNA - PKcs and Flag - ANT2 was disrupted by 10 Gy irradiation , but prevented by pre - treatment with 5 mM NAC . Quantification shown at the right . Data information : The bar graphs were generated from three independent biological replicates analyses ; data are represented as mean ± SD . Statistical significance was established using a two - way analysis of variance ( ANOVA , * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) . ( C ) Co - IP with endogenous DNA - PKcs show that the interaction between DNA - PKcs and Flag - ANT2 was temporarily disrupted by 10 Gy irradiation , whereas VDAC2 remained interaction with DNA - PKcs .

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Figure 6(B) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 was disrupted by 10 Gy irradiation, but prevented by pre-treatment with 5 mM NAC. Quantification shown at the right. Data information: The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).(C) Co-IP with endogenous DNA-PKcs show that the interaction between DNA-PKcs and Flag-ANT2 was temporarily disrupted by 10 Gy irradiation, whereas VDAC2 remained interaction with DNA-PKcs.


Figure 7 ( A , B ) Paired shGFP and shATM HeLa ( A ) and MCF7 cells ( B ) were subjected to ADP - ATP exchange assay using MgGreen 5K + fluorescent dye . The decreased free magnesium concentration was converted into efflux of ATP ( [ ATP ] e , mM ) after the addition of ADP . The bar graphs were generated from three independent biological replicates analyses ; , D ) PLA analysis show that the interaction between DNA - PKcs and Flag - ANT2 in HeLa ( C ) and MCF7 ( D ) was temporarily disrupted by treatment of 10Gy IR . Knockdown of ATM disrupted the dissociation . Quantification shown on the right . The bar graphs were generated from three independent biological replicates analyses ( E ) HeLa cells expressing Flag - tagged VDAC2 and V5 - tagged ANT2 were subjected to Co - IP using anti - Flag antibody and analyzed by western blot as indicated . ANT2 , but not VDAC2 , temporarily dissociated from DNA - PKcs after treatment of 10Gy IR . ATM knockdown and inhibition ( AZD1390 , 10 nM ) disrupted the dissociation .

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Figure 7(A, B) Paired shGFP and shATM HeLa (A) and MCF7 cells (B) were subjected to ADP-ATP exchange assay using MgGreen 5K+ fluorescent dye. The decreased free magnesium concentration was converted into efflux of ATP ([ATP]e, mM) after the addition of ADP. The bar graphs were generated from three independent biological replicates analyses;, D) PLA analysis show that the interaction between DNA-PKcs and Flag-ANT2 in HeLa (C) and MCF7 (D) was temporarily disrupted by treatment of 10Gy IR. Knockdown of ATM disrupted the dissociation. Quantification shown on the right. The bar graphs were generated from three independent biological replicates analyses(E) HeLa cells expressing Flag-tagged VDAC2 and V5-tagged ANT2 were subjected to Co-IP using anti-Flag antibody and analyzed by western blot as indicated. ANT2, but not VDAC2, temporarily dissociated from DNA-PKcs after treatment of 10Gy IR. ATM knockdown and inhibition (AZD1390, 10 nM) disrupted the dissociation.


Figure 8 ( A - G ) Non - transformed cell lines including ( A , B ) human retinal pigment epithelial - 1 ( RPE - 1 ) , ( C , D ) primary human skin fibroblasts ( HSF ) , ( E ) hTERT immortalized human skin fibroblasts 48BR , ( F ) primary mouse embryonic fibroblasts ( MEF ) RPE - 1 , and ( G ) A - T patient derived skin fibroblasts GM02052 were subjected to ADP - ATP exchange assay at various time points ( up to 16 hrs ) in response to 100 μM of H2O2 or 10 Gy IR as indicated . ATM inhibitor ( AZD1390 , 10 nM ) was included in parallel analyses . The grafts display sham ( white ) , bottom ( red ) , and recovery ( blue ) of ADP - ATP exchange activities after treatments . The bar graphs were generated from three independent biological replicates analyses ; data are represented as mean ± SD .

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Figure 8(A-G) Non-transformed cell lines including (A, B) human retinal pigment epithelial-1 (RPE-1), (C, D) primary human skin fibroblasts (HSF), (E) hTERT immortalized human skin fibroblasts 48BR, (F) primary mouse embryonic fibroblasts (MEF) RPE-1, and (G) A-T patient derived skin fibroblasts GM02052 were subjected to ADP-ATP exchange assay at various time points (up to 16 hrs) in response to 100 μM of H2O2 or 10 Gy IR as indicated. ATM inhibitor (AZD1390, 10 nM) was included in parallel analyses. The grafts display sham (white), bottom (red), and recovery (blue) of ADP-ATP exchange activities after treatments. The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD.


Figure 9 ( A ) Western blot results show knockdown of ATM attenuated 100 μM H2O2 induced T2609 and T2647 phosphorylation of DNA - PKcs in HeLa cells . ( C , D ) PLA Analysis shows that the interaction between DNA - PKcs and ANT2 in MEF cells was temporarily disrupted by treatment of 10Gy IR . DNA - PKcs3A / 3A remained interaction with ANT2 after IR . The bar graphs were generated from three independent biological replicates analyses ; data are represented as mean ± SD . Statistical significance was established using a two - way analysis of variance ( ANOVA , * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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Figure 9(A) Western blot results show knockdown of ATM attenuated 100 μM H2O2 induced T2609 and T2647 phosphorylation of DNA-PKcs in HeLa cells.(C,D) PLA Analysis shows that the interaction between DNA-PKcs and ANT2 in MEF cells was temporarily disrupted by treatment of 10Gy IR. DNA-PKcs3A/3A remained interaction with ANT2 after IR. The bar graphs were generated from three independent biological replicates analyses; data are represented as mean ± SD. Statistical significance was established using a two-way analysis of variance (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001).


Figure 1A T98G cells were challenged or not with dsDNAs or with 2 ' 3 ' cGAMP for 6 h prior to whole cell extraction and Western Blot ( WB ) analysis using indicated antibodies . C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies . F T98G cells were challenged or not with dsDNA for 6 h , in the presence or not of the NU7441 DNA - PKcs inhibitor , prior WB analysis using indicated antibodies . I T98G cells were treated or not with 0 . 16 µM camptothecin ( CPT ) for 48 h prior to immunofluorescence analysis using dsDNA - and 53BP1 - specific antibodies , and DAPI nuclear staining ( n = 3 independent experiments ) . J Whole cell extracts from T98G cells treated or not for 72 h with 0 . 16 µM CPT were analyzed by WB using indicated antibodies .

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Figure 1A T98G cells were challenged or not with dsDNAs or with 2'3'cGAMP for 6 h prior to whole cell extraction and Western Blot (WB) analysis using indicated antibodies.C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies.F T98G cells were challenged or not with dsDNA for 6 h, in the presence or not of the NU7441 DNA-PKcs inhibitor, prior WB analysis using indicated antibodies.I T98G cells were treated or not with 0.16 µM camptothecin (CPT) for 48 h prior to immunofluorescence analysis using dsDNA- and 53BP1-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).J Whole cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.


Figure 2A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA - PKcs - and pDNA - PKcs - specific antibodies , and DAPI nuclear staining ( n = 3 independent experiments ) . B Whole cell extracts from T98G cells were incubated with 80nt - long biotinylated ssDNA or dsDNA prior to pull - down using streptavidin - affinity beads . Input and eluates were analyzed by WB using indicated antibodies . C T98G cells were treated with non - targeting ( CTRL ) or KU70 - targeting siRNAs prior to whole cell extract preparation , and pull - down as in B . Inputs and eluates were analyzed by WB using indicated antibodies .

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Figure 2A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n=3 independent experiments).B Whole cell extracts from T98G cells were incubated with 80nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.C T98G cells were treated with non-targeting (CTRL) or KU70-targeting siRNAs prior to whole cell extract preparation, and pull-down as in B. Inputs and eluates were analyzed by WB using indicated antibodies.


Figure 3A T98G cells stably expressing cGAS ( T98GcGAS ) or not ( T98GEmpty ) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies . C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull - down using streptavidin - affinity beads . Inputs and eluates were analyzed by WB using indicated antibodies . J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441 , prior to WB analysis using indicated antibodies . K THP - 1 were transfected or not with dsDNA in presence or not of NU7441 , prior to WB analysis using indicated antibodies .

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Figure 3A T98G cells stably expressing cGAS (T98GcGAS) or not (T98GEmpty) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies.C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull-down using streptavidin-affinity beads. Inputs and eluates were analyzed by WB using indicated antibodies.J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


Figure 4F T98GEmpty and T98GcGAS stably expressing a GFP reporter ( T98G - GFPEmpty and T98G - GFPcGAS , respectively ) were xenotransplanted into the head of tg ( mfap4 : RFP ) zebrafish line at 3 days post fertilization ( dpf ) . Zebrafish embryos were imaged daily over 3 days . The graph represents the mean ( ± SEM ) percentage of tumor growth normalized by the area on the day of transplantation ( n = 21 T98G - GFPEmpty and n = 29 T98G - GFPcGAS embryos ) .

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Figure 4F T98GEmpty and T98GcGAS stably expressing a GFP reporter (T98G-GFPEmpty and T98G-GFPcGAS, respectively) were xenotransplanted into the head of tg(mfap4:RFP) zebrafish line at 3 days post fertilization (dpf). Zebrafish embryos were imaged daily over 3 days. The graph represents the mean (±SEM) percentage of tumor growth normalized by the area on the day of transplantation (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


Figure 5A Graph represents the mean ( ± SEM ) fold migration of THP - 1 cells through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h . CCL2 was used as positive control ( n = 8 biological replicates ) . E Graph presents the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D ( n = 21 T98G - GFPEmpty and n = 29 T98G - GFPcGAS embryos ) .

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Figure 5A Graph represents the mean (± SEM) fold migration of THP-1 cells through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h. CCL2 was used as positive control (n=8 biological replicates).E Graph presents the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).


Figure 6G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow / MB21D1low , PRKDChigh / MB21D1low and PRKDChigh / MB21D1high expression . H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A , based on tumor grade ( II to IV ) .

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Figure 6G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow/MB21D1low, PRKDChigh/MB21D1low and PRKDChigh/MB21D1high expression.H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A, based on tumor grade (II to IV).


Figure 1B ) Tumour growth curves for successive four - generation xenografts treated with 5 - Fu or PBS . Twelve mice per group . C ) Representative cases show the first and fourth generation xenograft H & E staining ( left ) and EBER - 1 ISH staining ( right ) . D ) The morphology of parental SNU719 and SNU - 4th cells . E ) Tumour sphere culture was performed to evaluate the sphere formation capacity in SNU719 and SNU - 4th cells , and the data are shown as the mean numbers and sizes of the tumour spheres . The central horizontal lines represent the median , the top and the bottom positions of the box represent upper and lower quartiles , error bars represent the mean ± SD , and the average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . F ) CCK8 assay of SNU719 and SNU - 4th cells to determine cell proliferation under the indicated culture conditions ( 10 % FBS or 0 . 1 % FBS ) . Expression of stemness markers ( Sox2 , Klf4 , Bmi1 , Oct4 ) and drug resistance genes ( ABCG2 , Mrp1 ) evaluated by real - time PCR ( G ) in SNU719 and SNU - 4th cells . Expression of stemness markers ( Sox2 , Klf4 , Bmi1 , Oct4 ) and drug resistance genes ( ABCG2 , Mrp1 ) evaluated by WB ( H ) in SNU719 and SNU - 4th cells . I ) Real - time PCR analysis of the expression of EMT - associated markers ( E - cad , vimentin ) and transcription factors ( Twist1 , Snail , Slug , Zeb1 ) in SNU719 and SNU - 4th cells . J ) WB analysis of the expression of E - cad , vimentin and Mmp7 in SNU719 and SNU - 4th cells . K ) Colony formation assay analysis of the colony - forming ability in SNU719 and SNU - 4th cells . L ) Transwell migration and the Matrigel invasion assay were performed to assess the migratory and invasive capability of SNU719 and SNU - 4th cells . M ) Representative images of the flow cytometric analysis of SP cells . N ) Representative images of the flow cytometric analysis of the surface antigen phenotype .

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Figure 1B) Tumour growth curves for successive four-generation xenografts treated with 5-Fu or PBS. Twelve mice per group.C) Representative cases show the first and fourth generation xenograft H&E staining (left) and EBER-1 ISH staining (right).D) The morphology of parental SNU719 and SNU-4th cells.E) Tumour sphere culture was performed to evaluate the sphere formation capacity in SNU719 and SNU-4th cells, and the data are shown as the mean numbers and sizes of the tumour spheres. The central horizontal lines represent the median, the top and the bottom positions of the box represent upper and lower quartiles, error bars represent the mean ± SD, and the average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates.F) CCK8 assay of SNU719 and SNU-4th cells to determine cell proliferation under the indicated culture conditions (10% FBS or 0.1% FBS).Expression of stemness markers (Sox2, Klf4, Bmi1, Oct4) and drug resistance genes (ABCG2, Mrp1) evaluated by real-time PCR (G) in SNU719 and SNU-4th cells.Expression of stemness markers (Sox2, Klf4, Bmi1, Oct4) and drug resistance genes (ABCG2, Mrp1) evaluated by WB (H) in SNU719 and SNU-4th cells.I) Real-time PCR analysis of the expression of EMT-associated markers (E-cad, vimentin) and transcription factors (Twist1, Snail, Slug, Zeb1) in SNU719 and SNU-4th cells.J) WB analysis of the expression of E-cad, vimentin and Mmp7 in SNU719 and SNU-4th cells.K) Colony formation assay analysis of the colony-forming ability in SNU719 and SNU-4th cells.L) Transwell migration and the Matrigel invasion assay were performed to assess the migratory and invasive capability of SNU719 and SNU-4th cells.M) Representative images of the flow cytometric analysis of SP cells.N) Representative images of the flow cytometric analysis of the surface antigen phenotype.


Figure 2A ) Heat map analysis of the expression of circRNAs ( both human circRNAs and ebv - circRNAs ) in the fourth passage xenografts treated with 5 - Fu or PBS . Red colour represents upregulated circRNAs , and green colour represents downregulated circRNAs . ( B ) Volcano plot representing the expression fold changes in circRNAs . The red dots represent upregulated circRNAs , and green dots represent downregulated circRNAs . ( C ) The back - spliced read distribution of ebv - circRNAs ( n = 261 ) . ( D ) The length distribution of ebv - circRNAs . ( E ) Genomic origin of ebv - circRNAs . ( F ) Number of ebv - circRNAs produced from one gene .

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Figure 2A) Heat map analysis of the expression of circRNAs (both human circRNAs and ebv-circRNAs) in the fourth passage xenografts treated with 5-Fu or PBS. Red colour represents upregulated circRNAs, and green colour represents downregulated circRNAs.(B) Volcano plot representing the expression fold changes in circRNAs. The red dots represent upregulated circRNAs, and green dots represent downregulated circRNAs. (C) The back-spliced read distribution of ebv-circRNAs (n=261). (D) The length distribution of ebv-circRNAs.(E) Genomic origin of ebv-circRNAs.(F) Number of ebv-circRNAs produced from one gene.


Figure 3 ( B ) Real - time PCR analysis of the expression of circLMP2A in SNU - 4th , SNU719 and YCCEL1 cells . ( C ) The existence of circLMP2A was validated by RT - PCR ( up ) and real - time PCR ( down ) in SNU - 4th , SNU719 and YCCEL1 cells treated with or without RNase R digestion . GAPDH was used as a negative control . ( D ) Real - time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU - 4th cells . ( E ) Real - time PCR analysis of circLMP2A and LMP2A expression in either the cytoplasm or nucleus in SNU - 4th cells . GAPDH and U6 were used as controls in cytoplasm and nucleus , respectively . ( F ) RNA ISH for circLMP2A . U6 and 18S are mainly localized in the nucleus and cytoplasm , respectively , and were used as controls . ( G ) Kaplan - Meier survival curve analysis showing the correlation between circLMP2A expression and OS . n = 69 , P < 0 . 001 , log - rank test . ( H ) Kaplan - Meier survival curve analysis showing the correlation between circLMP2A expression and progression - free survival ( PFS ) . n = 69 , P < 0 . 001 , log - rank test .

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Figure 3(B) Real-time PCR analysis of the expression of circLMP2A in SNU-4th, SNU719 and YCCEL1 cells.(C) The existence of circLMP2A was validated by RT-PCR (up) and real-time PCR (down) in SNU-4th, SNU719 and YCCEL1 cells treated with or without RNase R digestion. GAPDH was used as a negative control.(D) Real-time PCR analysis of circLMP2A and LMP2A expression after treatment with actinomycin D at the indicated time points in SNU-4th cells.(E) Real-time PCR analysis of circLMP2A and LMP2A expression in either the cytoplasm or nucleus in SNU-4th cells. GAPDH and U6 were used as controls in cytoplasm and nucleus, respectively.(F) RNA ISH for circLMP2A. U6 and 18S are mainly localized in the nucleus and cytoplasm, respectively, and were used as controls.(G) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and OS. n= 69, P < 0.001, log-rank test. (H) Kaplan-Meier survival curve analysis showing the correlation between circLMP2A expression and progression-free survival (PFS). n = 69, P < 0.001, log-rank test.


Figure 4 ( A ) Real - time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs . ( B ) Tumour sphere culture analysis of the tumour sphere formation in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . The central horizontal lines represent the median , top and bottom positions of the box represent upper and lower quartiles , and error bars represent the mean ± SD . The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . Scale bars = 100µm . EMT - related markers , stemness markers and drug resistance genes were evaluated by real - time PCR ( C ) in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . EMT - related markers , stemness markers and drug resistance genes were evaluated by WB ( D ) in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . ( E - F ) Transwell assay ( E ) and colony formation assay ( F ) analysis of the migratory and invasive capability and colony - forming ability in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . Scale bars = 100µm . ( G - H ) Flow cytometric analysis of the rate of SP cells ( G ) and apoptosis ( H ) in SNU - 4th cells transfected with sh - circLMP2A - 2 , sh - circLMP2A - 3 or sh - control . Subcutaneously established tumour xenografts in NOD / SCID mice . Tumour volume ( I ) of the xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group . Subcutaneously established tumour xenografts in NOD / SCID mice . tumour weight ( J ) of the xenografts were remarkably inhibited by down - regulation of circLMP2A . Six mice per group . ( K - L ) Representative images of bioluminescence ( K ) and lung metastases ( L ) are presented to measure the metastatic colonies . Six mice per group , scale bars = 50µm .

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 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 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 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 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

Figure 4(A) Real-time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs.(B) Tumour sphere culture analysis of the tumour sphere formation in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm.EMT-related markers, stemness markers and drug resistance genes were evaluated by real-time PCR (C) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.EMT-related markers, stemness markers and drug resistance genes were evaluated by WB (D) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.(E-F) Transwell assay (E) and colony formation assay (F) analysis of the migratory and invasive capability and colony-forming ability in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control. Scale bars =100µm.(G-H) Flow cytometric analysis of the rate of SP cells (G) and apoptosis (H) in SNU-4th cells transfected with sh-circLMP2A-2, sh-circLMP2A-3 or sh-control.Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group.Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were remarkably inhibited by down-regulation of circLMP2A. Six mice per group.(K-L) Representative images of bioluminescence (K) and lung metastases (L) are presented to measure the metastatic colonies. Six mice per group, scale bars =50µm.


Figure 5 ( A ) Real - time PCR confirmed the over - expression of circLMP2A in SNU719 and YCCEL1 cells after transfection . ( B ) Over - expression of circLMP2A induced the formation of tumour spheres in SNU719 and YCCEL1 cells . The central horizontal lines represent the median , top and bottom positions of the box represent upper and lower quartiles , and error bars represent the mean ± SD . The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment , n = 3 biological replicates . Scale bars = 100µm . EMT - related markers , stemness markers and drug resistance genes were assessed by real - time PCR ( C ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . EMT - related markers , stemness markers and drug resistance genes were assessed by WB ( D ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . ( E - F ) Flow cytometric analysis of the rate of SP cells ( E ) and apoptosis ( F ) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . ( G - H ) Transwell assay ( G ) and colony formation assay ( H ) analysis of the migratory and invasive capability and colony - forming ability in SNU719 and YCCEL1 cells transfected with circLMP2A or vector . Scale bars = 100µm . Subcutaneously established tumour xenografts in NOD / SCID mice . Tumour volume ( I ) of the xenografts were significantly increased by over - expression of circLMP2A . Six mice per group . Subcutaneously established tumour xenografts in NOD / SCID mice . tumour weight ( J ) of the xenografts were significantly increased by over - expression of circLMP2A . Six mice per group . ( K - L ) Representative images of bioluminescence ( K ) and lung metastases ( L ) are presented to measure the metastatic colonies . Six mice per group , scale bars = 50µm .

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 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 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 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 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

Figure 5(A) Real-time PCR confirmed the over-expression of circLMP2A in SNU719 and YCCEL1 cells after transfection.(B) Over-expression of circLMP2A induced the formation of tumour spheres in SNU719 and YCCEL1 cells. The central horizontal lines represent the median, top and bottom positions of the box represent upper and lower quartiles, and error bars represent the mean ± SD. The average number and maximal diameter of spheres were calculated under a microscope in five randomly chosen fields in each independent experiment, n=3 biological replicates. Scale bars =100µm.EMT-related markers, stemness markers and drug resistance genes were assessed by real-time PCR (C) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.EMT-related markers, stemness markers and drug resistance genes were assessed by WB (D) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.(E-F) Flow cytometric analysis of the rate of SP cells (E) and apoptosis (F) in SNU719 and YCCEL1 cells transfected with circLMP2A or vector.(G-H) Transwell assay (G) and colony formation assay (H) analysis of the migratory and invasive capability and colony-forming ability in SNU719 and YCCEL1 cells transfected with circLMP2A or vector. Scale bars =100µm.Subcutaneously established tumour xenografts in NOD/SCID mice. Tumour volume (I) of the xenografts were significantly increased by over-expression of circLMP2A. Six mice per group.Subcutaneously established tumour xenografts in NOD/SCID mice. tumour weight (J) of the xenografts were significantly increased by over-expression of circLMP2A. Six mice per group.(K-L) Representative images of bioluminescence (K) and lung metastases (L) are presented to measure the metastatic colonies. Six mice per group, scale bars =50µm.


Figure 6 ( B ) The relative levels of 7 miRNA candidates were detected by real - time PCR in SNU719 and SNU - 4th cells . ( C - E ) Real - time PCR analysis of 7 miRNA expression in SNU - 4th cells transfected with sh - circLMP2A - 2 or sh - control ( C ) , and SNU719 ( D ) and YCCEL1 cells ( E ) transfected with circLMP2A or vector . ( F - H ) circ LMP2Ain specific probe and detected by real - time PCR ( F ) and RT - PCR ( H ) . The relative levels of 5 miRNA candidates were detected by real - time PCR ( G ) and RT - PCR ( H ) . GAPDH was used as a negative control . ( I ) RNA ISH showing the co - localization between circLMP2A and miR - 3908 in SNU - 4th cells . circLMP2A probes were labelled with Cy3 , and miR - 3908 probes were labelled with FITC . Nuclei were stained with DAPI . ( K ) Dual luciferase reporter assay for luciferase activity of the indicated plasmids ( circLMP2A Wt , circLMP2A mut1 , circLMP2A mut2 , circLMP2Amut3 luciferase reporter ) in HEK - 293T cells transfected with mimic - NC or miR - 3908 .

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

Figure 6(B) The relative levels of 7 miRNA candidates were detected by real-time PCR in SNU719 and SNU-4th cells.(C-E) Real-time PCR analysis of 7 miRNA expression in SNU-4th cells transfected with sh-circLMP2A-2 or sh-control (C), and SNU719 (D) and YCCEL1 cells (E) transfected with circLMP2A or vector.(F-H) circ LMP2Ain specific probe and detected by real-time PCR (F) and RT-PCR (H). The relative levels of 5 miRNA candidates were detected by real-time PCR (G) and RT-PCR (H). GAPDH was used as a negative control.(I) RNA ISH showing the co-localization between circLMP2A and miR-3908 in SNU-4th cells. circLMP2A probes were labelled with Cy3, and miR-3908 probes were labelled with FITC. Nuclei were stained with DAPI.(K) Dual luciferase reporter assay for luciferase activity of the indicated plasmids (circLMP2A Wt, circLMP2A mut1, circLMP2A mut2, circLMP2Amut3 luciferase reporter) in HEK-293T cells transfected with mimic-NC or miR-3908.


Figure 7 ( A ) Tumour sphere culture analysis of the tumour sphere formation ability of SNU - 4th cells transfected with the above different mimics . Real - time PCR ( B ) analysis of EMT - related markers , stemness markers and drug resistance genes expression in SNU - 4th cells transfected with the above different mimics . WB ( C ) analysis of EMT - related markers , stemness markers and drug resistance genes expression in SNU - 4th cells transfected with the above different mimics . ( D - E ) Colony formation assay ( D ) and transwell assay ( E ) analysis of the colony - forming ability and migratory and invasive capability of SNU - 4th cells transfected with the above different mimics . ( F - G ) Flow cytometric analysis of the rate of apoptosis ( F ) and SP cells ( G ) in SNU - 4th cells transfected with the above different mimics . ( H ) Pearson correlation analysis of the correlation between the expression of circLMP2A and miR - 3908 . n = 69 , P = 0 . 039 , Pearson ' s correlation coefficient analysis . ( I ) Kaplan - Meier survival curves analysis of the correlation between miR - 3908 expression and OS . n = 69 , P = 0 . 049 , log - rank test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 7(A) Tumour sphere culture analysis of the tumour sphere formation ability of SNU-4th cells transfected with the above different mimics.Real-time PCR (B) analysis of EMT-related markers, stemness markers and drug resistance genes expression in SNU-4th cells transfected with the above different mimics.WB (C) analysis of EMT-related markers, stemness markers and drug resistance genes expression in SNU-4th cells transfected with the above different mimics.(D-E) Colony formation assay (D) and transwell assay (E) analysis of the colony-forming ability and migratory and invasive capability of SNU-4th cells transfected with the above different mimics.(F-G) Flow cytometric analysis of the rate of apoptosis (F) and SP cells (G) in SNU-4th cells transfected with the above different mimics.(H) Pearson correlation analysis of the correlation between the expression of circLMP2A and miR-3908. n = 69, P = 0.039, Pearson's correlation coefficient analysis.(I) Kaplan-Meier survival curves analysis of the correlation between miR-3908 expression and OS. n = 69, P = 0.049, log-rank test.


Figure 8 ( B ) Dual luciferase reporter assay for the luciferase activity of the indicated plasmids ( TRIM59 - Wt and TRIM59 - Mut luciferase reporter ) in HEK - 293T cells transfected with mimic - NC or miR - 3908 . ( C ) Real - time PCR analysis of the mRNA level of TRIM59 in SNU719 and SNU - 4th cells . ( D - F ) Real - time PCR analysis of the expression of TRIM59 in SNU - 4th ( D ) , SNU719 ( E ) and YCCEL1 cells ( F ) transfected with the above different mimics . ( G - I ) WB analysis of the expression of TRIM59 and p53 in SNU - 4th ( G ) , SNU719 ( H ) and YCCEL1 cells ( I ) transfected with the above different mimics . ( J ) Immunohistochemical staining showing that over - expression of circLMP2A could lead to increased expression of TRIM59 and ERG but decreased expression of p53 . ( K ) WB analysis of the expression of TRIM59 and p53 in tumour xenografts . ( L ) Immunohistochemical staining showing that high expression of circLMP2A in EBVaGC tissues resulted in significant increases in CD44 + CD24 - cells and significant decreases in p53 + cells .

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

Figure 8(B) Dual luciferase reporter assay for the luciferase activity of the indicated plasmids (TRIM59-Wt and TRIM59-Mut luciferase reporter) in HEK-293T cells transfected with mimic-NC or miR-3908.(C) Real-time PCR analysis of the mRNA level of TRIM59 in SNU719 and SNU-4th cells.(D-F) Real-time PCR analysis of the expression of TRIM59 in SNU-4th (D), SNU719 (E) and YCCEL1 cells (F) transfected with the above different mimics.(G-I) WB analysis of the expression of TRIM59 and p53 in SNU-4th (G), SNU719 (H) and YCCEL1 cells (I) transfected with the above different mimics.(J) Immunohistochemical staining showing that over-expression of circLMP2A could lead to increased expression of TRIM59 and ERG but decreased expression of p53.(K) WB analysis of the expression of TRIM59 and p53 in tumour xenografts.(L)Immunohistochemical staining showing that high expression of circLMP2A in EBVaGC tissues resulted in significant increases in CD44+ CD24- cells and significant decreases in p53+ cells.


Figure 1 ( A ) miR - 515 - 5p expression increases the area of the tubulin cytoskeleton . Tubulin ( green ) and cell nucleus ( blue ) . Objective x20 . Bar : 200 µm . ( B - C ) miR - 515 - 5p inhibits random ( B ) and directed ( C ) cell migration . The indicated cell lines were transfected with miR - 515 - 5p for 48 h before time - lapse imaging was performed for 18 h ( A ) or transwell migration assays were performed for 9 h ( C ) . ( B - left panel ) Plots show overlays of representative trajectories travelled . ( B - right panel ) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition . ( C - left panel ) Representative field of view from the bottom of the transwell chambers . Scale bar is 20 µm . ( C - right panel ) Data represent percentage of the average number migratory cells per field ( n = 5 fields per condition ) . Data are mean of three experiments ± SEM . ( B - C ) P values were calculated by t - test between miRNA ( 515 - 5p ) values and their respective non - targeting control miR ( NC ) values ( * * * , P < 0 . 001 ) . All data shown are representative of experiments performed at least in triplicate .

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

Figure 1(A) miR-515-5p expression increases the area of the tubulin cytoskeleton. Tubulin (green) and cell nucleus (blue). Objective x20. Bar: 200 µm.(B-C) miR-515-5p inhibits random (B) and directed (C) cell migration. The indicated cell lines were transfected with miR-515-5p for 48 h before time-lapse imaging was performed for 18 h (A) or transwell migration assays were performed for 9 h (C). (B-left panel) Plots show overlays of representative trajectories travelled. (B-right panel) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition. (C-left panel) Representative field of view from the bottom of the transwell chambers. Scale bar is 20 µm. (C-right panel) Data represent percentage of the average number migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (B-C) P values were calculated by t-test between miRNA (515-5p) values and their respective non-targeting control miR (NC) values (***, P<0.001). All data shown are representative of experiments performed at least in triplicate.


Figure 2 ( A ) RNA - seq of MCF7 and MDA - MB - 231 transfected with miR - 515 - 5p revealed the down - regulation of five transcripts , NRAS , FZD4 , CDC42BPA , PIK3C2B and MARK4 . ( B - D ) The effect of miR - 515 - 5p overexpression on NRAS , FZD4 , CDC42BPA , PIK3C2B and MARK4 mRNA levels in MCF7 ( B ) , MDA - MB - 231 ( C ) , A549 and H1299 ( D ) was determined by qPCR 48 h following transfection of the indicated cell line with non - targeting control miR ( NC ) or miR - 515 - 5p . Data is displayed as the normalised mean ± SEM . P values were calculated by t - test between miRNA conditions and their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) . ( E - F ) miR - 515 - 5p directly interacts with NRAS , MARK4 and PI3KC2B ' s 3 ' UTR . Relative luciferase activity levels were measured 24 h after co - transfection of MCF - 7 ( E ) and MDA - MB - 231 ( F ) with 3 ' UTR - luciferase reporter constructs and either with miR - 515 - 5p or miR NC . Data shown are normalised mean of three independent experiments ± SEM . ( B - F ) P values were calculated by t - test between miRNA conditions and their respective NC conditions ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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

Figure 2(A) RNA-seq of MCF7 and MDA-MB-231 transfected with miR-515-5p revealed the down-regulation of five transcripts, NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4.(B-D) The effect of miR-515-5p overexpression on NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNA levels in MCF7 (B), MDA-MB-231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non-targeting control miR (NC) or miR-515-5p. Data is displayed as the normalised mean ± SEM. P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).(E-F) miR-515-5p directly interacts with NRAS, MARK4 and PI3KC2B's 3'UTR. Relative luciferase activity levels were measured 24 h after co-transfection of MCF-7 (E) and MDA-MB-231 (F) with 3'UTR-luciferase reporter constructs and either with miR-515-5p or miR NC. Data shown are normalised mean of three independent experiments ± SEM. (B-F) P values were calculated by t-test between miRNA conditions and their respective NC conditions (*, P<0.05; **, P<0.01; ***, P<0.001).


Figure 3MARK4 siRNA - mediated silencing increases the tubulin cytoskeletal area ( A ) . ( A ) Data shown are representative of experiments performed in triplicate . Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions . MARK4 siRNA - mediated silencing reduces random ( B ) migration in MDA - MB - 231 ( MDA ) , A549 and H1299 cells . Cells were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before time - lapse imaging was performed for 18 h ( B ) . ( B - top panel ) Plots show overlays of representative trajectories described . ( B - bottom panel ) The migration distance was quantified and represented as the normalised mean ± SEM . ( C - top panel ) Representative field of view from the bottom side of the transwell membrane . ( C - bottom panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the siRNA - transfected and the respective control condition ( * * * P < 0 . 001 ) . MARK4 siRNA - mediated silencing reduces directed ( C ) migration in MDA - MB - 231 ( MDA ) , A549 and H1299 cells . ( C - top panel ) . Cells were transfected either with siRNA targeting MARK4 ( M4 ) or a non - targeting control ( NC ) for 48 h before transwell assay for 9h ( C ) . Representative field of view from the bottom side of the transwell membrane . ( C - bottom panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . ( B - C ) Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the siRNA - transfected and the respective control condition ( * * * P < 0 . 001 ) .

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Figure 3MARK4 siRNA-mediated silencing increases the tubulin cytoskeletal area (A). (A) Data shown are representative of experiments performed in triplicate. Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with 3 technical replicates per conditions.MARK4 siRNA-mediated silencing reduces random (B) migration in MDA-MB-231 (MDA), A549 and H1299 cells.Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before time-lapse imaging was performed for 18 h (B). (B-top panel) Plots show overlays of representative trajectories described. (B-bottom panel) The migration distance was quantified and represented as the normalised mean ± SEM. (C-top panel) Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).MARK4 siRNA-mediated silencing reduces directed (C) migration in MDA-MB-231 (MDA), A549 and H1299 cells.(C-top panel). Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before transwell assay for 9h (C). Representative field of view from the bottom side of the transwell membrane. (C-bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). (B-C) Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the siRNA-transfected and the respective control condition (***P<0.001).