Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . D . IF analysis to detect dense fibrillar component protein , ribosome processing factor 1 ( RPF1 ) . E . Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei . 43 nuclei mock treated cells , 69 nuclei PIC treated cells . F . Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells .

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Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1). E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells. F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells.


Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . G . FISH analysis to detect poly ( A ) + RNA and nuclear speckle - localized MALAT1 RNA and IF analysis with anti - sc35 antibody to detect nuclear speckle protein SRRM2 . H . Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei . 51 nuclei mock treated cells , 56 nuclei PIC treated cells . I . Graph of the median and value of the volume of individual SRRM2 foci in nuclei . Mock 1526 SRRM2 foci in 51 nuclei . PIC 136 SRRM2 foci in 20 nuclei that contain < 10 SRRM2 foci .

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Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2. H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells. I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci.


Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus ( NLS - RL - WT ) either mock transfected or treated with poly ( I : C ) ( PIC ) for 4 to 5 hours . J . FISH analysis to detect poly ( A ) + RNA and IF analysis to detect Cajal body protein coilin . K . Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo ( dT ) with different distributions of coilin protein . 90 nuclei in mock treated cells , 192 nuclei in PIC treated cells .

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Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours. J. FISH analysis to detect poly(A)+ RNA and IF analysis to detect Cajal body protein coilin. K. Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo(dT) with different distributions of coilin protein. 90 nuclei in mock treated cells, 192 nuclei in PIC treated cells.


Analysis of A549 cells with NLS - RL - WT or NLS - RL - CM either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . A . FISH analysis to detect poly ( A ) + RNA and IF analysis against FUS protein . Images are a single 0 . 2 micron Z section .

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Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. A. FISH analysis to detect poly(A)+ RNA and IF analysis against FUS protein. Images are a single 0.2 micron Z section.


Analysis of A549 cells with NLS - RL - WT or NLS - RL - CM either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . B . Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo ( dT ) signal . All the nuclei ( total ) in which nuclear RNA was degraded ( N = 64 ) or the same nuclei classified as to whether or not they contained residual poly ( A ) foci . Nuclei with poly ( A ) foci ( N = 14 ) . Nuclei without poly ( A ) foci ( N = 50 ) .

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Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours. B. Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo(dT) signal. All the nuclei (total) in which nuclear RNA was degraded (N=64) or the same nuclei classified as to whether or not they contained residual poly(A) foci. Nuclei with poly(A) foci (N=14). Nuclei without poly(A) foci (N=50).


Analysis of A549 cells with NLS - RL - WT either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . C . FISH analysis to detect poly ( A ) + RNA and IF analysis against mediator complex protein , MED1 , to detect super - enhancer condensates . D . Number of MED1 foci in mock and PIC treated NLS - RL - WT cells . E . Average volume of individual MED1 foci in mock and PIC treated NLS - RL - WT cells .

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Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. C. FISH analysis to detect poly(A)+ RNA and IF analysis against mediator complex protein, MED1, to detect super-enhancer condensates. D. Number of MED1 foci in mock and PIC treated NLS-RL-WT cells. E. Average volume of individual MED1 foci in mock and PIC treated NLS-RL-WT cells.


Analysis of A549 cells with NLS - RL - WT either mock transfected or treated with poly ( I : C ) ( PIC ) for 5 hours . F . FISH analysis to detect poly ( A ) + RNA and IF analysis against BRD4 , to detect super - enhancer condensates . G . Number of BRD4 foci in mock and PIC treated NLS - RL - WT cells . H . Average volume of individual BRD4 foci in mock and PIC treated NLS - RL - WT cells .

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Analysis of A549 cells with NLS-RL-WT either mock transfected or treated with poly(I:C) (PIC) for 5 hours. F. FISH analysis to detect poly(A)+ RNA and IF analysis against BRD4, to detect super-enhancer condensates. G. Number of BRD4 foci in mock and PIC treated NLS-RL-WT cells. H. Average volume of individual BRD4 foci in mock and PIC treated NLS-RL-WT cells.


B ) Volcano plot of quantified proteins . Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p - values were calculated with a Welch t - test . Proteins above the threshold p = 0 . 05 , s0 = 1 , c = 0 . 5 ( dashed line ) are considered enriched . Abbreviations : LFQ : Label free

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B) Volcano plot of quantified proteins. Log2 fold change was determined as the difference between the mean LFQ intensity of the four replicates of telomeric to control sequence and p-values were calculated with a Welch t-test. Proteins above the threshold p=0.05, s0=1, c=0.5 (dashed line) are considered enriched. Abbreviations: LFQ: Label free quantification


D ) Heatmap of LFQ intensity of proteins enriched at DNA baits . Telomere interactors are indicated on the right side of the heatmap . Color code indicates measured log2 LFQ intensities in the telomere pull - down . RNA binding proteins are marked with a black box on the left side of the figure . Abbreviations : LFQ : Label free quantification RBP : RNA binding

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

D) Heatmap of LFQ intensity of proteins enriched at DNA baits. Telomere interactors are indicated on the right side of the heatmap. Color code indicates measured log2 LFQ intensities in the telomere pull-down. RNA binding proteins are marked with a black box on the left side of the figure. Abbreviations: LFQ: Label free quantification RBP: RNA binding protein


E ) Critically short telomeres arise after 90 population doublings in the absence of telomerase ( tlc1 ) . Genomic DNA was extracted at indicated population doublings and digested with XhoI . A radioactive probe was used to recognize telomeric DNA in a southern blot . Abbreviations : PD : population doubling .

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E) Critically short telomeres arise after 90 population doublings in the absence of telomerase (tlc1). Genomic DNA was extracted at indicated population doublings and digested with XhoI. A radioactive probe was used to recognize telomeric DNA in a southern blot. Abbreviations: PD: population doubling.


F ) tlc1 cells reach senescence at PD 80 - 100 . % viability of indicated strains after propagation in YPD is indicated at different population doublings n = 3 . Asterisk indicates population doubling used in G ) to determine binding of Npl3 to short telomeres by TAP - ChIP . 3 independent spores from the indicated genotypes were used . Population doublings are counted from germination of meiotic products and hence begin at approx . PD40 . Abbreviations : TAP : tandem affinity purification

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F) tlc1 cells reach senescence at PD 80-100. %viability of indicated strains after propagation in YPD is indicated at different population doublings n=3. Asterisk indicates population doubling used in G) to determine binding of Npl3 to short telomeres by TAP-ChIP. 3 independent spores from the indicated genotypes were used. Population doublings are counted from germination of meiotic products and hence begin at approx. PD40. Abbreviations: TAP: tandem affinity purification tag


G ) Npl3 - TAP associates strongly to short telomeres . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . PD90 refers to 90 population doublings in the absence of TLC1 ( telomerase RNA subunit ) . The no tag control serves as an indicator of non - specific background signal . Abbreviations : TAP : tandem affinity purification tag , PD : population doubling .

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G) Npl3-TAP associates strongly to short telomeres. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). PD90 refers to 90 population doublings in the absence of TLC1 (telomerase RNA subunit). The no tag control serves as an indicator of non-specific background signal. Abbreviations: TAP: tandem affinity purification tag, PD: population doubling.


A ) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A , B and using tlc1 protein extracts . Interactors were identified using untreated protein extracts from tlc1 cells ( left panel ) or digested with RNase A and RNase H ( right panel ) .

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A) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A, B and using tlc1 protein extracts. Interactors were identified using untreated protein extracts from tlc1 cells (left panel) or digested with RNase A and RNase H (right panel).


B ) Npl3 associates to TERRA . Cross - linked samples from the indicated strains were subjected to Npl3 - TAP RIP . Enrichment of the indicated RNAs was determined by quantitative PCR on pulled - down reverse - transcribed RNA . Data represents mean % input + / - SEM n = 3 ( unpaired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 ) .

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B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01).


Npl3 associates strongly to telomeres in the absence of Rat1 . C ) The Rat1 - AID tagged protein is strongly degraded following 1h of 1mM IAA treatment . Protein levels are determined using anti - FLAG ( Rat1 - AID ) and anti - actin antibodies . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


Npl3 associates strongly to telomeres in the absence of Rat1 . D ) TERRA levels increase in cells expressing the Rat1 - AID variant after 1h treatment with 1mM IAA . TERRA levels were determined by quantitative PCR on reverse - transcribed RNAs after phenol - chloroform extraction . RNA levels are normalized to 7S ncRNA levels . Data represents fold increase to corresponding - IAA samples n = 2 . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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Npl3 associates strongly to telomeres in the absence of Rat1. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


Npl3 associates strongly to telomeres in the absence of Rat1 . E ) Npl3 - TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1 - AID . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Npl3 associates strongly to telomeres in the absence of Rat1. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


F - G ) Npl3 associates to telomeres in an R - loop dependent manner . E ) RNH1 was overexpressed from a galactose - inducible promoter . Protein levels are determined using anti - HA and anti - actin antibodies . F ) Npl3 - TAP associates to telomeres in an R - loop - dependent manner . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : eV : empty vector control , oE :

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F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: eV: empty vector control, oE: overexpression


A - B ) Npl3 associates to telomeres in early S . A ) Cell cycle profiles of indicated strains . Cells were synchronized in G1 with alpha - factor for 2 . 5h and released into the cell cycle at 30 ° C in pre - warmed medium supplemented with the indicated HU concentrations . B ) Npl3 - TAP associates strongly to telomeres in early S ( 250 mM HU ) . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM relative to cells arrested in alpha factor n = 3 ( paired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 Abbreviations : ns : not significant HU : hydroxyurea . * p < 0 . 05 , * * p < 0 .

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A-B) Npl3 associates to telomeres in early S. A) Cell cycle profiles of indicated strains. Cells were synchronized in G1 with alpha-factor for 2.5h and released into the cell cycle at 30°C in pre-warmed medium supplemented with the indicated HU concentrations. B) Npl3-TAP associates strongly to telomeres in early S (250 mM HU). Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to cells arrested in alpha factor n=3 (paired t-test two tailed *p<0.05, **p<0.01 Abbreviations: ns: not significant HU: hydroxyurea. *p<0.05, **p<0.01


C ) NPL3 over expression stabilizes telomeric R - loops . The indicated strains were grown on 1 % raffinose 2 % galactose to induce NPL3 over expression . Cross - linked samples were subjected to DRIP . DNA - RNA hybrids were immunoprecipitated using the S9 . 6 antibody . R - loop levels were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM relative to 250 mM HU eV n = 3 ( paired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations eV : empty vector control , oE : over expression HU : hydroxyurea . * p < 0 . 05 , * * p < 0 .

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C) NPL3 over expression stabilizes telomeric R-loops. The indicated strains were grown on 1% raffinose 2% galactose to induce NPL3 over expression. Cross-linked samples were subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to 250 mM HU eV n=3 (paired t-test two tailed *p<0.05, **p<0.01). Abbreviations eV: empty vector control, oE: over expression HU: hydroxyurea. *p<0.05, **p<0.01


D ) Deletion of NPL3 decreases telomeric R - loops . The indicated strains were crosslinked and subjected to DRIP . DNA - RNA hybrids were immunoprecipitated using the S9 . 6 antibody . R - loop levels were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 . Specificity of S9 . 6 antibody was determined treating a fraction of the samples with RNase H .

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D) Deletion of NPL3 decreases telomeric R-loops. The indicated strains were crosslinked and subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3. Specificity of S9.6 antibody was determined treating a fraction of the samples with RNase H.


E - G ) NPL3 stabilizes R - loops . E ) R - loop dot blot . The indicated strains were grown on 1 % raffinose 2 % galactose for 72h and subsequently released into 1 % raffinose 2 % galactose or 2 % glucose medium for 6 PDs . R - loop levels and dsDNA levels were determined using the S9 . 6 antibody and anti - dsDNA antibodies . Specificity of the S9 . 6 antibody was confirmed by treatment with RNase H . Loaded genomic DNA content is indicated at the bottom of the figure . F ) R - loop dot blot quantification . Data represents R - loop signal as the S9 . 6 antibody signal relative to corresponding dsDNA signal n = 3 ( unpaired t - test two tailed * p < 0 . 05 ) . G ) Serial dilutions of indicated strains were assayed on 1 % raffinose 2 % galactose media . Plates were imaged after 72h incubation at indicated temperatures . Abbreviations : V : empty vector control , oE : over expression , ds : double - strand ,

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E-G) NPL3 stabilizes R-loops. E) R-loop dot blot. The indicated strains were grown on 1% raffinose 2% galactose for 72h and subsequently released into 1% raffinose 2% galactose or 2% glucose medium for 6 PDs. R-loop levels and dsDNA levels were determined using the S9.6 antibody and anti-dsDNA antibodies. Specificity of the S9.6 antibody was confirmed by treatment with RNase H. Loaded genomic DNA content is indicated at the bottom of the figure. F) R-loop dot blot quantification. Data represents R-loop signal as the S9.6 antibody signal relative to corresponding dsDNA signal n=3 (unpaired t-test two tailed *p<0.05). G) Serial dilutions of indicated strains were assayed on 1% raffinose 2% galactose media. Plates were imaged after 72h incubation at indicated temperatures. Abbreviations: V: empty vector control, oE: over expression, ds: double-strand,


A ) Serial dilutions of indicated strains were assayed on YPD media . Cells were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

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A) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


B ) Serial dilutions of indicated strains were assayed on YPD media . Cells were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 48h growth at 30 ° C .

B) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 48h growth at 30°C.


C ) Serial dilutions of indicated strains were assayed on SC - His media . Cells were plated after 30 - 50 generations propagated on SC - His agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

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C) Serial dilutions of indicated strains were assayed on SC-His media. Cells were plated after 30-50 generations propagated on SC-His agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


Representative images of colon tumors were shown . Scale bars , 10 mm . Tumor number , tumor size and tumor load in each mouse was measured .

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Representative images of colon tumors were shown. Scale bars, 10 mm. Tumor number, tumor size and tumor load in each mouse was measured.


Histological analysis of colon tumors was shown by hematoxylin and eosin ( HE ) staining . Tumors were microscopically analyzed and classified as low or high grade . Histological score was assessed by a pathologist . Scale bars , 25 µm .

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Histological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm.


Tumor tissues were stained for Ki67 and p - STAT3 . The percentage of Ki67 - positive and p - STAT3 - positive cells was quantified . Scale bars , 25 µm .

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Tumor tissues were stained for Ki67 and p-STAT3. The percentage of Ki67-positive and p-STAT3-positive cells was quantified. Scale bars, 25 µm.


Proportion of CD11b + F4 / 80 + and RORγt + / CD45 + lin - cells was analyzed in colonic LP cells and mLNs among tumor - bearing WT and Dectin - 3 - / - mice by flow cytometry .

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Proportion of CD11b+F4/80+ and RORγt+/CD45+lin- cells was analyzed in colonic LP cells and mLNs among tumor-bearing WT and Dectin-3-/- mice by flow cytometry.


Relative expression of Il - 6 , Il - 22 , Cxcl1 and Il - 17a in LP cells from tumor - bearing WT and Dectin - 3 - / - mice were detected using qPCR .

Relative expression of Il-6, Il-22, Cxcl1 and Il-17a in LP cells from tumor-bearing WT and Dectin-3-/- mice were detected using qPCR.


Tumor number , tumor size , and tumor load in colons were measured .

Tumor number, tumor size, and tumor load in colons were measured.


Tumors were microscopically analyzed and classified as low or high grade . Histological score was assessed by a pathologist . Tumor tissues were stained for Ki67 . The percentages of Ki67 - positive tumor cells were quantified . Scale bars , 25 µm .

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Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Tumor tissues were stained for Ki67. The percentages of Ki67-positive tumor cells were quantified. Scale bars, 25 µm.


Mice were treated as described in Fig 1A and Fig 2A . Feces were collected from colons in each mouse . Total fungal burden in feces were quantified using qPCR .

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Mice were treated as described in Fig 1A and Fig 2A. Feces were collected from colons in each mouse. Total fungal burden in feces were quantified using qPCR.


Mice were treated as described in Fig 1A . Fungal ITS2 rDNA gene sequence was performed in each group . Fungal - taxon - based analysis at the phylum level in feces of mice . Color from blue to red indicates enrichment of fungus .

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Mice were treated as described in Fig 1A. Fungal ITS2 rDNA gene sequence was performed in each group. Fungal-taxon-based analysis at the phylum level in feces of mice. Color from blue to red indicates enrichment of fungus.


Specific fungal burden of C . albicans in the feces were quantified using qPCR .

Specific fungal burden of C.albicans in the feces were quantified using qPCR.


GF mice were orally gavaged with C . albicans ( twice a week , 1 × 107 ) during administration with AOM - DSS ( n = 8 , each group ) . Mice were euthanized on Day 100 , tumor load in each mouse were measured .

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GF mice were orally gavaged with C.albicans (twice a week, 1×107) during administration with AOM-DSS (n=8, each group). Mice were euthanized on Day 100, tumor load in each mouse were measured.


ELISA results for binding assays of Dectin - 3 with C . albicans . Plates were coated with C . albicans and then were added with 100 ml / well recombinant Dectin - 3 at indicated concentrations .

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ELISA results for binding assays of Dectin-3 with C. albicans. Plates were coated with C.albicans and then were added with 100 ml/well recombinant Dectin-3 at indicated concentrations.


GFP - C . albicans ( 5 × 106 ) were added onto 1 × 106 BMDMs , and incubated at 37 ° C for 1h . Wells were washed and fresh media containing fluconazole ( 300 μg / ml ) was added . At 6 hours and 18 hours , C . albicans CFU inside BMDMs were photographed and calculated by plating on YPD agar . Scale bars , 25 µm .

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GFP-C. albicans (5×106) were added onto 1×106 BMDMs, and incubated at 37°C for 1h. Wells were washed and fresh media containing fluconazole (300 μg/ml) was added. At 6 hours and 18 hours, C. albicans CFU inside BMDMs were photographed and calculated by plating on YPD agar. Scale bars, 25 µm.


Mice were treated as described in Fig . 2A . LP cells were isolated from each mouse and were culture for 48h . Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay . Color from blue to red indicates enrichment of gene expression .

Mice were treated as described in Fig.2A. LP cells were isolated from each mouse and were culture for 48h. Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay. Color from blue to red indicates enrichment of gene expression.


mRNA expressions of IL - 22 , β - defensin , Reg3g , and Cxcl1 in LP cells were detected by qPCR .

mRNA expressions of IL-22, β-defensin, Reg3g, and Cxcl1 in LP cells were detected by qPCR.


LP cells and mLNs were isolated from each mouse and were culture for 48h . Production of IL - 22 in LP cells and mLNs were detected by ELISA . LP cells were isolated from each mouse and were culture for 48h . Expression of Il - 17 in LP cells were detected by qPCR . Production of IL - 17 in LP cells were detected by ELISA .

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LP cells and mLNs were isolated from each mouse and were culture for 48h. Production of IL-22 in LP cells and mLNs were detected by ELISA. LP cells were isolated from each mouse and were culture for 48h. Expression of Il-17 in LP cells were detected by qPCR. Production of IL-17 in LP cells were detected by ELISA.


Protein level of p - STAT3 and STAT3 in colonic epithelial cells was detected by using Western Blot ( one mouse per lane ) . The relative expression of p - STAT3 was calculated .

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Protein level of p-STAT3 and STAT3 in colonic epithelial cells was detected by using Western Blot (one mouse per lane). The relative expression of p-STAT3 was calculated.


WT and Dectin - 3 - / - mice were intraperitoneally treated with anti - IL22 antibody or anti - IgG antibody as control during AOM - DSS administration ( n = 5 , each group ) . Mice were euthanized on Day 100 , tumor number , tumor size , and tumor load in colons were measured .

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WT and Dectin-3-/- mice were intraperitoneally treated with anti-IL22 antibody or anti-IgG antibody as control during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, tumor number, tumor size, and tumor load in colons were measured.


Mice were treated as described in Fig . 1A . LP cells were isolated from each mouse . The proportions of IL - 22 + cells in CD3 - RORγt + , CD3 - RORγt - , CD3 + RORγt - and CD3 + RORγt + were determined by flow cytometry .

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Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in CD3-RORγt+, CD3-RORγt-, CD3+RORγt- and CD3+RORγt+ were determined by flow cytometry.


Mice were treated as described in Fig . 1A . LP cells were isolated from each mouse . The proportions of IL - 22 + cells in NKp46 - RORγt + , NKp46 + RORγt - and NKp46 + RORγt + were determined by flow cytometry .

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Mice were treated as described in Fig.1A. LP cells were isolated from each mouse. The proportions of IL-22+ cells in NKp46-RORγt+, NKp46+RORγt- and NKp46+RORγt+ were determined by flow cytometry.


WT and Dectin - 3 - / - mice were intraperitoneally treated with anti - CD90 antibody or anti - IgG antibody during AOM - DSS administration ( n = 5 , each group ) . Mice were euthanized on Day 100 , feces were collected in colon from each mouse . Total fungal burden in feces were quantified using 18S rDNA qPCR .

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WT and Dectin-3-/- mice were intraperitoneally treated with anti-CD90 antibody or anti-IgG antibody during AOM-DSS administration (n=5, each group). Mice were euthanized on Day 100, feces were collected in colon from each mouse. Total fungal burden in feces were quantified using 18S rDNA qPCR.


Mice were treated as described in Fig . 4E and colonic LP cells were isolated . Numbers of IL - 22 + cells in LP cells were determined by flow cytometry .

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Mice were treated as described in Fig.4E and colonic LP cells were isolated. Numbers of IL-22+ cells in LP cells were determined by flow cytometry.


Mice were treated as described in Fig . 4E . Tumor number , tumor size , tumor load and histological score in colons were measured .

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Mice were treated as described in Fig.4E. Tumor number, tumor size, tumor load and histological score in colons were measured.


Mice were treated as described in Fig . 2A and Fig . 2I . Colonic LP cells were isolated . Proportion and absolute number of IL - 22 + cells in LP cells were detected by flow cytometry .

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Mice were treated as described in Fig.2A and Fig.2I. Colonic LP cells were isolated. Proportion and absolute number of IL-22+ cells in LP cells were detected by flow cytometry.


Primary macrophages were isolated from LP cells in WT and Dectin - 3 - / - tumor - bearing mice . mRNA expression of glycolysis - relative genes detected by using qPCR .

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Primary macrophages were isolated from LP cells in WT and Dectin-3-/- tumor-bearing mice. mRNA expression of glycolysis-relative genes detected by using qPCR.


BMDM cells were stimulated with C . albicans ( 5 × 106 ) , Curdlan , and α - mannan for 24h . Glucose uptake , pyruvate level , lactate production , and ATP level were determined using assay kit .

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BMDM cells were stimulated with C.albicans (5×106), Curdlan, and α-mannan for 24h. Glucose uptake, pyruvate level, lactate production, and ATP level were determined using assay kit.


BMDM cells were stimulated with C . albicans ( 5 × 106 ) for 24h , and ECAR and OCR were examined .

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BMDM cells were stimulated with C.albicans (5×106) for 24h, and ECAR and OCR were examined.


BMDMs acquired from WT and Dectin - 3 - / - mice were stimulated with C . albicans for 24h . Cytokine and chemokine production of BMDMs were detected using multiplex cytokine assay .

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BMDMs acquired from WT and Dectin-3-/- mice were stimulated with C.albicans for 24h. Cytokine and chemokine production of BMDMs were detected using multiplex cytokine assay.


WT - derived BMDMs were stimulated with C . albicans , curldan , or α - mannan in combination with or without 2 - DG ( 2 . 5mM ) for 24h . mRNA expression of Il - 7 were detected by using qPCR . BMDMs were acquired from WT and HIF - 1 - / - mice and were stimulated with C . albicans , curldan , or α - mannan for 24h . mRNA expression of Il - 7 were detected by using qPCR .

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WT-derived BMDMs were stimulated with C.albicans, curldan, or α-mannan in combination with or without 2-DG (2.5mM) for 24h. mRNA expression of Il-7 were detected by using qPCR. BMDMs were acquired from WT and HIF-1-/- mice and were stimulated with C.albicans, curldan, or α-mannan for 24h. mRNA expression of Il-7 were detected by using qPCR.


Primary ILC3 cells were sorted from colonic LP cells and were stimulated with indicated cytokines . Productions of IL - 22 were detected by ELISA . mRNA expressions of Il - 22 were detected by qPCR .

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Primary ILC3 cells were sorted from colonic LP cells and were stimulated with indicated cytokines. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 were detected by qPCR.


BMDMs were stimulated with C . albicans for 24h and the supernatant was isolated and added to primary ILC3 cells . ( 1 ) Supernatant from BMDMs without stimulation . ( 2 ) Supernatant from BMDMs with stimulation of C . albicans . ( 3 ) Supernatant from BMDMs with stimulation of C . albicans and IL - 7 antibody . Productions of IL - 22 by ILC3 cells were detected by ELISA . mRNA expressions of Il - 22 in ILC3 cells were detected by qPCR .

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BMDMs were stimulated with C.albicans for 24h and the supernatant was isolated and added to primary ILC3 cells. (1) Supernatant from BMDMs without stimulation. (2) Supernatant from BMDMs with stimulation of C.albicans. (3) Supernatant from BMDMs with stimulation of C.albicans and IL-7 antibody. Productions of IL-22 by ILC3 cells were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR.


Primary ILC3 cells were sorted from LPs and were stimulated with ( IL - 7 group ) or without IL - 7 ( None group ) . mRNA expressions of relative genes were detected by qPCR .

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Primary ILC3 cells were sorted from LPs and were stimulated with (IL-7 group) or without IL-7 (None group). mRNA expressions of relative genes were detected by qPCR.


Primary ILC3 cells were sorted in LPs from STAT3fl / fl and STAT3fl / fl VillinCre mice and were stimulated with IL - 7 . Productions of IL - 22 were detected by ELISA . mRNA expressions of Il - 22 in ILC3 cells were detected by qPCR .

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Primary ILC3 cells were sorted in LPs from STAT3fl/fl and STAT3fl/fl VillinCre mice and were stimulated with IL-7. Productions of IL-22 were detected by ELISA. mRNA expressions of Il-22 in ILC3 cells were detected by qPCR.


Primary ILC3 cells were sorted from LPs and were stimulated with IL - 7 in combined with FIZC and AhR Inh . Cells without stimulation were used as control ( None ) . Production of IL - 22 was detected by ELISA .

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Primary ILC3 cells were sorted from LPs and were stimulated with IL-7 in combined with FIZC and AhR Inh. Cells without stimulation were used as control (None). Production of IL-22 was detected by ELISA.


HEK293T cells were transiently transfected with the indicated expression constructs ( Ctl or AhR ) and were stimulated with ( IL - 7 group ) or without IL - 7 ( none group ) . ChIP analysis of the interaction of AhR and STAT3 to binding sites in the il22 promoter with or without IL - 7 , respectively .

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HEK293T cells were transiently transfected with the indicated expression constructs (Ctl or AhR) and were stimulated with (IL-7 group) or without IL-7 (none group). ChIP analysis of the interaction of AhR and STAT3 to binding sites in the il22 promoter with or without IL-7, respectively.


HEK293T cells were transiently transfected with the indicated expression constructs . Whole cell extracts were subjected to anti - HA immunoprecipitation , and were then immunoblotted with HA antibody and anti - STAT3 antibody .

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HEK293T cells were transiently transfected with the indicated expression constructs. Whole cell extracts were subjected to anti-HA immunoprecipitation, and were then immunoblotted with HA antibody and anti-STAT3 antibody.


EL4 cells were transduced with retroviruses encoding AhR and STAT3 . IL - 22 mRNA expression was analyzed by qPCR .

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EL4 cells were transduced with retroviruses encoding AhR and STAT3. IL-22 mRNA expression was analyzed by qPCR.


Patients with colon cancer were divided into four groups based on tumor stage . mRNA expressions of Dectin - 3 in tumor tissues were detected by qPCR . The box represents the 25th to 75th percentile , and the whisker plots represent the minimum and maximum percentiles . Patients with colon cancer were divided into two groups based on fungal burden in the feces . mRNA expressions of Dectin - 3 was compared between these two groups .

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Patients with colon cancer were divided into four groups based on tumor stage. mRNA expressions of Dectin-3 in tumor tissues were detected by qPCR. The box represents the 25th to 75th percentile, and the whisker plots represent the minimum and maximum percentiles. Patients with colon cancer were divided into two groups based on fungal burden in the feces. mRNA expressions of Dectin-3 was compared between these two groups.


Overall survival rate was shown by K - M survival curve .

Overall survival rate was shown by K-M survival curve.


Tumor tissues were stained for IL - 22 and p - STAT3 . The percentages of IL - 22 - positive and p - STAT3 - positive cells were quantified . mRNA expressions of IL22 and STAT3 were detected by qPCR . Scale bars , 25 µm .

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Tumor tissues were stained for IL-22 and p-STAT3. The percentages of IL-22-positive and p-STAT3-positive cells were quantified. mRNA expressions of IL22 and STAT3 were detected by qPCR. Scale bars, 25 µm.


B . Blood stage growth rate of mutated parasite lines . A single parasite was injected i . v . into a mouse and the growth rate was calculated from parasitemia at day 6 - 9 for each mouse ( black dots ) . Bars represent the mean of 4 - 7 mice . Significance for ( B ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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B. Blood stage growth rate of mutated parasite lines. A single parasite was injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9 for each mouse (black dots). Bars represent the mean of 4-7 mice. Significance for (B) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


C . Infected midgut showing oocysts stained with mercurochrome ; scale bar , 100 μm . The graph shows oocyst numbers per infected mosquito . Numbers of mosquitoes that were analyzed are indicated above the graph . Data derived from at least two independent cage feeds originating from independently infected mice . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance for ( C ) determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

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C. Infected midgut showing oocysts stained with mercurochrome; scale bar, 100 μm. The graph shows oocyst numbers per infected mosquito. Numbers of mosquitoes that were analyzed are indicated above the graph. Data derived from at least two independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (C) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


D . Ratio of salivary gland to midgut sporozoites of the indicated parasite lines . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Significance for , ( D ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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D. Ratio of salivary gland to midgut sporozoites of the indicated parasite lines. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for , (D) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


E . Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging . Arrow indicates direction of a motile sporozoite . The graph shows the fraction of motile hemolymph sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Significance for ( E ) determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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E. Hemolymph sporozoites were isolated and observed on glass slides by live cell imaging. Arrow indicates direction of a motile sporozoite. The graph shows the fraction of motile hemolymph sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Significance for (E) determined by One-way analysis of variance with Tukeys Multiple Comparison test.


F . qRT - PCR analysis of schizonts and oocysts of the PbMyoA 3 ' dhfs parasite line . Individual data points correspond to the mean of three technical replicates .

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F. qRT-PCR analysis of schizonts and oocysts of the PbMyoA 3'dhfs parasite line. Individual data points correspond to the mean of three technical replicates.


C . Blood stage growth rate of clonal parasite lines . A single parasite or 100 iRBCs were injected i . v . into a mouse and the growth rate was calculated from parasitemia at day 6 - 9 . Black dots : individual mice ; bars : mean . Statistical analysis : ( C ) One - way analysis of variance with Tukeys multiple comparison test .

C. Blood stage growth rate of clonal parasite lines. A single parasite or 100 iRBCs were injected i.v. into a mouse and the growth rate was calculated from parasitemia at day 6-9. Black dots: individual mice; bars: mean. Statistical analysis: (C) One-way analysis of variance with Tukeys multiple comparison test.


D . Proportion of motile ookinetes . Black dots : Individual movie ; bars : mean . Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment . Statistical analysis : ( D ) Kruskal - Wallis test with ( D ) Dunn ' s multiple comparisons

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D. Proportion of motile ookinetes. Black dots: Individual movie; bars: mean. Data pooled from three independent biological replicates with 1 - 3 movies taken per experiment. Statistical analysis: (D) Kruskal-Wallis test with (D) Dunn's multiple comparisons test


E . Speed of ookinetes of the different lines . Numbers of analysed parasites are shown above the graph . Data pooled from three independent experiments ( biological replicates ) . Violin plots show median ( line ) and quartiles ( dashed lines ) .

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E. Speed of ookinetes of the different lines. Numbers of analysed parasites are shown above the graph. Data pooled from three independent experiments (biological replicates). Violin plots show median (line) and quartiles (dashed lines).


F . Number of oocysts per infected mosquito . Numbers of analyzed mosquitoes are shown above the graph . Data pooled from at least three independent cage feeds originating from independently infected mice . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Statistical analysis : ( F ) Kruskal - Wallis test with ( F ) Bonferroni ' s Multiple Comparison test .

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F. Number of oocysts per infected mosquito. Numbers of analyzed mosquitoes are shown above the graph. Data pooled from at least three independent cage feeds originating from independently infected mice. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Statistical analysis: (F) Kruskal-Wallis test with (F) Bonferroni's Multiple Comparison test.


A . Ratio of hemolymph to midgut sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . B . Ratio of salivary gland to midgut sporozoites . Black dots : individual experiments with 10 - 20 mosquitoes . Bars : mean . Statistical analysis : ( A ) and ( B ) One - way analysis of variance with Tukeys Multiple Comparison test .

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A. Ratio of hemolymph to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. B. Ratio of salivary gland to midgut sporozoites. Black dots: individual experiments with 10-20 mosquitoes. Bars: mean. Statistical analysis: (A) and (B) One-way analysis of variance with Tukeys Multiple Comparison test.


C . Blood stage infection of mice after transmission by mosquito bite . Shown is the mean ± standard deviation . D . Blood stage infection of mice after i . v . injection of 1000 sporozoites .

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C. Blood stage infection of mice after transmission by mosquito bite. Shown is the mean ± standard deviation. D. Blood stage infection of mice after i.v. injection of 1000 sporozoites.


AFraction ( left ) and speed ( right ) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance for ( A left ) and ( B left ) determined by One - way analysis of variance with Tukeys Multiple Comparison test . Significance for ( A right ) and ( B right ) determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

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AFraction (left) and speed (right) of motile hemolymph sporozoites on glass . Dots correspond to individual experiments and bars indicate the mean. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance for (A left) and (B left) determined by One-way analysis of variance with Tukeys Multiple Comparison test. Significance for (A right) and (B right) determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min . Scale bar , 10 μm . D . Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min . Scale bar , 10 μm .

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C. Selected trajectories of 20 manually tracked sporozoites isolated from hemolymph and moving over a period of 3 min. Scale bar, 10 μm. D. Selected trajectories of 20 manually tracked sporozoites isolated from salivary glands and moving over a period of 3 min. Scale bar, 10 μm.


A . Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap . The bead is placed at the apical end of the sporozoite . The graph shows the fraction of sporozoites that pulled the bead out of the optical trap . Black dots : individual experiments . Bars : mean . 73 - 131 sporozoites were probed for each line . Significance determined by One - way analysis of variance with Tukeys Multiple Comparison test .

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A. Scheme depicting the setup of optical tweezers for measuring the forces that a sporozoite can generate to pull a bead out of an optical trap. The bead is placed at the apical end of the sporozoite. The graph shows the fraction of sporozoites that pulled the bead out of the optical trap. Black dots: individual experiments. Bars: mean. 73-131 sporozoites were probed for each line. Significance determined by One-way analysis of variance with Tukeys Multiple Comparison test.


B . Scheme showing a sporozoite moving through a polymeric network in 3D . The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel . Box ‐ and ‐ whisker plots depict the 25 % quantile , median , 75 % quantile , and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Significance determined by Kruskal - Wallis test with Bonferroni ' s Multiple Comparison test .

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B. Scheme showing a sporozoite moving through a polymeric network in 3D. The graph shows sporozoite speeds as measured by manual tracking from imaging of sporozoites in a 3D hydrogel. Box‐and‐whisker plots depict the 25% quantile, median, 75% quantile, and nearest observations within 1.5 times the interquartile range (whiskers). Significance determined by Kruskal-Wallis test with Bonferroni's Multiple Comparison test.


C . Selected trajectories of 20 moving sporozoites observed over a period of 3 min . Scale bar , 10 μm .

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C. Selected trajectories of 20 moving sporozoites observed over a period of 3 min. Scale bar, 10 μm.


A Representative pictures of embryos at 18 . 5 days postcoitum ( E18 . 5 ) and hematoxylin and eosin staining of whole - mount embryo sections from PR - SET7loxp / Alfp - Cre ( PR - SET7ΔHepE ) mice and control littermates ( WT ) .

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A Representative pictures of embryos at 18.5 days postcoitum (E18.5) and hematoxylin and eosin staining of whole-mount embryo sections from PR-SET7loxp/Alfp-Cre (PR-SET7ΔHepE) mice and control littermates (WT).


B Hematoxylin and eosin staining of liver sections .

B Hematoxylin and eosin staining of liver sections.


C Immunohistochemical staining of liver sections with albumin antibody .

C Immunohistochemical staining of liver sections with albumin antibody.


D qPCR analysis of Albumin , Afp and HNF4 mRNA levels . Bars represent mean values of mRNA levels normalized to glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) mRNA and SEM from samples of four individual mice . * P - value < 0 . 01 .

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D qPCR analysis of Albumin,Afp and HNF4 mRNA levels. Bars represent mean values of mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and SEM from samples of four individual mice. *P-value < 0.01.


E Immunohistochemical staining of liver sections with cyclin B1 antibody .

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E Immunohistochemical staining of liver sections with cyclin B1 antibody.


F Immunohistochemical staining of liver sections with γH2AX antibody .

F Immunohistochemical staining of liver sections with γH2AX antibody.


A Macroscopic appearance of livers in 120 - day - old ( P120 ) wild - type ( WT ) and PR - SET7loxp / Alb - Cre ( KO ) mice . Note , small adenomatous foci in KO livers .

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A Macroscopic appearance of livers in 120-day-old (P120) wild-type (WT) and PR-SET7loxp/Alb-Cre (KO) mice. Note, small adenomatous foci in KO livers.


B Representative hematoxylin and eosin staining of liver sections from P120 wild - type ( WT ) and PR - SET7loxp / Alb - Cre ( PR - SET7ΔHepA ) mice . Arrows show three areas containing morphologically different hepatocytes . Right panels : zoom - in to Area - A - ' normal zone ' , to Area - B - ' necrotic zone ' and to Area - C - ' regenerative zone ' .

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B Representative hematoxylin and eosin staining of liver sections from P120 wild-type (WT) and PR-SET7loxp/Alb-Cre (PR-SET7ΔHepA) mice. Arrows show three areas containing morphologically different hepatocytes. Right panels: zoom-in to Area-A-'normal zone', to Area-B-'necrotic zone' and to Area-C-'regenerative zone'.


C Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with HNF4 antibody .

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C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with HNF4 antibody.


D TUNEL staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) . Note that cells containing enlarged nuclei ( white arrows ) are TUNEL negative .

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D TUNEL staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT). Note that cells containing enlarged nuclei (white arrows) are TUNEL negative.


E Immunohistological staining with γH2AX and albumin ( Alb ) antibodies .

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E Immunohistological staining with γH2AX and albumin (Alb) antibodies.


A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR - SET7ΔHepA livers and normal hepatocytes in control littermates ( WT ) . White arrows indicate : ( Nuc ) nuclei ; ( ER ) endoplasmic reticulum ; ( Mit ) mitochondrium ; ( PM ) plasma membrane ; ( a ) swollen mitochondria ; ( b ) swollen endoplasmic reticulum ; ( c ) disorganized endoplasmic reticulum ; ( d ) disrupted plasma membrane ; and ( e ) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes .

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A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR-SET7ΔHepA livers and normal hepatocytes in control littermates (WT). White arrows indicate: (Nuc) nuclei; (ER) endoplasmic reticulum; (Mit) mitochondrium; (PM) plasma membrane; (a) swollen mitochondria; (b) swollen endoplasmic reticulum; (c) disorganized endoplasmic reticulum; (d) disrupted plasma membrane; and (e) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes.


B Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with H4K20Me1 and H4K20Me3 antibodies . Normal ( Area - A ) , Necrotic ( Area - B ) and Regenerative ( Area - C ) hepatocyte - containing areas are indicated . Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei .

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B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with H4K20Me1 and H4K20Me3 antibodies. Normal (Area-A), Necrotic (Area-B) and Regenerative (Area-C) hepatocyte-containing areas are indicated. Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei.


C - F Partial ( 2 / 3rd ) hepatectomy was performed in 45 - day - old PR - SET7ΔHepA mice and control littermates ( WT ) . Two weeks later , at postnatal day 60 ( P60 ) liver sections were stained with hematoxylin and eosin ( C ) or γH2AX ( D ) or H4K20Me1 ( E ) or HNF4 and F4 / 80 ( F ) antibodies .

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C-F Partial (2/3rd) hepatectomy was performed in 45-day-old PR-SET7ΔHepA mice and control littermates (WT). Two weeks later, at postnatal day 60 (P60) liver sections were stained with hematoxylin and eosin (C) or γH2AX (D) or H4K20Me1 (E) or HNF4 and F4/80 (F) antibodies.


A , B Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with Ki67 , albumin ( Alb ) and A6 or HNF4 antibodies as indicated . Arrows depict the different areas in the sections as indicated above ( Fig . 1B ) . Asterisks ( * ) indicate Ki67 + hepatocytes . Small arrows ( > ) indicate Ki67 + non - hepatic cells . Note the lack of co - staining of cells with HNF4 and A6 antibodies .

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A, B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with Ki67, albumin (Alb) and A6 or HNF4 antibodies as indicated. Arrows depict the different areas in the sections as indicated above (Fig.1B). Asterisks (*) indicate Ki67+ hepatocytes. Small arrows (>) indicate Ki67+ non-hepatic cells. Note the lack of co-staining of cells with HNF4 and A6 antibodies.


C Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with FGF7 and A6 antibodies .

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C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with FGF7 and A6 antibodies.


D Relative mRNA levels of FGF7 . Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120 - day - old mice . The data are presented as fold over values obtained with wild - type P45 samples . * P - value < 0 . 01 .

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D Relative mRNA levels of FGF7. Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120-day-old mice. The data are presented as fold over values obtained with wild-type P45 samples. *P-value < 0.01.


A Analysis of reactive oxygen species ( ROS ) accumulation in frozen liver sections from 120 - day - old ( P120 ) and 240 - day - old ( P240 ) PR - SET7ΔHepA mice and control littermates ( WT ) .

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A Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT).


B Immunohistological staining of liver sections from P240 animals with phospho - STAT3 antibody .

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B Immunohistological staining of liver sections from P240 animals with phospho-STAT3 antibody.


C Western blot analysis of liver extracts from P45 , P120 and P240 mice with STAT3 and phospho - STAT3 antibodies .

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C Western blot analysis of liver extracts from P45, P120 and P240 mice with STAT3 and phospho-STAT3 antibodies.


D Macroscopic appearance of livers in P240 PR - SET7ΔHepA mice and control littermates ( WT ) .

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D Macroscopic appearance of livers in P240 PR-SET7ΔHepA mice and control littermates (WT).


E Serum alanine aminotransferase ( ALT ) levels in P45 , P120 and P240 mice . Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice . * P - value < 0 . 01 .

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E Serum alanine aminotransferase (ALT) levels in P45, P120 and P240 mice. Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice. *P-value < 0.01.


F Representative hematoxylin and eosin staining of liver sections from P240 PR - SET7ΔHepA mice and control littermates ( WT ) . Two different magnifications are shown .

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F Representative hematoxylin and eosin staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT). Two different magnifications are shown.


G Relative mRNA levels of selected oncofetal genes . Bars represent mean Afp , H19 , GPC - 3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240 - day - old mice . The data are presented as fold over values obtained with wild - type samples . * P - value < 0 . 01 .

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G Relative mRNA levels of selected oncofetal genes. Bars represent mean Afp, H19, GPC-3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240-day-old mice. The data are presented as fold over values obtained with wild-type samples. *P-value < 0.01.


A , B Immunohistological staining of liver sections from P240 PR - SET7ΔHepA mice and control littermates ( WT ) with Afp and HNF4 ( A ) or Ki - 67 and albumin ( Alb ) ( B ) antibodies .

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A, B Immunohistological staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT) with Afp and HNF4 (A) or Ki-67 and albumin (Alb) (B) antibodies.


C - E Double staining of liver sections with A6 and HNF4 ( C ) or Sox9 and Alb ( D ) or CD133 and HNF4 ( E ) antibodies . Right panels show larger magnifications of stainings performed in PR - SET7ΔHepA liver sections .

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C-E Double staining of liver sections with A6 and HNF4 (C) or Sox9 and Alb (D) or CD133 and HNF4 (E) antibodies. Right panels show larger magnifications of stainings performed in PR-SET7ΔHepA liver sections.


A P240 PR - SET7ΔHepA liver sections were double - stained with A6 and Sox9 ( upper panel ) or CD133 and Sox9 ( lower panel ) . Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI - stained cells in 5 high - power fields ( HPF ) in liver sections of 3 different mice .

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A P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice.


B Cell duplication rate of primary hepatocytes from P240 PR - SET7ΔHepA livers in culture .

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B Cell duplication rate of primary hepatocytes from P240 PR-SET7ΔHepA livers in culture.


C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR - SET7ΔHepA livers or Hepa 1 - 6 cells . Right panel shows average tumor volumes ( n = 3 ) dissected at the indicated time points following injection .

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C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers or Hepa 1-6 cells. Right panel shows average tumor volumes (n = 3) dissected at the indicated time points following injection.


D , E Hematoxylin and eosin and immunohistological staining with A6 , HNF4 , Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR - SET7ΔHepA livers .

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D, E Hematoxylin and eosin and immunohistological staining with A6, HNF4, Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers.


B . T6SS secretion assay of A . tumefaciens strains : wild type C58 , various mutants lacking one , two , or three toxin - immunity gene pairs , and a mutant lacking tssL .

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B. T6SS secretion assay of A. tumefaciens strains: wild type C58, various mutants lacking one, two, or three toxin-immunity gene pairs, and a mutant lacking tssL.


C . T6SS secretion assay of various A . tumefaciens strains : wild type C58 , ΔtssL , the tde double deletion mutant ( Δtdei ) containing pRL662 and pTrc200 empty vector ( V ) only or expression of pTdei1 ( tde1 - tdi1 expressed from pTrc200 ) , pTde2 * ( catalytic site mutated tde2 expressed on pRL662 ) , or pTdei1 + pTde2 * .

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C. T6SS secretion assay of various A. tumefaciens strains: wild type C58, ΔtssL, the tde double deletion mutant (Δtdei) containing pRL662 and pTrc200 empty vector (V) only or expression of pTdei1 (tde1-tdi1 expressed from pTrc200), pTde2* (catalytic site mutated tde2 expressed on pRL662), or pTdei1+ pTde2*.


D . A . tumefaciens antibacterial activity assay against E . coli . The strains of A . tumefaciens were co - cultured at a ratio of 30 : 1 with E . coli DH10B ( + pRL662 ) on LB agar . The survival of target E . coli cells was quantified by counting CFUs on gentamicin - containing LB agar plates . Data represent mean ± SEM of 6 biological replicates from three independent experiments . One - way ANOVA followed by Turkey HSD test was used for statistical analysis . Two groups with significant differences ( P = 0 . 0010053 ) are indicated with different letters ( a and b ) .

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

D. A. tumefaciens antibacterial activity assay against E. coli. The strains of A. tumefaciens were co-cultured at a ratio of 30:1 with E. coli DH10B (+ pRL662) on LB agar. The survival of target E. coli cells was quantified by counting CFUs on gentamicin-containing LB agar plates. Data represent mean ± SEM of 6 biological replicates from three independent experiments. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Two groups with significant differences (P=0.0010053) are indicated with different letters (a and b).


A . T6SS secretion assay of A . tumefaciens strains : wild type C58 , ΔtssL , Δtap - 1 , and Δatu3641 harboring a pTrc200 vector ( V ) or derivatives pTap - 1 ( tap - 1 expressed from pTrc200 ) , or p3641 ( atu3641 expressed from pTrc200 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O

A. T6SS secretion assay of A. tumefaciens strains: wild type C58, ΔtssL, Δtap-1, and Δatu3641 harboring a pTrc200 vector (V) or derivatives pTap-1 (tap-1 expressed from pTrc200), or p3641 (atu3641 expressed from pTrc200).


B . T6SS secretion assay of A . tumefaciens C58 Δtap - 1Δatu3641 containing an empty pTrc200 vector ( V ) or its derivatives expressing tap - 1 , atu3641 , tde1 - tdi1 , or tde2 - tdi2 .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O

B. T6SS secretion assay of A. tumefaciens C58 Δtap-1Δatu3641 containing an empty pTrc200 vector (V) or its derivatives expressing tap-1, atu3641, tde1-tdi1, or tde2-tdi2.


C . T6SS secretion assay of A . tumefaciens C58ΔG2op mutant encoding full - length or truncated VgrG1 proteins .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

C. T6SS secretion assay of A. tumefaciens C58ΔG2op mutant encoding full-length or truncated VgrG1 proteins.


A . Representative images of cells of A . tumefaciens C58 strains ΔtssB , ΔtssLΔtssB , and ΔtdeiΔtssB cells each expressing TssB - GFP from pTrc200 . Upper panel : phase contrast images ; Lower panel : green fluorescence images . Examples of GFP streaks were indicated by white arrowheads . Scale bar : 2 μm .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative images of cells of A. tumefaciens C58 strains ΔtssB, ΔtssLΔtssB, and ΔtdeiΔtssB cells each expressing TssB-GFP from pTrc200. Upper panel: phase contrast images; Lower panel: green fluorescence images. Examples of GFP streaks were indicated by white arrowheads. Scale bar: 2 μm.


B . Quantification of TssB - GFP foci in different genetic background . The number of the fluorescent foci were counted using the " analysis particle " function in Fiji with a manually set threshold . The number of cells were counted manually . For each strain , a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis . One - way ANOVA followed by Turkey HSD test was used for statistical analysis . Data are mean ± SEM and two groups with significant differences ( P = 0 . 000272 ) are indicated with different letters ( a and b ) . Similar results are reproduced in two independent experiments .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of TssB-GFP foci in different genetic background. The number of the fluorescent foci were counted using the "analysis particle" function in Fiji with a manually set threshold. The number of cells were counted manually. For each strain, a total of three images were obtained from one independent experiment and each image contained more than 300 cells for quantification and statistical analysis. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Data are mean ± SEM and two groups with significant differences (P=0.000272) are indicated with different letters (a and b). Similar results are reproduced in two independent experiments.


C . Western blots of the isolated sheaths , which were prepared via ultracentrifugation of cell lysates from A . tumefaciens C58 wild type , ΔtssL , and Δtdei . T : total proteins from the cell lysate . P : pellet samples containing TssBC sheaths after ultracentrifugation . S : supernatant after ultracentrifugation . Proteins were analyzed in western blots probed with indicated antibodies . Fha serves as a cytoplasmic control . Molecular weight markers ( in kDa ) are indicated on the left . Similar results were obtained from at least two independent experiments . Data are from one independent experiment and reproduced in at least two independent experiments .

O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blots of the isolated sheaths, which were prepared via ultracentrifugation of cell lysates from A. tumefaciens C58 wild type, ΔtssL, and Δtdei. T: total proteins from the cell lysate. P: pellet samples containing TssBC sheaths after ultracentrifugation. S: supernatant after ultracentrifugation. Proteins were analyzed in western blots probed with indicated antibodies. Fha serves as a cytoplasmic control. Molecular weight markers (in kDa) are indicated on the left. Similar results were obtained from at least two independent experiments. Data are from one independent experiment and reproduced in at least two independent experiments.


D . Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy ( TEM ) . Sheaths are indicated by red arrowheads and flagella , which are distinguishable on the basis of smoother , more solid , and thinner structures ( ~ 13 nm in width ) , are indicated by green arrowheads [ 33 ] . Data are from one independent experiment and reproduced in at least two independent experiments . Scale bar : 0 . 5 μm ( upper panel ) , 100 nm ( upper panel ) .

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

D. Pellet samples of wild type and Δtdei were visualized with transmission electron microscopy (TEM). Sheaths are indicated by red arrowheads and flagella, which are distinguishable on the basis of smoother, more solid, and thinner structures (~13 nm in width), are indicated by green arrowheads [33]. Data are from one independent experiment and reproduced in at least two independent experiments. Scale bar: 0.5 μm (upper panel), 100 nm (upper panel).


B . Secretion assay of wild type and various mutants of 12D1 , 1D1108 and 15955 .

B. Secretion assay of wild type and various mutants of 12D1, 1D1108 and 15955.


C . Antibacterial activity assay of wild type and mutants of 12D1 , 1D1108 and 15955 was carried out in a ratio of 30 : 1 against E . coli harboring the plasmid pRL662 . The target E . coli cells were serially - diluted and grown overnight on gentamicin - containing LB agar prior to photographing . Each competition was done at least four times and reproduced in three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Antibacterial activity assay of wild type and mutants of 12D1, 1D1108 and 15955 was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in three independent experiments.


D . Secretion assay of wild type 12D1 , ΔtssL , Δv1 , Δv3 - 4 containing pRL662 empty vector ( V ) or its derivatives with v1 or v3 - 4 were analyzed for secretion .

O O O O O 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-CONTROLLED_VAR O O O O O O O

D. Secretion assay of wild type 12D1, ΔtssL, Δv1, Δv3-4 containing pRL662 empty vector (V) or its derivatives with v1 or v3-4 were analyzed for secretion.


E . Secretion assay of wild type 1D1108 , ΔtssL , Δv4 , Δv6 containing pRL662 empty vector ( V ) or its derivatives with v4 or v6 .

O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O

E. Secretion assay of wild type 1D1108, ΔtssL, Δv4, Δv6 containing pRL662 empty vector (V) or its derivatives with v4 or v6.


A . T6SS secretion assay of A . tumefaciens wild type C58 , ΔtssL , and various single , double , and triple toxin - immunity deletion strains .

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

A. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, and various single, double, and triple toxin-immunity deletion strains.


B . T6SS secretion assay of A . tumefaciens wild type C58 , ΔtssL , Δtdei , and Δ3TIs harboring the indicated plasmids .

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. T6SS secretion assay of A. tumefaciens wild type C58, ΔtssL, Δtdei, and Δ3TIs harboring the indicated plasmids.


A . T6SS secretion assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains .

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

A. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains.


B . T6SS secretion assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains harboring the indicated plasmids .

O O O 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. T6SS secretion assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids.


C . Antibacterial activity assay of A . tumefaciens 12D1 wild type , ΔtssL , and various toxin - immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30 : 1 against E . coli harboring the plasmid pRL662 . The target E . coli cells were serially - diluted and grown overnight on gentamicin - containing LB agar prior to photographing . Each competition was done at least four times and reproduced in two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Antibacterial activity assay of A. tumefaciens 12D1 wild type, ΔtssL, and various toxin-immunity deletion strains harboring the indicated plasmids was carried out in a ratio of 30:1 against E. coli harboring the plasmid pRL662. The target E. coli cells were serially-diluted and grown overnight on gentamicin-containing LB agar prior to photographing. Each competition was done at least four times and reproduced in two independent experiments.


D . T6SS secretion assay of A . tumefaciens C58 wild type , ΔtssL , and various mutant strains .

D. T6SS secretion assay of A. tumefaciens C58 wild type, ΔtssL, and various mutant strains.


( a ) Intracellular levels of spermidine ( Spd ) in chronologically ageing wild - type yeast . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 and * P 0 . 001 ) .

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

(a) Intracellular levels of spermidine (Spd) in chronologically ageing wild-type yeast. Data represent means ± s.e.m. (n = 3; *P 0.05 and *P 0.001).


( b ) Survival determined by clonogenicity during chronological ageing of wild - type yeast ( BY4741 ) cells with ( o ) and without ( ▪ ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival determined by clonogenicity during chronological ageing of wild-type yeast (BY4741) cells with (o) and without (▪) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 5).


( c ) Intracellular levels of spermidine in chronologically ageing wild - type yeast cells cultured with ( open bar ) or without ( closed bar ) spermidine ( 4 mM ) for 5 days . Data represent means ± s . e . m . ( n = 3 ; * * P 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Intracellular levels of spermidine in chronologically ageing wild-type yeast cells cultured with (open bar) or without (closed bar) spermidine (4 mM) for 5 days. Data represent means ± s.e.m. (n = 3; **P 0.001).


( d ) Replicative lifespan analysis of BY4741 wild - type yeast cells after separation into old ( fraction V ) and young ( fraction II ) cells by elutriation centrifugation . The remaining lifespan with or without ( control ) spermidine ( 1 mM , applied after elutriation ) on 2 % glucose synthetic complete medium is shown .

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

(d) Replicative lifespan analysis of BY4741 wild-type yeast cells after separation into old (fraction V) and young (fraction II) cells by elutriation centrifugation. The remaining lifespan with or without (control) spermidine (1 mM, applied after elutriation) on 2% glucose synthetic complete medium is shown.


( e ) Survival determined by the age - specific number of dead individuals of female Drosophila with and without ( control ) supplementation of food with various concentrations of spermidine ( as indicated ) . A representative ageing experiment of at least 50 flies per sample is shown .

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

(e) Survival determined by the age-specific number of dead individuals of female Drosophila with and without (control) supplementation of food with various concentrations of spermidine (as indicated). A representative ageing experiment of at least 50 flies per sample is shown.


( f ) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine , compared with normal food ( control ) . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 01 ) .

(f) Endogenous spermidine of female Drosophila fed for 48 h with food supplemented by 1 mM spermidine, compared with normal food (control). Data represent means ± s.e.m. (n = 3; *P 0.01).


( g ) Survival determined by annexin V / 7 - AAD co - staining ( unstained cells were considered as viable ) of human immune cells ( PBMC ) cultured for 6 and 12 days in the absence ( black bar ) or presence ( white bars ) of various spermidine concentrations ( as indicated ) . Data represent means ± s . e . m . of 3 independent experiments ( each performed on PBMC from different donors ) . * P 0 . 05 and * * P 0 . 01 .

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

(g) Survival determined by annexin V/7-AAD co-staining (unstained cells were considered as viable) of human immune cells (PBMC) cultured for 6 and 12 days in the absence (black bar) or presence (white bars) of various spermidine concentrations (as indicated). Data represent means ± s.e.m. of 3 independent experiments (each performed on PBMC from different donors). *P 0.05 and **P 0.01.


( h , i ) Free thiol group ( RSH ) concentration in serum ( h ) and intracellular spermidine concentration in hepatocytes ( i ) of ageing mice with ( open bars ) or without ( closed bar ) supplementation of drinking water with spermidine ( 0 . 3 and 3 mM ) for 200 days . Data represent mean ± s . e . m . ( n = 3 ; * P 0 . 05 and * * P 0 . 01 ) .

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

(h, i) Free thiol group (RSH) concentration in serum (h) and intracellular spermidine concentration in hepatocytes (i) of ageing mice with (open bars) or without (closed bar) supplementation of drinking water with spermidine (0.3 and 3 mM) for 200 days. Data represent mean ± s.e.m. (n = 3; *P 0.05 and **P 0.01).


( a ) Intracellular spermidine of five - day - oldΔspe1 cells , compared with wild - type cells . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

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

(a) Intracellular spermidine of five-day-oldΔspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001).


( b ) Chronological ageing of wild - type ( ▪ , o ) and polyamine - depleted Δspe1 ( ▴ , ▿ ) yeast cells with ( open symbols ) and without ( closed symbols ) supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ) . Cells were tested for cell death markers at day 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3.


( c ) Fluorescence microscopy of DHE → Eth conversion in wild - type and Δspe1 cells indicating ROS production . Scale bars , 10 μm .

(c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm.


( d ) Quantification ( FACS analysis ) of ROS accumulation using DHE → Eth conversion of wild - type ( WT ) and Δspe1 cells with and without supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) .

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

(d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001).