( 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 ) .

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(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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(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.


( 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 ;

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(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

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, 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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(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.


( 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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(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.


( A ) Western blot results show knockdown of ATM attenuated 100 μM H2O2 induced T2609 and T2647 phosphorylation of DNA - PKcs in HeLa cells .

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(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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(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).


A 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 .

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A 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 .

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 .

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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 ) .

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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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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.


A 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 ) .

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A 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 .

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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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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.


A 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 .

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A 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 .

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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 .

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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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K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.


F 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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F 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).


A 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 ) .

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A 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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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).


G Graph presents the survival rate of glioblastoma patients from A that present PRKDClow / MB21D1low , PRKDChigh / MB21D1low and PRKDChigh / MB21D1high expression .

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G 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 ) .

H Violin plots present the expression of PRKDC and MB21D1 in datasets analyzed in A, based on tumor grade (II to IV).


B ) Tumour growth curves for successive four - generation xenografts treated with 5 - Fu or PBS . Twelve mice per group .

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B) 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 ) .

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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 .

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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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 .

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 .

M) Representative images of the flow cytometric analysis of SP cells.


N ) Representative images of the flow cytometric analysis of the surface antigen phenotype .

N) Representative images of the flow cytometric analysis of the surface antigen phenotype.


A ) 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 .

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A) 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 ) .

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(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 .

(D) The length distribution of ebv-circRNAs.


( E ) Genomic origin of ebv - circRNAs .

(E) Genomic origin of ebv-circRNAs.


( F ) Number of ebv - circRNAs produced from one gene .

(F) Number of ebv-circRNAs produced from one gene.


( B ) Real - time PCR analysis of the expression of circLMP2A in SNU - 4th , SNU719 and YCCEL1 cells .

(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 .

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(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 .

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(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 .

(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 .

(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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(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.


( A ) Real - time PCR analysis of the interfering efficacy and linear LMP2A mRNA levels after transfection of three circLMP2A shRNAs .

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(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 .

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(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 .

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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 .

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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 .

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(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 .

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(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 .

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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 .

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 .

(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.


( A ) Real - time PCR confirmed the over - expression of circLMP2A in SNU719 and YCCEL1 cells after transfection .

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(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 .

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(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 .

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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 .

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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 .

(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 .

(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 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 .

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.


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( B ) The relative levels of 7 miRNA candidates were detected by real - time PCR in SNU719 and SNU - 4th cells .

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(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 .

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(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 .

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(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 .

(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 .

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(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.


( A ) Tumour sphere culture analysis of the tumour sphere formation ability of SNU - 4th cells transfected with the above different mimics .

(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 .

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 .

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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 .

(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 .

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(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 .

(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 .

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(I) Kaplan-Meier survival curves analysis of the correlation between miR-3908 expression and OS. n = 69, P = 0.049, log-rank test.


( 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 .

(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 .

(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 .

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(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 .

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(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 .

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(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 .

(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 .

(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.


( A ) miR - 515 - 5p expression increases the area of the tubulin cytoskeleton . Tubulin ( green ) and cell nucleus ( blue ) . Objective x20 . Bar : 200 µm .

(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 .

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(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.


( 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 .

(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 ) .

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(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 ) .

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(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).


MARK4 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 .

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MARK4 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 ) .

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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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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).


( A - B ) The indicated cell lines were transfected with either miR - 515 - 5p or a non - targeting miR ( miR NC ) together with a MARK4 expressing or an empty vector ( EV ) plasmid DNA . MARK4 over - expression rescues the inhibition by miR - 515 - 5p of random ( A ) and directed ( B ) migration of the indicated cell lines . ( A - left panel ) Plots show overlays of representative trajectories described for MDA - MB - 231 cells ( n = 30 ) . ( A - right panel ) The migration distance was quantified and represented as the normalised mean ± SEM . ( B - left panel ) Representative field of view from the bottom side of the transwell membrane for MDA - MB - 231 . ( B - right panel ) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field ( n = 5 fields per condition ) . Data are mean of three experiments ± SEM . ( A - C ) P values were calculated by t - test between the transfected and the respective control condition ( EV + miR - NC ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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(A-B) The indicated cell lines were transfected with either miR-515-5p or a non-targeting miR (miR NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 over-expression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A-left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n=30). (A-right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B-left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B-right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n=5 fields per condition). Data are mean of three experiments ± SEM. (A-C) P values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*,P<0.05; **,P<0.01; ***,P<0.001).


( A - B ) Overall survival of ER - negative metastatic breast cancer ( n = 226 ) ( A ) and lung adenocarcinoma ( n = 719 ) ( B ) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier - plotter [ 25 ] .

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(A-B) Overall survival of ER-negative metastatic breast cancer (n=226) (A) and lung adenocarcinoma (n=719) (B) patients according to MARK4 mRNA levels found in their tumors was represented using the Kaplan Meier-plotter [25].


( C ) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression . Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie - chart .

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(C) A cDNA array for 24 matched lung cancers and surrounding normal tissue was analysed for MARK4 expression. Expression levels from tumors were normalised to their respective normal control and the percentage of cases for the indicated fold expression in the tumor represented as a pie-chart.


( D - F ) MARK4 protein expression was determined by histoimmunochemistry in ( D - E ) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or ( F ) a home - made tissue microarray comprising 100 matched cases of primary and metastatic tumors . ( D - E ) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples ( D ) or in lung tumors according to their histological subtypes ( E ) is represented . ( F ) The percentage of cases with increased , decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented .

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(D-F) MARK4 protein expression was determined by histoimmunochemistry in (D-E) a tissue microarray comprising 90 lung tumor and 10 normal lung samples or (F) a home-made tissue microarray comprising 100 matched cases of primary and metastatic tumors. (D-E) The percentage of positivity for MARK4 staining in lung tumors versus normal lung samples (D) or in lung tumors according to their histological subtypes (E) is represented. (F) The percentage of cases with increased, decreased or unchanged levels of MARK4 in metastatic lesions as compared to the corresponding primary tumors is represented.


( A - D ) Low expression of miR - 515 - 5p is linked to survival in both Lung and Breast cancer . ( A ) Survival analysis of miR - 515 - 5p from 195 lung adenocarcinoma samples from the TCGA . ( B ) Survival analysis of miR - 515 - p from 34 lung squamous cell carcinoma from the TCGA . ( C ) Survival analysis of miR - 515 - 5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study ( GSE37405 ) . ( D ) Survival analysis of miR - 515 - 5p from 33 Breast cancer from the GSE7405 Breast study . Analysis and plot were obtained with SurvMicro http : / / bioinformatica . mty . itesm . mx : 8080 / Biomatec / Survmicro . jsp for ( A - C ) and with MIRUMIR for ( D ) .

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(A-D) Low expression of miR-515-5p is linked to survival in both Lung and Breast cancer. (A) Survival analysis of miR-515-5p from 195 lung adenocarcinoma samples from the TCGA. (B) Survival analysis of miR-515-p from 34 lung squamous cell carcinoma from the TCGA. (C) Survival analysis of miR-515-5p from 60 Breast cancer samples from the Lyng Ditzel Breast Study (GSE37405). (D) Survival analysis of miR-515-5p from 33 Breast cancer from the GSE7405 Breast study. Analysis and plot were obtained with SurvMicro http://bioinformatica.mty.itesm.mx:8080/Biomatec/Survmicro.jsp for (A-C) and with MIRUMIR for (D).


( E ) Comparison of miR - 515 - 5p expression in 18 primary and matched metastatic lesions from breast cancer patients . miR - 515 - 5p levels were quantified by TaqMan qRT - PCR . Absolute values were normalized to u6 snRNA values and displayed as a dot - plot . Bar ; Mean , Whiskers ; SEM .

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(E) Comparison of miR-515-5p expression in 18 primary and matched metastatic lesions from breast cancer patients. miR-515-5p levels were quantified by TaqMan qRT-PCR. Absolute values were normalized to u6 snRNA values and displayed as a dot-plot. Bar; Mean, Whiskers; SEM.


( F ) MDA - MB - 231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [ 22 ] . miR - 515 - 5p and MARK4 levels were then determined in the primary and indicated secondary lesions . Data are average ± SEM of technical triplicates .

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(F) MDA-MB-231 cells were inoculated into the mammary fat pads of nude mice and allowed to form metastatic deposits [22]. miR-515-5p and MARK4 levels were then determined in the primary and indicated secondary lesions. Data are average ± SEM of technical triplicates.


( G ) Luciferase - expressing A549 cells overexpressing miR - 515 - 5p or a non - targeting control ( miR - NC ) were injected in the tail - vein of SCID mice and the development of tumor burden monitored by whole animal - imaging in response to luciferin administration at week 1 and 4 following injection . ( G - left panel ) Representative imaging of the same animals for the indicated conditions at week 1 and 4 . ( G - Right panel ) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1 . Data represented are representative of three independent experiments . ( E - G ) P values were calculated by t - test ( * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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(G) Luciferase-expressing A549 cells overexpressing miR-515-5p or a non-targeting control (miR-NC) were injected in the tail-vein of SCID mice and the development of tumor burden monitored by whole animal-imaging in response to luciferin administration at week 1 and 4 following injection. (G-left panel) Representative imaging of the same animals for the indicated conditions at week 1 and 4. (G-Right panel) The emitted photons were quantified and the signals normalised to the corresponding readings at week 1. Data represented are representative of three independent experiments. (E-G) P values were calculated by t-test (*, P<0.05; **, P<0.01; ***, P<0.001).


A549 - Luc cells transfected either with miR - 515 - 5p precursor ( miR ) , siRNAs to MARK4 ( siMARK4 ) or the corresponding control sequences ( C and NT , respectively ) were injected in Scid mice through the tail vein . 4 weeks later , the mice were sacrificed , their lungs extracted and fixed prior to staining for Luciferase expression . ( A ) Representative pictures showing the presence of A549 - Luc cells in the lungs of NT mice either as single cells ( upper panel ) or as a tumour mass ( lower panel ) . ( B ) Percentage of the area covered by tumour cells on randomly observed fields of views . Quantification was performed using Photoshop CS4 . Each spot corresponds to quantification of an individual 1 mm x 1 . 5 mm field . Transversal bar ; Median . Statistics : Student t - test . * , p ≤ 0 . 05 .

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A549-Luc cells transfected either with miR-515-5p precursor (miR), siRNAs to MARK4 (siMARK4) or the corresponding control sequences (C and NT, respectively) were injected in Scid mice through the tail vein. 4 weeks later, the mice were sacrificed, their lungs extracted and fixed prior to staining for Luciferase expression. (A) Representative pictures showing the presence of A549-Luc cells in the lungs of NT mice either as single cells (upper panel) or as a tumour mass (lower panel). (B) Percentage of the area covered by tumour cells on randomly observed fields of views. Quantification was performed using Photoshop CS4. Each spot corresponds to quantification of an individual 1 mm x 1.5 mm field. Transversal bar; Median. Statistics: Student t-test. *, p≤0.05.


B , qRT - PCR of piwi - 1 relative to GAPDH in RNAi animals as indicated , 2dpi . Circles represent individual biological replicates averaged from 3 technical replicates .

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B, qRT-PCR of piwi-1 relative to GAPDH in RNAi animals as indicated, 2dpi. Circles represent individual biological replicates averaged from 3 technical replicates.


D , Percentage of X1 cells from control and atm ( RNAi ) animals , 1dpi or unirradiated .

D, Percentage of X1 cells from control and atm(RNAi) animals, 1dpi or unirradiated.


E , qRT - PCR of piwi - 1 relative to GAPDH in RNAi animals as indicated , 2dpi . Data are averages ± SD ; circles represent biological replicates ; statistics represent ordinary one - way ANOVA compared to controls , * * * * P < 0 . 0001 , ns = not significant .

E, qRT-PCR of piwi-1 relative to GAPDH in RNAi animals as indicated, 2dpi. Data are averages ± SD; circles represent biological replicates; statistics represent ordinary one-way ANOVA compared to controls, ****P<0.0001, ns=not significant.


F , piwi - 1 fluorescent in situ hybridization ( FISH ) of RNAi animals as indicated , 2 dpi .

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F, piwi-1 fluorescent in situ hybridization (FISH) of RNAi animals as indicated, 2 dpi.


A , Top : Strategy for EdU pulse and animal fixation . Bottom : Confocal images of EdU - stained atm and control RNAi animals . B , Quantification by area of EdU + cells from A . Circles represent individual animals . Inset , expanded scale showing compressed data below . Data includes two independent experiments .

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A, Top: Strategy for EdU pulse and animal fixation. Bottom: Confocal images of EdU-stained atm and control RNAi animals. B, Quantification by area of EdU+ cells from A. Circles represent individual animals. Inset, expanded scale showing compressed data below. Data includes two independent experiments.


C , Graph showing percentage of G2 / M cells in RNAi animals on indicated dpi .

C, Graph showing percentage of G2/M cells in RNAi animals on indicated dpi.


D , Confocal images of phosphohistone H3 ( pH3 ) - stained RNAi animals after radiation exposure . Blue , DAPI . E and F , Quantification by area of pH3 cells in radiated ( E ) or unirradiated ( F ) animals after atm ( solid line ) and control ( dashed line ) RNAi .

D, Confocal images of phosphohistone H3 (pH3)-stained RNAi animals after radiation exposure. Blue, DAPI. E and F, Quantification by area of pH3 cells in radiated (E) or unirradiated (F) animals after atm (solid line) and control (dashed line) RNAi.


A , Confocal images of piwi - 1 FISH in RNAi animals after radiation exposure . Scale bars = 250µm . B and C , Percentage of animal area occupied by piwi - 1 FISH divided by total animal area , in radiated ( B ) or unirradiated ( C ) RNAi animals . D , Total number of piwi - 1 + cells in RNAi animals , 7 dpi . Experiments in ( A ) - ( D ) were repeated twice .

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A, Confocal images of piwi-1 FISH in RNAi animals after radiation exposure. Scale bars=250µm. B and C, Percentage of animal area occupied by piwi-1 FISH divided by total animal area, in radiated (B) or unirradiated (C) RNAi animals. D, Total number of piwi-1+ cells in RNAi animals, 7 dpi. Experiments in (A) - (D) were repeated twice.


E , Schematic of experimental design . Below , Kaplan - Meier survival curves of intact ( solid line ) and decapitated ( dashed line ) animals after RNAi as indicated . n = 45 animals , repeated twice .

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E, Schematic of experimental design. Below, Kaplan-Meier survival curves of intact (solid line) and decapitated (dashed line) animals after RNAi as indicated. n = 45 animals, repeated twice.


A - C , Kaplan - Meier survival curves of intact RNAi animals after radiation , n = 50 animals , repeated twice . Experiments in A and B were performed simultaneously but split into two graphs for clarity ; control and atm ( RNAi ) values are replicated in both panels .

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A - C, Kaplan-Meier survival curves of intact RNAi animals after radiation, n = 50 animals, repeated twice. Experiments in A and B were performed simultaneously but split into two graphs for clarity; control and atm(RNAi) values are replicated in both panels.


D , Confocal images of piwi - 1 FISH in RNAi animals after exposure to 20Gy , 2dpi . Scale bars = 250µm . E , Quantification by area of piwi - 1 + cells in RNAi animals after 20Gy exposure , 7dpi .

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D, Confocal images of piwi-1 FISH in RNAi animals after exposure to 20Gy, 2dpi. Scale bars=250µm. E, Quantification by area of piwi-1+ cells in RNAi animals after 20Gy exposure, 7dpi.


Figure 1 . Helix - 7 dynamically interacts with the miRNA seed region . A ) Cartoon representation of the Ago2 - miRNA crystal structure . Ago2 is colored gray except for helix - 7 , which is yellow . miRNA guide colored red . C ) Close - up view of the unpaired complex shows helix - 7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7 .

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Figure 1. Helix-7 dynamically interacts with the miRNA seed region. A) Cartoon representation of the Ago2-miRNA crystal structure. Ago2 is colored gray except for helix-7, which is yellow. miRNA guide colored red.C) Close-up view of the unpaired complex shows helix-7 breaks nucleobase stacking in the miRNA seed region by intercalating between g6 and g7.


B ) Crystal structure of the Ago2 - miRNA - target RNA ternary complex . Target RNA is colored blue . D ) Close - up view of the Ago2 - miRNA - target complex shows helix - 7 docks into the minor groove of the guide : target duplex , directly contacting base pairs at positions g6 and g7 .

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B) Crystal structure of the Ago2-miRNA-target RNA ternary complex. Target RNA is colored blue. D) Close-up view of the Ago2-miRNA-target complex shows helix-7 docks into the minor groove of the guide:target duplex, directly contacting base pairs at positions g6 and g7.


Figure 2 . Helix - 7 is not required for target recognition . A ) Plot of g2 - g8 target RNA bound ( 0 . 1 nM ) versus Ago2 - miR122 concentration for wild type ( WT ) , helix - 7 double point mutant ( MI - AA ) , and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± standard deviation ( SD ) are plotted . Top panel shows target RNA paired to seed region of the guide RNA ( miRNA - 122 ) .

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Figure 2. Helix-7 is not required for target recognition. A) Plot of g2-g8 target RNA bound (0.1 nM) versus Ago2-miR122 concentration for wild type (WT), helix-7 double point mutant (MI-AA), and Ηhelix-7 Ago2. Average values from at least three independent experiments ± standard deviation (SD) are plotted. Top panel shows target RNA paired to seed region of the guide RNA (miRNA-122).


B ) Dissociation of a 32P - labeled target RNA ( 0 . 1 nM ) from the Ago2 - miR122 complex ( 1 nM ) was monitored in the presence of unlabeled target RNA ( 100 nM ) . Fraction of the target RNA bound to Ago2 - miR122 is plotted as a function of time for WT and Ηhelix - 7 Ago2 . Average values from at least three independent experiments ± SD were fit to single exponential decays .

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B) Dissociation of a 32P-labeled target RNA (0.1 nM) from the Ago2-miR122 complex (1 nM) was monitored in the presence of unlabeled target RNA (100 nM). Fraction of the target RNA bound to Ago2-miR122 is plotted as a function of time for WT and Ηhelix-7 Ago2. Average values from at least three independent experiments ± SD were fit to single exponential decays.


C ) Representative fluorescence time traces ( with a resolution of 100 ms for wt , 300 ms for MI - AA and Ηhelix - 7 ) show docking and dissociation of wild type ( top ) , MI - AA ( middle ) , and Ηhelix - 7 ( bottom ) Ago2 - miRNA at single spots in the microfluidic chamber .

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C) Representative fluorescence time traces (with a resolution of 100 ms for wt, 300 ms for MI-AA and Ηhelix-7) show docking and dissociation of wild type (top), MI-AA (middle), and Ηhelix-7 (bottom) Ago2-miRNA at single spots in the microfluidic chamber.


D ) Dwell time histograms obtained for N = 4 , 5 and 6 , with wild type Ago2 ; histograms were fit with single exponential decay . The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution . The bin size is 0 . 5 sec for N = 4 and 1 sec for N = 5 and 6 .

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D) Dwell time histograms obtained for N = 4, 5 and 6, with wild type Ago2; histograms were fit with single exponential decay. The first column of the data was not included in the analysis to avoid potential artifacts arising from the limit of the time resolution. The bin size is 0.5 sec for N =4 and 1 sec for N =5 and 6.


E ) Bar plot showing the dependence of dwell time on N for wild type Ago2 . The dashed red line indicates the observation time limit ( 300 s ) , which is constrained by photobleaching . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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E) Bar plot showing the dependence of dwell time on N for wild type Ago2. The dashed red line indicates the observation time limit (300 s), which is constrained by photobleaching.For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


F ) Dwell time histogram for different N ' s with the MI - AA helix - 7 mutant . The dwell time histogram for N = 6 fit with double exponential decay ( green ; R2 = 0 . 98 ) ; The dwell time for N = 6 with single exponential fit was 15 . 2 ± 3 . 41 sec , R2 = 0 . 96 ) .

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F) Dwell time histogram for different N's with the MI-AA helix-7 mutant. The dwell time histogram for N = 6 fit with double exponential decay (green; R2 = 0.98); The dwell time for N = 6 with single exponential fit was 15.2 ± 3.41 sec, R2 = 0.96).


G ) Bar plot for dwell times of MI - AA Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

G) Bar plot for dwell times of MI-AA Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


H ) Dwell time histogram for different N ' s with the ∆ helix - 7 mutant . The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays ( green ; R2 = 0 . 97 for N = 5 and R2 = 0 . 96 for N = 6 ) ; The dwell time for N = 5 and N = 6 with single exponential fit was 9 . 1 ± 0 . 91 sec ( R2 = 0 . 86 ) and 22 . 3 ± 5 . 08 sec ( R2 = 0 . 90 ) respectively ) .

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H) Dwell time histogram for different N's with the ∆helix-7 mutant. The dwell time histograms for N = 5 and N = 6 were fit with double exponential decays (green; R2 = 0.97 for N = 5 and R2 = 0.96 for N = 6); The dwell time for N = 5 and N = 6 with single exponential fit was 9.1 ± 0.91 sec (R2 = 0.86) and 22.3 ± 5.08 sec (R2 = 0.90) respectively).


I ) Bar plot for dwell times of ∆ helix - 7 Ago2 as a function of N . For all bar plots ( E , G and I ) error bars are the SD of three independent experiments carried out on different days .

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I) Bar plot for dwell times of ∆helix-7 Ago2 as a function of N. For all bar plots (E, G and I) error bars are the SD of three independent experiments carried out on different days.


Figure 4 . Helix - 7 discourages non - Watson Crick pairing at g6 and g7 . A - C ) Plots of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 ( wild type and Ηhelix - 7 ) concentration for targets with an increasing number of G : U wobble pairs in the seed 3 ' end . D ) Plot of target RNA ( 0 . 1 nM ) bound to Ago2 versus Ago2 - miR122 concentration for a target with mismatches at positions g6 - g8 . For A - E , average values from at least three independent experiments ± SD are plotted .

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Figure 4. Helix-7 discourages non-Watson Crick pairing at g6 and g7. A-C) Plots of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 (wild type and Ηhelix-7) concentration for targets with an increasing number of G:U wobble pairs in the seed 3' end. D) Plot of target RNA (0.1 nM) bound to Ago2 versus Ago2-miR122 concentration for a target with mismatches at positions g6-g8. For A-E, average values from at least three independent experiments ± SD are plotted.


E ) Bar plots of the Kd of wild type ( left ) Ηhelix - 7 ( right ) Ago2 - miR122 for indicated target RNAs . For A - E , average values from at least three independent experiments ± SD are plotted .

E) Bar plots of the Kd of wild type (left) Ηhelix-7 (right) Ago2-miR122 for indicated target RNAs. For A-E, average values from at least three independent experiments ± SD are plotted.


Figure 5 . ΗHelix - 7 Ago2 is sensitive to inhibition by off - target RNAs . A ) Ago2 - miRNA122 ( 1 nM ) was incubated with a 32P - labeled , seed - matched target RNA ( 0 . 1 nM ) in the presence of increasing concentrations of unlabeled a competitor RNA ( top panel ) . Fraction target RNA bound to Ago2 is plotted as a function of off - target competitor RNA concentration . B ) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA . For A - B , average values from at least three independent experiments ± SD are plotted .

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Figure 5. ΗHelix-7 Ago2 is sensitive to inhibition by off-target RNAs. A) Ago2-miRNA122 (1 nM) was incubated with a 32P-labeled, seed-matched target RNA (0.1 nM) in the presence of increasing concentrations of unlabeled a competitor RNA (top panel). Fraction target RNA bound to Ago2 is plotted as a function of off-target competitor RNA concentration. B) Fraction of target RNA bound to Ago2 in the presence of increasing concentrations of unlabeled total cellular RNA. For A-B, average values from at least three independent experiments ± SD are plotted.


Figure 6 . Helix - 7 mutants display reduced target - binding rates . A ) Ago2 - miR122 complexes were mixed with a 32P - labeled seed matched target RNA ; bound and free RNAs were then separated using a filter - binding apparatus at various times . Representative time course for target RNA ( 0 . 1 nM ) binding to wild type , MI - AA , and Δhelix - 7 Ago2 ( 1 nM ) . Average values from at least three independent experiments ± SD are plotted .

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Figure 6. Helix-7 mutants display reduced target-binding rates. A) Ago2-miR122 complexes were mixed with a 32P-labeled seed matched target RNA; bound and free RNAs were then separated using a filter-binding apparatus at various times. Representative time course for target RNA (0.1 nM) binding to wild type, MI-AA, and Δhelix-7 Ago2 (1 nM). Average values from at least three independent experiments ± SD are plotted.


B ) Observed binding rates ( kon , obs ) plotted as a function of Ago2 - miR122 concentration . Average values ± SD are plotted .

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B) Observed binding rates (kon, obs) plotted as a function of Ago2-miR122 concentration. Average values ± SD are plotted.


C ) Arrival time distribution of single Ago2 - miRNA complexes binding immobilized targets in a microfluidic chamber . The value is the average of three independent measurements .

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C) Arrival time distribution of single Ago2-miRNA complexes binding immobilized targets in a microfluidic chamber. The value is the average of three independent measurements.


Figure 7 . Structure of a helix - 7 Ago2 mutant . A ) Superposition of wild type ( white ) and MI - AA ( gray ) Ago2 . Helix - 7 colored yellow ; ordered region of the guide RNAs colored in red .

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Figure 7. Structure of a helix-7 Ago2 mutant. A) Superposition of wild type (white) and MI-AA (gray) Ago2. Helix-7 colored yellow; ordered region of the guide RNAs colored in red.


B ) Close - up view shows helix - 7 tilts away from the seed region in the MI - AA mutant .

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B) Close-up view shows helix-7 tilts away from the seed region in the MI-AA mutant.


C ) Side - by - side comparison of the seed region in wild type ( left ) and MI - AA ( right ) Ago2 crystal structures .

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C) Side-by-side comparison of the seed region in wild type (left) and MI-AA (right) Ago2 crystal structures.


A . Cryo - sections of human organoids were analyzed for the presence of enterocytes ( E - cad ) , Goblet cells ( Muc - 2 ) and tight junctions ( ZO - 1 ) by indirect immunofluorescence . Nuclei are stained with DAPI . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test .

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A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.


B . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . 16hpi organoids were frozen , cryo - sectioned , and HAstV1 infected cells were visualized by indirect immunofluorescence ( HAstV1 ( red ) , nuclei ( DAPI , blue ) . Scale bar 25 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test .

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B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.


C . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells ( green ) were visualized by indirect immunofluorescence . Apical and basolateral membranes were immunostained for actin ( magenta ) and Laminin ( red ) respectively . Nuclei are stained with DAPI ( blue ) . Scale bar is 20 μm . Data information : Three biological replicates were performed for each experiment . Representative immunofluorescence images are shown . Error bars indicate the standard deviation . Statistics are from unpaired t - test .

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

C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.


E . Human intestinal organoids were infected with HAstV1 . At indicated time post - infection the increase in viral copy number was determined by q - RT - PCR .

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E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR.


F . Human intestinal organoids were incubated with media ( mock ) or infected with HAstV1 . At 24 hpi the presence of IFNλ in the media was tested by ELISA . Dotted line indicates detection limit of the assay .

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F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay.


G . Same as E but for the induced levels of either type I IFN ( IFNβ1 ) or type III IFN ( IFNλ ) .

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G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ).


H . Human intestinal organoids were pre - treated for 24h with 2000IU / mL of IFNβ1 or 300ng / mL of IFNλ1 - 3 . Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q - RT - PCR .

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H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.


J - M . Percentage of stem cells ( OLFM4 ) goblet cell ( FGCBP ) , mature enterocytes ( APOA4 ) and cells of the enterocyte lineage ( FGCBP ) from the total organoid cells population ( black bar ) in mock - treated organoids and after HAstV1 infection . The percentage of infected cells ( red bars ) and bystander cells ( blue bars ) within each cell type is shown . The data was obtained from multiplex in situ RNA hybridization in 2D organoids . n = 10 , mean ± SEM . Statistics show comparison of infected cells between mock - treated , 4h pi and 16h pi organoids . An Ordinary one - way ANOVA and Tukey ' s multiple comparisons test was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.


A . One image per condition was used to show the multiplex in situ RNA FISH . A representative region of the images was chosen and cropped in order to have a zoom - in of a specific area for better visualization . Shown are the enterocyte lineage marker FABP6 ( green ) , HAstV1 infection ( white ) , and CCL2 gene expression ( red ) of mock - treated and infected organoids . DAPI is in blue . White arrows show co - localization of FABP6 and CCL2 with HAstV1 signal . Scale bar 100µm .

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A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.


B - D . Fluorescence intensity ( arbitrary units ) of innate immune marker expression in stem cells ( OLFM4 positive ) , goblet cells ( FCGBP positive ) , enterocyte lineage cells ( FABP6 positive ) , and mature enterocytes ( APOA4 positive ) . Bystander cells are in blue and infected cells in red . 10 - 90 percentile box plots , each dot represents one cell ( mock N = 2274 , 4h N = 3682 , 16h N = 1510 . C . mock N = 2693 , 4h N = 3706 , 16h N = 1921 . D . mock N = 2274 , 4h N = 3706 , 16h N = 1514 . ) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages . Ordinary one - way ANOVA and Tukey ' s multiple comparisons test were used . Grey statistics show the comparison between infected and bystander cells within each cell lineage . Unpaired t - test with Welch ' s correction was used . n . s non significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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

B-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Total leukocyte and red blood cell count in PB . CoPP increases the number of all WBC populations . Granulocyte and monocyte percentage are increasing , whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP increases the concentrations of a group of cytokines ( red box ) .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box).


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Selected cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP increases concentrations of G - CSF , MCP - 1 , IP - 10 , IL - 6 and IL - 5 . Results are shown as mean + SEM , one - way Anova with Bonferroni post - test , n = 5 mice per group .

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C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . ( A ) Cell numbers of main leukocyte populations in PB measured by flow cytometry . Both G - CSF and CoPP increase numbers of CD45 + cells in blood . G - CSF and CoPP similarly increase monocyte numbers , but the increase of granulocytes is higher after G - CSF . Lymphocytes , including T cells , are increased only by G - CSF and not by CoPP .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP induces a group of cytokines ( red box ) which are not induced by G - CSF .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Plasma concentrations of selected cytokines and growth factors . CoPP highly increases concentration of endogenous G - CSF and IL - 6 that are not increased by G - CSF .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Treatment with G - CSF and CoPP decreases percentage of Ly6G + macrophages in the

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BM


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Treatment with G - CSF and CoPP increases relative spleen weight .

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C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP increases relative spleen weight.