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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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.


( A ) HEK GFP - LC3 cells were transfected with siRNA pools targeting PX domain proteins and starved ( large circles ) or not starved ( small circles ) for 2 h , followed by fixation and counterstaining of the nuclei by Hoechst . The images were processed for high - content image analysis to quantify the number of GFP - LC3 spots per cell . The graph shows the average of

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK GFP-LC3 cells were transfected with siRNA pools targeting PX domain proteins and starved (large circles) or not starved (small circles) for 2 h, followed by fixation and counterstaining of the nuclei by Hoechst. The images were processed for high-content image analysis to quantify the number of GFP-LC3 spots per cell. The graph shows the average of three


( B ) Selected PX domain proteins were depleted by transfection of HEK GFP - LC3 with the four individual siRNA oligos of the pools used in A . The cells were treated , imaged , and analyzed as in A . The area of each circle corresponds to the relative amount of target mRNA as measured by qPCR . The graph shows the average of two independent experiments in triplicate with a total number of 18 , 000 cells . Coloring is as in A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Selected PX domain proteins were depleted by transfection of HEK GFP-LC3 with the four individual siRNA oligos of the pools used in A. The cells were treated, imaged, and analyzed as in A. The area of each circle corresponds to the relative amount of target mRNA as measured by qPCR. The graph shows the average of two independent experiments in triplicate with a total number of 18,000 cells. Coloring is as in A.


( C ) Micrographs of starved HEK GFP - LC3 cells treated with nontargeting or siSNX18 - 3 siRNA . ( left and middle ) 3 × 3 fields of cells . ( right ) Close - up of a few cells from the respective population . The images in the right panels are shown again in Fig . S1 B alongside cells treated with other SNX18 - targeting siRNAs . Bars , 10 µm . See also Table S1 and Table S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Micrographs of starved HEK GFP-LC3 cells treated with nontargeting or siSNX18-3 siRNA. (left and middle) 3 × 3 fields of cells. (right) Close-up of a few cells from the respective population. The images in the right panels are shown again in Fig. S1 B alongside cells treated with other SNX18-targeting siRNAs. Bars, 10 µm. See also Table S1 and Table S2.


( A ) HEK GFP - LC3 cells transfected with control ( siCtrl ) or two different SNX18 siRNA oligos ( siSNX18 - 3 and siSNX18 - 5 ) were starved or not starved for 2 h in the presence or absence of BafA1 . GFP - LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting . The graph shows the average GFP - LC3 - II relative to actin normalized to siCtrl - 1 fed ± SEM ( error bars ) , n = 4 .

O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O 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) HEK GFP-LC3 cells transfected with control (siCtrl) or two different SNX18 siRNA oligos (siSNX18-3 and siSNX18-5) were starved or not starved for 2 h in the presence or absence of BafA1. GFP-LC3 lipidation and SNX18 protein knockdown were monitored by immunoblotting. The graph shows the average GFP-LC3-II relative to actin normalized to siCtrl-1 fed ± SEM (error bars), n = 4.


( B ) The degradation of long - lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3 - MA and normalized to the degradation in fed cells ( mean ± SEM [ error bars ] , n = 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(B) The degradation of long-lived proteins in HeLa cells transfected with control or SNX18 siRNA was quantified after 4 h of starvation in the absence or presence of 3-MA and normalized to the degradation in fed cells (mean ± SEM [error bars], n = 3).


( C ) HEK GFP - LC3 cells were transfected with control or SNX18 siRNA and then with a myc control or siRNA - resistant myc - SNX18 plasmid . The number of GFP - LC3 spots per cell was quantified ( graph shows mean ± SEM [ error bars ] , n = 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

(C) HEK GFP-LC3 cells were transfected with control or SNX18 siRNA and then with a myc control or siRNA-resistant myc-SNX18 plasmid. The number of GFP-LC3 spots per cell was quantified (graph shows mean ± SEM [error bars], n = 3).


( D ) HEK GFP - LC3 cells were transfected with myc - SNX18 or a myc control plasmid and treated as in A . The graph shows the average LC3 - II levels relative to actin normalized to myc - transfected fed cells , ± SEM ( error bars ) . n = 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

(D) HEK GFP-LC3 cells were transfected with myc-SNX18 or a myc control plasmid and treated as in A. The graph shows the average LC3-II levels relative to actin normalized to myc-transfected fed cells, ±SEM (error bars). n = 3.


( E ) HEK GFP - LC3 cells were transfected with control or Atg7 siRNA and then with myc - SNX18 or a myc control plasmid , followed by quantification of the number of GFP - LC3 spots per cell ( graphs show mean ± SEM [ error bars ] , n = 3 ) . Representative images are shown . Bars , 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK GFP-LC3 cells were transfected with control or Atg7 siRNA and then with myc-SNX18 or a myc control plasmid, followed by quantification of the number of GFP-LC3 spots per cell (graphs show mean ± SEM [error bars], n = 3). Representative images are shown. Bars, 5 µm.


( F ) The degradation of long - lived protein in HeLa cells transfected with myc - SNX18 or a myc control plasmid was quantified as in B ( graph shows mean ± SEM [ error bars ] , n = 4 ) . * , P < 0 . 05 ; * * , P < 0 . 01 . See also Figs . S1 and S2 .

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

(F) The degradation of long-lived protein in HeLa cells transfected with myc-SNX18 or a myc control plasmid was quantified as in B (graph shows mean ± SEM [error bars], n = 4). *, P < 0.05; **, P < 0.01. See also Figs. S1 and S2.


( A ) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting . Migration of different organelles is indicated , based on the positions in the gradient of calnexin ( ER ) , GM130 ( Golgi stack / CGN ) , SNX9 ( plasma membrane ) , AP - 1 , TfR and Rab11a ( Endosomes / TGN ) , and LC3 - II and GABARAP - II ( autophagosomes ) .

(A) The total membrane fractions from fed or starved HeLa cells were analyzed by density gradient centrifugation and probed for the indicated proteins by immunoblotting. Migration of different organelles is indicated, based on the positions in the gradient of calnexin (ER), GM130 (Golgi stack/CGN), SNX9 (plasma membrane), AP-1, TfR and Rab11a (Endosomes/TGN), and LC3-II and GABARAP-II (autophagosomes).


( B ) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130 . Panels to the right show enlarged views of the boxed regions .

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(B) HeLa cells were starved or not starved for 2 h and immunostained for SNX18 and GM130. Panels to the right show enlarged views of the boxed regions.


( C ) HeLa cells were transfected with GFP - Atg14L and immunostained for SNX18 and calnexin . The boxed regions are enlarged on the top and bottom .

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(C) HeLa cells were transfected with GFP-Atg14L and immunostained for SNX18 and calnexin. The boxed regions are enlarged on the top and bottom.


( D and E ) HEK GFP - DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 ( D ) or WIPI2 ( E ) . Results shown are mean ± SEM ( error bars ) , n = 3 with 30 cells in each experiment . * , P < 0 . 05 .

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(D and E) HEK GFP-DFCP1 cells were starved or not starved for 50 min and immunostained for SNX18 and LC3 (D) or WIPI2 (E). Results shown are mean ± SEM (error bars), n = 3 with 30 cells in each experiment. *, P < 0.05.


( F ) HeLa cells were transfected with control or SNX18 siRNA , starved for 2 h , and immunostained against SNX18 and Atg16L1 .

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(F) HeLa cells were transfected with control or SNX18 siRNA, starved for 2 h, and immunostained against SNX18 and Atg16L1.


( G ) HEK GFP - DFCP1 cells were transfected with control or SNX18 siRNA , starved for 50 min , and immunostained for Atg16L1 . All images are from confocal microscopy . Inset panels show enlarged views of the boxed regions . Bars , 5 µm . See also Fig . S3 .

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(G) HEK GFP-DFCP1 cells were transfected with control or SNX18 siRNA, starved for 50 min, and immunostained for Atg16L1. All images are from confocal microscopy. Inset panels show enlarged views of the boxed regions. Bars, 5 µm. See also Fig. S3.


( A ) HeLa GFP - LC3 cells were transfected to express myc - SNX18 , immunostained against myc , and analyzed by confocal imaging . Asterisks indicate a nontransfected cell . Bars : ( main panels ) 20 µm ; ( inset ) 10 µm .

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(A) HeLa GFP-LC3 cells were transfected to express myc-SNX18, immunostained against myc, and analyzed by confocal imaging. Asterisks indicate a nontransfected cell. Bars: (main panels) 20 µm; (inset) 10 µm.


( B ) HeLa cells transfected with flag - Atg16L1 and myc - SNX18 were starved or not starved for 2 h before immunostaining with anti - myc and anti - flag antibodies and confocal microscopy . Bars , 5 µm .

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(B) HeLa cells transfected with flag-Atg16L1 and myc-SNX18 were starved or not starved for 2 h before immunostaining with anti-myc and anti-flag antibodies and confocal microscopy. Bars, 5 µm.


( D ) HeLa cells were transfected with the indicated myc - SNX18 plasmids for 6 h and starved for an additional 2 h before harvest . Equal parts of cytosol ( C ) and membrane ( M ) fractions were analyzed by Western blotting . The graph shows the average membrane - bound myc - SNX18 ± SEM ( error bars ) , n = 3 .

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(D) HeLa cells were transfected with the indicated myc-SNX18 plasmids for 6 h and starved for an additional 2 h before harvest. Equal parts of cytosol (C) and membrane (M) fractions were analyzed by Western blotting. The graph shows the average membrane-bound myc-SNX18 ± SEM (error bars), n = 3.


( E and F ) HEK GFP - LC3 cells were transfected with the indicated mCherry - SNX18 ( E ) or mycSNX18 ( F ) constructs for 16 h . The number of GFP - LC3 spots per transfected cell ( E ; graph shows mean ± SEM [ error bars ] , n = 5 ) or ratio of LC3 - II to actin determined by Western blotting ( F ) was quantified .

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(E and F) HEK GFP-LC3 cells were transfected with the indicated mCherry-SNX18 (E) or mycSNX18 (F) constructs for 16 h. The number of GFP-LC3 spots per transfected cell (E; graph shows mean ± SEM [error bars], n = 5) or ratio of LC3-II to actin determined by Western blotting (F) was quantified.


( G and H ) HEK GFP - LC3 cells transfected with myc - SNX18 or a myc control plasmid alone ( G ) or together with CFP - FRB and mRFP - FKBP or mRFP - FKBP 5pase ( H ) for 6 h were treated or not treated with 5 µM ionomycin ( G ) or 2 . 5 µM A / C heterodimerizer ( rapalog ; H ) for the last 3 h before analysis of the total intensity of GFP - LC3 spots per transfected cell ( graph shows mean ± SEM [ error bars ] , n = 3 ) .

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(G and H) HEK GFP-LC3 cells transfected with myc-SNX18 or a myc control plasmid alone (G) or together with CFP-FRB and mRFP-FKBP or mRFP-FKBP 5pase (H) for 6 h were treated or not treated with 5 µM ionomycin (G) or 2.5 µM A/C heterodimerizer (rapalog; H) for the last 3 h before analysis of the total intensity of GFP-LC3 spots per transfected cell (graph shows mean ± SEM [error bars], n = 3).


( I ) MEFs were transfected with myc , myc - SNX18 WT , or KR for 16 h , starved or not starved for 2 h , and immunostained for endogenous Atg16L1 . Insets show enlarged views of the boxed regions . Bars , 5 µm .

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(I) MEFs were transfected with myc, myc-SNX18 WT, or KR for 16 h, starved or not starved for 2 h, and immunostained for endogenous Atg16L1. Insets show enlarged views of the boxed regions. Bars, 5 µm.


( J ) HeLa cells were transfected for 12 h with the indicated constructs and starved for 90 min . Cells were solubilized and immunoprecipitated with anti - flag or an unrelated antibody of the same isotype ( mock ab ) . Immunoprecipitates and 2 % of the lysates were analyzed by immunoblotting against myc and flag . * , P < 0 . 05 ; * * , P < 0 . 01 . See also Fig . S3 .

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(J) HeLa cells were transfected for 12 h with the indicated constructs and starved for 90 min. Cells were solubilized and immunoprecipitated with anti-flag or an unrelated antibody of the same isotype (mock ab). Immunoprecipitates and 2% of the lysates were analyzed by immunoblotting against myc and flag. *, P < 0.05; **, P < 0.01. See also Fig. S3.


( A ) HeLa GFP - SNX18 cells were starved for the indicated time , followed by incubation in full medium ( Refeed ) where indicated . Cell lysates were analyzed for LC3 by immunoblotting , or immunoprecipitated with GFP - Trap before immunoblotting against SNX18 ( pS233 ) and GFP . The ratio of pS233 to GFP , and the ratio of LC3 - II to LC3 - I , was quantified , and normalized to 1 for the zero - time sample in both cases ( graph shows mean ± SEM [ error bars ] , n = 4 ; * , P < 0 . 05 ) .

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(A) HeLa GFP-SNX18 cells were starved for the indicated time, followed by incubation in full medium (Refeed) where indicated. Cell lysates were analyzed for LC3 by immunoblotting, or immunoprecipitated with GFP-Trap before immunoblotting against SNX18(pS233) and GFP. The ratio of pS233 to GFP, and the ratio of LC3-II to LC3-I, was quantified, and normalized to 1 for the zero-time sample in both cases (graph shows mean ± SEM [error bars], n = 4; *, P < 0.05).


( B ) The indicated myc - SNX18 PX - BAR constructs were transfected into HeLa cells , and their tubulation efficiency was measured by scoring the percentage of cells displaying more than three elongated structures longer than 5 µm ( n = 300 ) . The graph shows the mean of two independent experiments ± range ( error bars ) . Bars , 10 µm .

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(B) The indicated myc-SNX18 PX-BAR constructs were transfected into HeLa cells, and their tubulation efficiency was measured by scoring the percentage of cells displaying more than three elongated structures longer than 5 µm (n = 300). The graph shows the mean of two independent experiments ± range (error bars). Bars, 10 µm.


( C ) The indicated myc - tagged SNX18 constructs were transfected into HEK GFP - LC3 cells , and the number of GFP - LC3 spots per cell was quantified . The graph shows mean ± SEM ( error bars ) , n = 5 .

(C) The indicated myc-tagged SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 5.


( D ) HeLa GFP - LC3 cells were transfected with the indicated myc - tagged SNX18 constructs for 16 h , starved for 2 h , and analyzed by confocal imaging . Bars , 10 µm .

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(D) HeLa GFP-LC3 cells were transfected with the indicated myc-tagged SNX18 constructs for 16 h, starved for 2 h, and analyzed by confocal imaging. Bars, 10 µm.


( E ) HEK GFP - LC3 cells were transfected with myc - SNX18 WT or myc - SNX18 PX - BAR constructs , and the number of GFP - LC3 spots per cell was quantified . The graph shows mean ± SEM ( error bars ) , n = 3 .

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(E) HEK GFP-LC3 cells were transfected with myc-SNX18 WT or myc-SNX18 PX-BAR constructs, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3.


( F ) HeLa GFP - LC3 cells were transfected with siRNA against Atg16L1 and later with myc - SNX18 constructs before immunostaining and confocal imaging . Bar , 5 µm .

(F) HeLa GFP-LC3 cells were transfected with siRNA against Atg16L1 and later with myc-SNX18 constructs before immunostaining and confocal imaging. Bar, 5 µm.


( A ) GFP , GFP - LC3 , or GFP - LC3 G120A were immunoprecipitated from transfected HeLa cells . The immunoprecipitates and 2 % of the lysate were analyzed by immunoblotting against SNX18 . The GFP proteins were detected by Ponceau staining .

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(A) GFP, GFP-LC3, or GFP-LC3 G120A were immunoprecipitated from transfected HeLa cells. The immunoprecipitates and 2% of the lysate were analyzed by immunoblotting against SNX18. The GFP proteins were detected by Ponceau staining.


( B ) Lysates from fed or starved MDBK cells were incubated with the indicated glutathione - Sepharose - bound GST - tagged proteins . SNX18 was detected by immunoblotting . Ponceau staining shows the GST proteins .

(B) Lysates from fed or starved MDBK cells were incubated with the indicated glutathione-Sepharose-bound GST-tagged proteins. SNX18 was detected by immunoblotting. Ponceau staining shows the GST proteins.


( C ) In vitro translated GFP - p62 or GFP - SNX18 were incubated with the indicated GST - tagged Atg8 family proteins . Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining .

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(C) In vitro translated GFP-p62 or GFP-SNX18 were incubated with the indicated GST-tagged Atg8 family proteins. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.


( A ) GFP - p62 or - SNX18 full - length , SH3 - LC , or PX - BAR regions were in vitro translated and incubated with GST - LC3B or - GABARAP . The resulting pulldowns were separated by SDS - PAGE . Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-p62 or -SNX18 full-length, SH3-LC, or PX-BAR regions were in vitro translated and incubated with GST-LC3B or -GABARAP. The resulting pulldowns were separated by SDS-PAGE. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.


( B ) 18 - mer peptides covering the entire sequence of the SNX18 SH3 - LC region were spotted on a membrane that was incubated with GST or GST - GABARAP , which were detected by immunoblotting against GST . The peptide sequences that specifically bound GST - GABARAP are shown with the common WDDEW motif in bold .

(B) 18-mer peptides covering the entire sequence of the SNX18 SH3-LC region were spotted on a membrane that was incubated with GST or GST-GABARAP, which were detected by immunoblotting against GST. The peptide sequences that specifically bound GST-GABARAP are shown with the common WDDEW motif in bold.


( C ) Peptides with the sequence YGGYQASQGSDDDWDDEWDDSSTVADEPGAL ( SNX18 WT ) or with the first ( SNX18 W154S ) , the second ( SNX18 W158S ) , or both ( SNX18 W154S / W158S ) W mutated to S were spotted on membranes that were incubated with GST , GST - LC3B , or GST - GABARAP . Binding was analyzed as in B .

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(C) Peptides with the sequence YGGYQASQGSDDDWDDEWDDSSTVADEPGAL (SNX18 WT) or with the first (SNX18 W154S), the second (SNX18 W158S), or both (SNX18 W154S/W158S) W mutated to S were spotted on membranes that were incubated with GST, GST-LC3B, or GST-GABARAP. Binding was analyzed as in B.


( D ) GFP - SNX18 WT and the indicated mutants were in vitro translated and incubated with GST - LC3B or - GABARAP , and their binding was analyzed as in A .

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(D) GFP-SNX18 WT and the indicated mutants were in vitro translated and incubated with GST-LC3B or -GABARAP, and their binding was analyzed as in A.


( E ) HeLa GFP - LC3 cells were transfected with myc - SNX18 W154S / W158S mutant , starved for 2 h , immunostained against myc , and analyzed by confocal imaging . Bar , 10 µm . ( F ) Indicated myc - SNX18 constructs were transfected into HEK GFP - LC3 cells , and the number of GFP - LC3 spots per cell was quantified . The graph shows mean ± SEM ( error bars ) , n = 3 . * , P < 0 . 05 .

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(E) HeLa GFP-LC3 cells were transfected with myc-SNX18 W154S/W158S mutant, starved for 2 h, immunostained against myc, and analyzed by confocal imaging. Bar, 10 µm. (F) Indicated myc-SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3. *, P < 0.05.


SNX18 tubules are positive for TfR . HeLa GFP - LC3 cells were transfected with myc - SNX18 or a control myc plasmid and then immunostained for myc and TfR ( A ) or Rab11 ( B ) . Bars : ( A ) 10 µm ; ( B ) 5 µm . Insets show enlarged views of the boxed regions .

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SNX18 tubules are positive for TfR. HeLa GFP-LC3 cells were transfected with myc-SNX18 or a control myc plasmid and then immunostained for myc and TfR (A) or Rab11 (B). Bars: (A) 10 µm; (B) 5 µm. Insets show enlarged views of the boxed regions.


( C , i ) Clusters of tubulo - vesicular structures together with small multivesicular body - like vesicles were observed upon plastic embedding of HeLa GFP - LC3 cells overexpressing myc - SNX18 . ( ii - vi ) Immuno - EM showed that similar clusters labeled strongly for GFP - LC3 ( ii , and enlarged in iii from the boxed region ) , myc - SNX18 ( v ) , and TfR ( vi ) . Strong GFP - LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected ( iv ) . See also Fig . S4 .

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(C, i) Clusters of tubulo-vesicular structures together with small multivesicular body-like vesicles were observed upon plastic embedding of HeLa GFP-LC3 cells overexpressing myc-SNX18. (ii-vi) Immuno-EM showed that similar clusters labeled strongly for GFP-LC3 (ii, and enlarged in iii from the boxed region), myc-SNX18 (v), and TfR (vi). Strong GFP-LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected (iv). See also Fig. S4.


Recycling endosomes provide membranes for SNX18 - mediated GFP - LC3 membrane trafficking and remodeling . ( A ) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18 , Rab11 , and TfR . ( B ) Cells were transfected to express flag - Atg16L1 , starved , and immunostained against Rab11 or TfR and flag . ( C ) Cells were starved and immunostained against TfR , Atg16L1 , and SNX18 . ( D ) Cells were transfected with control or SNX18 siRNA , starved , and immunostained against SNX18 and Rab11 . Inset panels show enlarged views of the boxed regions . ( E ) Cells were transfected with control or Rab11 a + b siRNA , starved , and immunostained against SNX18 and Atg16L1 . Cells were imaged by confocal microscopy . Bars , 5 µm .

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Recycling endosomes provide membranes for SNX18-mediated GFP-LC3 membrane trafficking and remodeling. (A) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18, Rab11, and TfR. (B) Cells were transfected to express flag-Atg16L1, starved, and immunostained against Rab11 or TfR and flag. (C) Cells were starved and immunostained against TfR, Atg16L1, and SNX18. (D) Cells were transfected with control or SNX18 siRNA, starved, and immunostained against SNX18 and Rab11. Inset panels show enlarged views of the boxed regions. (E) Cells were transfected with control or Rab11 a+b siRNA, starved, and immunostained against SNX18 and Atg16L1. Cells were imaged by confocal microscopy. Bars, 5 µm.


( B and C ) RNAi against SH3PX1 ( GFP - positive clones ) decreases formation of LTR punctae in response to 4 h of starvation in 20 % sucrose ( B ) and inhibits formation of mCherry - Atg8a - positive autophagosomes in response to 2 h starvation ( C ) . Genotypes : ( B and D ) hs - flp ; UAS - dicer / + ; Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ; ( C and E ) hs - flp ; UAS - dicer / + ; r4 : mCherry - Atg8a Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ;

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(B and C) RNAi against SH3PX1 (GFP-positive clones) decreases formation of LTR punctae in response to 4 h of starvation in 20% sucrose (B) and inhibits formation of mCherry-Atg8a-positive autophagosomes in response to 2 h starvation (C). Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;


( D and E ) RNAi against SH3PX1 inhibits formation of LTR ( D ) and mCherry - Atg8a - positive structures ( E ) at early stages of developmental autophagy in fat bodies of 110 - h - old larvae . Genotypes : ( B and D ) hs - flp ; UAS - dicer / + ; Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ; ( C and E ) hs - flp ; UAS - dicer / + ; r4 : mCherry - Atg8a Act > CD2 > GAL4 UAS - GFPnls / UAS - SH3PX1 RNAi ;

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(D and E) RNAi against SH3PX1 inhibits formation of LTR (D) and mCherry-Atg8a-positive structures (E) at early stages of developmental autophagy in fat bodies of 110-h-old larvae. Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi;


( F ) There was no change in mCherry - Atg8a in SH3PX1 mutant clones ( GFP negative , outlined ) in well - fed larvae . Genotypes : ( F and G ) hs - flp ; Cg - GAL4 UAS - mChAtg8a / + ; FRT80B UAS - 2XeGFP / FRT80B SH3PX1HK62b .

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(F) There was no change in mCherry-Atg8a in SH3PX1 mutant clones (GFP negative, outlined) in well-fed larvae. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B UAS-2XeGFP/FRT80B SH3PX1HK62b.


( G ) Decreased formation of mCherry - Atg8a - positive autophagosomes in SH3PX1 mutant clones ( GFP negative , outlined ) in fat bodies from larvae starved in 20 % sucrose for 3 h . Bars , 20 µm . ( H ) The graph shows quantification of total mCherry - Atg8a spot intensity per cell for WT and SH3PX1 mutant cells . The graph shows mean ± SEM ( error bars ) ; * * * , P < 0 . 001 . Genotypes : ( F and G ) hs - flp ; Cg - GAL4 UAS - mChAtg8a / + ;

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(G) Decreased formation of mCherry-Atg8a-positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B


( A ) Detection of VgpA in supernatants from indicated strains by immunoblotting ( IB ) with anti - VgpA antisera .

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(A) Detection of VgpA in supernatants from indicated strains by immunoblotting (IB) with anti-VgpA antisera.


( B ) T3SS2 translocation of VgpA into Caco - 2 cells using CyaA - based assay ; VopV is a known T3SS2 effector . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) .

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(B) T3SS2 translocation of VgpA into Caco-2 cells using CyaA-based assay; VopV is a known T3SS2 effector. Error bars represent mean ± standard deviation (n=3 biologically independent experiments).


( C ) Localization of translocated VgpA ( upper panel ) and VgpAL10A ( lower panel ) during infection .

(C) Localization of translocated VgpA (upper panel) and VgpAL10A (lower panel) during infection.


( A ) Silver - stained PAGE gel of proteins from a nuclear extract that interact with either GST - VgpA or GST glutathione agarose . The protein bound by GST - VgpA ( red arrow ) , but not GST , was identified as EBP2 by mass spectrometry ; GST - VgpA is labeled with a blue arrow .

(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow.


( B ) Western blot using anti - EBP2 as antibody confirmed that EBP2 binds VgpA .

(B) Western blot using anti-EBP2 as antibody confirmed that EBP2 binds VgpA.


( C ) Interaction of VgpA or VgpA mutants ( VgpAΔ10 and VgpAL10A ) with EBP2 .

(C) Interaction of VgpA or VgpA mutants (VgpAΔ10 and VgpAL10A) with EBP2.


( D ) Localization of VgpA ( upper panel ) or in VgpAL10A ( lower panel ) in HeLa cells after transfection . EBP2 was detected with anti - EBP2 antibody ( red ) , VgpA and VgpAL10A were detected with anti - VgpA sera ( green ) and nuclei were stained with DAPI ( blue ) . White arrows indicate co - localization of EBP2 and VgpA ( upper panel ) . VgpAL10A localizes primarily to the nucleoplasm ( white arrow , lower 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

(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes primarily to the nucleoplasm (white arrow, lower panel).


( D ) Localization of VgpA ( upper panel ) or in VgpAL10A ( lower panel ) in HeLa cells after transfection . EBP2 was detected with anti - EBP2 antibody ( red ) , VgpA and VgpAL10A were detected with anti - VgpA sera ( green ) and nuclei were stained with DAPI ( blue ) . White arrows indicate co - localization of EBP2 and VgpA ( upper panel ) . VgpAL10A

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes


( A ) Fluorescence micrographs of uninfected Caco - 2 cells or Caco - 2 cells infected with the indicated V . parahaemolyticus strains . Cells were stained with DAPI ( blue ) and anti - BrdU antibody ( green ) .

O O O O O O O O O O O O O O O O O O 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) Fluorescence micrographs of uninfected Caco-2 cells or Caco-2 cells infected with the indicated V. parahaemolyticus strains. Cells were stained with DAPI (blue) and anti-BrdU antibody (green).


( B ) Fluorescence micrographs of Caco - 2 cells transfected with siRNA targeting EBP2 ( siEBP2 ) or control siRNA ( siNC ) and subsequently infected with vopZ ' ΔvscN1 . Cells were stained with anti - BrdU antibody ( green ) and DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence micrographs of Caco-2 cells transfected with siRNA targeting EBP2 (siEBP2) or control siRNA (siNC) and subsequently infected with vopZ'ΔvscN1. Cells were stained with anti-BrdU antibody (green) and DAPI (blue).


( C ) The percentage of BrdU - positive cells ( green ) among the total cells ( green + blue ) was analyzed for 100 cells in three experiments . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) . One - way analysis of variance ( ANOVA ) was used for statistical analysis . * P < 0 . 05 ( when compared to the uninfected Caco - 2 cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The percentage of BrdU-positive cells (green) among the total cells (green + blue) was analyzed for 100 cells in three experiments. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way analysis of variance (ANOVA) was used for statistical analysis. *P<0.05 (when compared to the uninfected Caco-2 cells).


( D ) The percentage of BrdU + cells was analyzed as above for cells treated with siNC or siEBP2 . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) . Two - way ANOVA was used for statistical analysis . * P < 0 . 05 ( when compared to siNC - transfected cells that are infected with vopZ ' ΔvscN1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The percentage of BrdU+ cells was analyzed as above for cells treated with siNC or siEBP2. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). Two-way ANOVA was used for statistical analysis. *P<0.05 (when compared to siNC-transfected cells that are infected with vopZ'ΔvscN1).


( E ) Immunoblot of EBP2 and Actin in Caco - 2 cells transfected with siEBP2 or siNC .

(E) Immunoblot of EBP2 and Actin in Caco-2 cells transfected with siEBP2 or siNC.


( A ) Localization of c - Myc in uninfected Caco - 2 cells ( first row ) , Caco - 2 cells infected with V . parahaemolyticus vopZ ' ΔvscN1 ( wild type VgpA , second row ) , Caco - 2 cells infected with vopZ ' ΔvscN1vgpA ' ( third row ) , Caco - 2 cells infected with vopZ ' ΔvscN1vgpA ' : pvgpA ( fourth row ) and Caco - 2 cells infected with vopZ ' ΔvscN1vgpA ' : pvgpAL10A ( fifth row ) . Fixed cells were stained with DAPI ( blue ) , and anti - EBP2 ( red ) , and anti c - Myc ( green ) antibodies . White arrows are examples indicating the co - localization of EBP2 and c - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc.


( B Detection of c - Myc or EBP2 in nuclear extracts from uninfected Caco - 2 cells or after infection with indicated strains . Following infection , nuclear extracts were obtained and loaded on protein A / G beads cross - linked with anti - EBP2 ( B , IP : anti - EBP2 ) Input and eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band intensity ( normalized to uninfected ) from three independent experiments are presented under the signals of the blot of IP samples . n . d . , not determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked with anti-EBP2 (B, IP: anti-EBP2) Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


C ) Detection of c - Myc or EBP2 in nuclear extracts from uninfected Caco - 2 cells or after infection with indicated strains . Following infection , nuclear extracts were obtained and loaded on protein A / G beads cross - linked anti - C - Myc ( C , IP : anti - c - Myc ) antibodies , respectively . Input and eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band intensity ( normalized to uninfected ) from three independent experiments are presented under the signals of the blot of IP samples . n . d . , not determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked anti-C-Myc (C, IP: anti-c-Myc) antibodies, respectively. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


( D ) Purified proteins EBP2 , c - Myc and GST - VgpA or GST - VgpAL10A were mixed and then pulled down with protein A / G beads cross - linked with anti - EBP2 . Input and eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band intensity ( normalized to EBP2 + c - Myc sample without GST - VgpA and GST - VgpAL10A ) from three independent experiments are presented under the signals of the blot of IP samples . n . d . , not determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Purified proteins EBP2, c-Myc and GST-VgpA or GST-VgpAL10A were mixed and then pulled down with protein A/G beads cross-linked with anti-EBP2. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to EBP2+c-Myc sample without GST-VgpA and GST-VgpAL10A) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


( E ) Caco - 2 cells were infected with the indicated strains and treated with cycloheximide ( CHX ) for the indicated time . Whole cells lysates were analyzed by western blot using anti - c - Myc and anti - Actin antibody .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_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

(E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody.


( A ) Amounts of rRNA were from uninfected Caco - 2 cells or cells infected with indicated strains . * P < 0 . 05 ( when compared to the uninfected Caco - 2 cells ) .

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

(A) Amounts of rRNA were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).


( B ) Amounts of rRNA in uninfected or infected Caco - 2 cells transfected with control ( siNC ) or EBP2 - targeting ( siEBP2 ) siRNAs . * P < 0 . 05 ( when compared to the siNC - transfected cells that are infected with vopZ ' ΔvscN1 ) .

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

(B) Amounts of rRNA in uninfected or infected Caco-2 cells transfected with control (siNC) or EBP2-targeting (siEBP2) siRNAs. *P<0.05 (when compared to the siNC-transfected cells that are infected with vopZ'ΔvscN1).


( C ) Amounts of RNA polymerase I ( POLR1A ) were from uninfected Caco - 2 cells or cells infected with indicated strains . * P < 0 . 05 ( when compared to the uninfected Caco - 2 cells ) .

O O O O O O O O O O O O O O O O O O O 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) Amounts of RNA polymerase I (POLR1A) were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).


( D ) . Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection . * P < 0 . 05 ( when compared to the uninfected rabbits at 18 hours ) . ΔP < 0 . 05 ( when compared to the uninfected rabbits at 38 hours post infection ) .

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

(D). Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection. *P<0.05 (when compared to the uninfected rabbits at 18 hours). ΔP<0.05 (when compared to the uninfected rabbits at 38 hours post infection).


( A ) VgpA ( upper panel : animals infected with WT ) and VgpAL10A ( lower panel : animals infected with vgpAL10A ) localization in intestinal epithelial cells of infected animals . Arrows in upper panel indicate VgpA localized as punctae in the nucleus ; the arrow in the lower panel indicates VgpAL10A localized diffusively in the nucleoplasm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) VgpA (upper panel: animals infected with WT) and VgpAL10A (lower panel: animals infected with vgpAL10A) localization in intestinal epithelial cells of infected animals. Arrows in upper panel indicate VgpA localized as punctae in the nucleus; the arrow in the lower panel indicates VgpAL10A localized diffusively in the nucleoplasm.


( B ) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V . parahaemolyticus . Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains . Outputs represent eluates from protein A / G beads cross - linked with anti - EBP2 ( IP : anti - EBP2 ) or anti - VgpA ( IP : anti - VgpA ) antibodies and analyzed with anti - VgpA and anti - EBP2 antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V. parahaemolyticus. Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains. Outputs represent eluates from protein A/G beads cross-linked with anti-EBP2 (IP: anti-EBP2) or anti-VgpA (IP: anti-VgpA) antibodies and analyzed with anti-VgpA and anti-EBP2 antibodies.


( C ) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti - Ki67 antibodies to detect actively dividing cells ( green ) , phalloidin to visualize F - actin ( red ) and DAPI to detect nuclei ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti-Ki67 antibodies to detect actively dividing cells (green), phalloidin to visualize F-actin (red) and DAPI to detect nuclei (blue).


( D ) Eighteen hours after infant rabbits were infected with WT ( middle panel ) or vgpAL10A ( lower panel ) V . parahaemolyticus , fixed sections from the small intestine were stained with antibodies to c - Myc ( green ) , EBP2 ( red ) , and DAPI ( blue ) . Uninfected rabbits were processed similarly and were included as a control ( upper panel ) . White arrows indicate co - localization of c - Myc and EBP2 ( middle 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

(D) Eighteen hours after infant rabbits were infected with WT (middle panel) or vgpAL10A (lower panel) V. parahaemolyticus, fixed sections from the small intestine were stained with antibodies to c-Myc (green), EBP2 (red), and DAPI (blue). Uninfected rabbits were processed similarly and were included as a control (upper panel). White arrows indicate co-localization of c-Myc and EBP2 (middle panel).


( A ) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18 , 28 and 38 h following inoculation . Black horizontal line indicates mean CFU ( n = 7 - 10 animals / group ) . One - way ANOVA was used for statistical analysis . * P < 0 . 05 for the indicated comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18, 28 and 38 h following inoculation. Black horizontal line indicates mean CFU (n=7-10 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 for the indicated comparison.


( B ) H & E stained sections from the distal small intestine from the rabbits infected with the indicated strains 18 hours after infection . Black arrows point to bacterial colonies closely attached to the epithelium .

O O O O O O O O O O O O O O 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) H&E stained sections from the distal small intestine from the rabbits infected with the indicated strains 18 hours after infection. Black arrows point to bacterial colonies closely attached to the epithelium.


( C ) Fluid accumulation in rabbits infected with the indicated strains 38 hours post inoculation . Error bars represent mean ± standard deviation ( n = 7 animals / group ) . One - way ANOVA was used for statistical analysis . * P < 0 . 05 ( when compared to the rabbits infected with WT ) .

(C) Fluid accumulation in rabbits infected with the indicated strains 38 hours post inoculation. Error bars represent mean ± standard deviation (n=7 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 (when compared to the rabbits infected with WT).


( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products ( ARPs ) are also indicated , as are the different species of catenated dimers : nicked - nicked catenanes , ' n - nCats ' ; nicked - supercoiled catenanes , ' n - scCats ' ; supercoiled - supercoiled catenanes ' sc - scCats ' . ( C ) Quantification of total DNA synthesis from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( D ) Quantification of total DNA synthesis from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to the maximum recorded signal within each individual experiment . ( E ) Quantification of supercoiled circular monomers ( ' scCMs ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( F ) Quantification of scCMs from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . ( G ) Quantification of linears ( ' Lins ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( H ) Quantification of Lins from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Data information : For ( E ) - ( H ) , abundance of the indicated species was calculated as a percentage of total lane signal ( % of total ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples from (A) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products (ARPs) are also indicated, as are the different species of catenated dimers: nicked-nicked catenanes, 'n-nCats'; nicked-supercoiled catenanes, 'n-scCats'; supercoiled-supercoiled catenanes 'sc-scCats'. (C) Quantification of total DNA synthesis from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (D) Quantification of total DNA synthesis from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (E) Quantification of supercoiled circular monomers ('scCMs') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (F) Quantification of scCMs from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. (G) Quantification of linears ('Lins') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (H) Quantification of Lins from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Data information: For (E)-(H), abundance of the indicated species was calculated as a percentage of total lane signal (% of total).


( A ) Plasmid DNA was replicated in the presence of [ α - 32P ] dATP and vehicle control , etoposide , or doxorubicin . Purified DNA intermediates were digested with XmnI , which cuts the plasmid template once , to allow for identification of DNA breaks . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . ( C ) Quantification of DNA breaks from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of breaks was calculated as a percentage of total lane signal ( % of total ) . ( D ) Quantification of linears ( ' Lins ' ) from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of linears ( AU ) was normalized to the maximum recorded signal within each individual experiment .

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 B-CONTROLLED_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 O O O O O O O

(A) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, etoposide, or doxorubicin. Purified DNA intermediates were digested with XmnI, which cuts the plasmid template once, to allow for identification of DNA breaks. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of DNA breaks from (B). Mean ± SD, n=3 independent experiments. Abundance of breaks was calculated as a percentage of total lane signal (% of total). (D) Quantification of linears ('Lins') from (B). Mean ± SD, n=3 independent experiments. Abundance of linears (AU) was normalized to the maximum recorded signal within each individual experiment.


( F ) Samples from ( E ) were separated on native agarose gels and visualized by autoradiography . ( G ) Quantification of DPC - containing molecules from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of the DPC - containing molecules was calculated as a percentage of total lane signal ( % of total ) . ( H ) Quantification of the amount of supercoiled circular monomers ( ' scCMs ' ) from ' + ProK ' samples from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers ( AU ) was normalized to the maximum recorded signal within each individual experiment .

(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. (G) Quantification of DPC-containing molecules from (F). Mean ± SD, n=3 independent experiments. Abundance of the DPC-containing molecules was calculated as a percentage of total lane signal (% of total). (H) Quantification of the amount of supercoiled circular monomers ('scCMs') from '+ProK' samples from (F). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers (AU) was normalized to the maximum recorded signal within each individual experiment.


( A ) Plasmid DNA was replicated in Mock - or TOP2α - immunodepleted extracts in the presence or absence of vehicle control , etoposide , or doxorubicin . In mock - depleted extracts ( ΔMock ) , the final replication product are supercoiled circular monomers ( ' scCMs ' ) . In TOP2α - depleted extracts ( ΔTOP2α ) , the final replication product are catenanes ( ' Cats ' ) because TOP2α is required for DNA unlinking . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . Linear molecules , arising from DNA breaks , are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes . ( C ) Quantification of scCMs from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( D ) Quantification of Cats from ( B ) lanes 16 - 30 . Mean ± SD , n = 3 independent experiments . Abundance of catenanes was calculated as a percentage of total lane signal ( % of total ) .

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 B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in Mock- or TOP2α-immunodepleted extracts in the presence or absence of vehicle control, etoposide, or doxorubicin. In mock-depleted extracts (ΔMock), the final replication product are supercoiled circular monomers ('scCMs'). In TOP2α-depleted extracts (ΔTOP2α), the final replication product are catenanes ('Cats') because TOP2α is required for DNA unlinking. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. Linear molecules, arising from DNA breaks, are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes. (C) Quantification of scCMs from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (D) Quantification of Cats from (B) lanes 16-30. Mean ± SD, n=3 independent experiments. Abundance of catenanes was calculated as a percentage of total lane signal (% of total).


( F ) Samples from ( E ) were separated on native agarose gels and visualized by autoradiography . Linear molecules are indicated with a red arrow . ( G ) Quantification of scCMs from + ProK gel in ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) . ( H ) Quantification of DPC Containing Molecules from ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of DPC - containing molecules was calculated as a percentage of total lane signal ( % of total ) .

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(F) Samples from (E) were separated on native agarose gels and visualized by autoradiography. Linear molecules are indicated with a red arrow. (G) Quantification of scCMs from + ProK gel in (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (H) Quantification of DPC Containing Molecules from (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of DPC-containing molecules was calculated as a percentage of total lane signal (% of total).


( A ) Plasmid DNA was replicated in the presence or absence of etoposide and p97 - i , which inhibits removal of replisomes ( green ) from DNA . At different timepoints chromatin - bound proteins were recovered via plasmid pulldowns . ( B ) Proteins from ( A ) were recovered and analyzed via Western blotting . Input corresponds to 1 % of total reaction . Modified TOP2α species are also indicated . ( C ) Quantification of TOP2α ( both modified and unmodified species ) from ( B ) lanes 1 - 8 . ( D ) Quantification of TOP2α ( both modified and unmodified species ) from ( B ) lanes 9 - 16 . ( E ) Quantification of CDC45 from ( B ) lanes 1 - 8 . ( F ) Quantification of CDC45 from ( B ) lanes 9 - 16 . Data information : ( C - F Mean ± SD , n = 3 independent experiments . Protein abundance ( AU ) was normalized to the maximum recorded signal within each individual experiment .

O O O O O O O O O O O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in the presence or absence of etoposide and p97-i, which inhibits removal of replisomes (green) from DNA. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (B) Proteins from (A) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. Modified TOP2α species are also indicated. (C) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 1-8. (D) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 9-16. (E) Quantification of CDC45 from (B) lanes 1-8. (F) Quantification of CDC45 from (B) lanes 9-16. Data information: (C-F Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


( G ) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin ( Aph ) , which prevents DNA synthesis but still permits unwinding by replisomes . At different timepoints chromatin - bound proteins were recovered via plasmid pulldowns . ( H ) Proteins from ( G ) were recovered and analyzed via Western blotting . Input corresponds to 1 % of total reaction . ( I ) Quantification of RPA30 from ( H ) lanes 9 - 16 . ( J ) Quantification of RPA30 from ( H ) lanes 1 - 8 . ( K ) Quantification of TOP2α ( both modified and unmodified species ) from ( H ) lanes 1 - 4 and 9 - 12 . ( L ) Quantification of TOP2α ( both modified and unmodified species ) from ( H ) lanes 5 - 8 and 13 - 16 . Data information : I - L ) Mean ± SD , n = 3 independent experiments . Protein abundance ( AU ) was normalized to the maximum recorded signal within each individual experiment .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin (Aph), which prevents DNA synthesis but still permits unwinding by replisomes. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (H) Proteins from (G) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. (I) Quantification of RPA30 from (H) lanes 9-16. (J) Quantification of RPA30 from (H) lanes 1-8. (K) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 1-4 and 9-12. (L) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 5-8 and 13-16. Data information: I-L) Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


( A ) Plasmid DNA was replicated in the presence of [ α - 32P ] dATP and vehicle control , aclarubicin , or doxorubicin . ( B ) Samples from ( A ) were separated on a native agarose gel and visualized by autoradiography . ( C ) Quantification of supercoiled circular monomers from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a percentage of total lane signal ( % of total ) .

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 B-CONTROLLED_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

(A) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, aclarubicin, or doxorubicin. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of supercoiled circular monomers from (B). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).


( E ) Samples from ( D ) were separated on a native agarose gel and visualized by autoradiography . ( F ) Quantification of fork merger from ( E ) lanes 1 - 8 . Mean ± SD , n = 3 independent experiments . ( G ) Quantification of fork merger from ( E ) lanes 9 - 17 . Mean ± SD , n = 3 independent experiments . Fork merger was calculated as a percentage of total lane signal ( % of total ) for ( F ) - ( G ) .

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(E) Samples from (D) were separated on a native agarose gel and visualized by autoradiography. (F) Quantification of fork merger from (E) lanes 1-8. Mean ± SD, n=3 independent experiments. (G) Quantification of fork merger from (E) lanes 9-17. Mean ± SD, n=3 independent experiments. Fork merger was calculated as a percentage of total lane signal (% of total) for (F)-(G).


S2 cells were incubated at 21 % or 1 % O2 concentrations for 24h hours . A ) Puromycin was added 20 min . before cell harvesting . Total puromycin incorporation and Actin level were revealed by western blot .

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S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. A) Puromycin was added 20 min. before cell harvesting. Total puromycin incorporation and Actin level were revealed by western blot.


S2 cells were incubated at 21 % or 1 % O2 concentrations for 24h hours . ( C ) LDH and RpL32 protein levels were detected by western blot in total cell extracts from S2 cells exposed to normoxic conditions or to 1 % O2 environment for 24 and 48 hours .

S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. (C) LDH and RpL32 protein levels were detected by western blot in total cell extracts from S2 cells exposed to normoxic conditions or to 1% O2 environment for 24 and 48 hours.


S2 cells were incubated at 21 % or 1 % O2 concentrations for 24h hours . ( E ) One representative experiment performed as in ( D ) is shown .

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S2 cells were incubated at 21% or 1% O2 concentrations for 24h hours. (E) One representative experiment performed as in (D) is shown.


S2 cells were transiently transfected with the indicated constructs and exposed to 1 % O2 for 24h . ( A , B ) ( A ) Activities of Firefly ( Fluc ) and Renilla ( Rluc ) luciferase , produce from reporter genes under control of the Ldh gene promoter , were measured in total cell extracts by dual luciferase assay and the ratio of both measures was calculated for each transfection . ( B ) Relative FLuc and RLuc mRNA levels were measured by RT - qPCR .

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S2 cells were transiently transfected with the indicated constructs and exposed to 1% O2 for 24h. (A, B) (A) Activities of Firefly (Fluc) and Renilla (Rluc) luciferase, produce from reporter genes under control of the Ldh gene promoter, were measured in total cell extracts by dual luciferase assay and the ratio of both measures was calculated for each transfection. (B) Relative FLuc and RLuc mRNA levels were measured by RT-qPCR.


S2 cells were transiently transfected with the indicated constructs and exposed to 1 % O2 for 24h . ( D ) Indicated stably transfected cell lines were exposed to 21 % or 1 % O2 for 24h before fractionation of cell extracts by ultracentrifugation on linear sucrose gradients ( 15 % - 50 % The distribution of FLuc mRNA , produced from reporter genes under control of the pMT promoter was measured by northern blot in each cell types . Fractions numbers from top to bottom of the gradient are indicated .

S2 cells were transiently transfected with the indicated constructs and exposed to 1% O2 for 24h. (D) Indicated stably transfected cell lines were exposed to 21% or 1% O2 for 24h before fractionation of cell extracts by ultracentrifugation on linear sucrose gradients (15%-50% The distribution of FLuc mRNA, produced from reporter genes under control of the pMT promoter was measured by northern blot in each cell types. Fractions numbers from top to bottom of the gradient are indicated.


( A ) Control ( TM3 > UAS - eIF4EHP _ RNAi ) or eIF4EHP KD ( Tub - GAL4 > UAS - eIF4EHP _ RNAi ) flies were maintained in either normoxia or hypoxia ( 1 % O2 , 24h ) . Protein extracts were prepared from whole flies and eIF4EHP , LDH and Actin were detected by western blot . ( B ) Quantification of LDH / Actin ratio in 3 independent experiments performed as in A . Values represent mean + / - SD , n = 3 biological replicates , P - value of two - ways ANOVA .

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(A) Control (TM3>UAS-eIF4EHP_RNAi) or eIF4EHP KD (Tub-GAL4>UAS-eIF4EHP_RNAi) flies were maintained in either normoxia or hypoxia (1%O2, 24h). Protein extracts were prepared from whole flies and eIF4EHP, LDH and Actin were detected by western blot. (B) Quantification of LDH/Actin ratio in 3 independent experiments performed as in A. Values represent mean +/- SD, n=3 biological replicates, P-value of two-ways ANOVA.


( C ) S2 cells lines inactivated for Yellow or eIF4EHP genes were established by a CRISPR - Cas9 strategy as described in the methods section . After exposure to normoxic or hypoxic conditions , cells were lysed and Actin , LDH and eIF4EHP protein levels were detected by western blot . ( D ) Quantification of LDH / Actin ratio in 6 independent experiments performed as in C . Values represent mean + / - SD , P - values of two - way ANOVA .

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(C) S2 cells lines inactivated for Yellow or eIF4EHP genes were established by a CRISPR-Cas9 strategy as described in the methods section. After exposure to normoxic or hypoxic conditions, cells were lysed and Actin, LDH and eIF4EHP protein levels were detected by western blot. (D) Quantification of LDH/Actin ratio in 6 independent experiments performed as in C. Values represent mean +/- SD, P-values of two-way ANOVA.


( E ) Cell extracts from Yellow , eIF4EHP or eIF4E6 KO cells incubated at 21 or 1 % O2 were separated by centrifugation on linear sucrose gradients and Ldh mRNA was detected by northern blot in the different fractions . Fractions numbers from top to bottom of the gradient are indicated .

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(E) Cell extracts from Yellow, eIF4EHP or eIF4E6 KO cells incubated at 21 or 1% O2 were separated by centrifugation on linear sucrose gradients and Ldh mRNA was detected by northern blot in the different fractions. Fractions numbers from top to bottom of the gradient are indicated.


( G ) Indicated S2 cell lines were cultivated at 1 % O2 for 24h and labelled with puromycin ( 5µg / ml ) for 20 min . before harvesting . Puromycin signal in total cell extracts was quantified by western blot and expressed relative to the level detected in wild - type cells ( n = 6 biological replicates , t - test , two - sided ) .

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(G) Indicated S2 cell lines were cultivated at 1% O2 for 24h and labelled with puromycin (5µg/ml) for 20 min. before harvesting. Puromycin signal in total cell extracts was quantified by western blot and expressed relative to the level detected in wild-type cells (n=6 biological replicates, t-test, two-sided).


B ) Polysome profiles of FLuc - Ldh 3 ' UTR and Fluc - CA - rich 3 ' UTR reporter mRNA in control ( Yellow KO ) and eIF4EHP KO S2 cells in hypoxia .

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B) Polysome profiles of FLuc-Ldh 3'UTR and Fluc-CA-rich 3'UTR reporter mRNA in control (Yellow KO) and eIF4EHP KO S2 cells in hypoxia.


( D ) WT and eIF4EHP KO cells were co - transfected with plasmids encoding V5 - tagged D . persimilis eIF4EHP ( Dp eIF4EHP ) or GFP , along with FLuc - Ldh3 ' UTR and RLuc reporters . eIF4EHP proteins were detected by western blot using eIF4EHP antibody ( cross - reacting to Dp eIF4EHP ) .

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(D) WT and eIF4EHP KO cells were co-transfected with plasmids encoding V5-tagged D. persimilis eIF4EHP (Dp eIF4EHP) or GFP, along with FLuc-Ldh3'UTR and RLuc reporters. eIF4EHP proteins were detected by western blot using eIF4EHP antibody (cross-reacting to Dp eIF4EHP).


( E , F ) 24h after transfection , cells were cultivated in hypoxia ( 1 % O2 ) for an additional 24h before measurement of luciferase activity by dual luciferase reporter assay ( D ) and luciferase mRNA levels by qPCR ( E ) . Values represent mean ± SD , n = 3 biological replicates , P - value of one - way ANOVA , * - P ≤ 0 . 05 , ns - P > 0 . 05 .

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(E, F) 24h after transfection, cells were cultivated in hypoxia (1%O2) for an additional 24h before measurement of luciferase activity by dual luciferase reporter assay (D) and luciferase mRNA levels by qPCR (E). Values represent mean ± SD, n=3 biological replicates, P-value of one-way ANOVA, *-P ≤ 0.05, ns- P>0.05.


( C ) Combined detection of eIF4EHP by immunofluorescence ( green ) and polyA mRNA by oligo - dT FISH ( red ) in normoxic and hypoxic WT S2 cells . nuclei were counterstained with DAPI ( blue ) . Red and green signal intensities were quantified with the Zen software ( Zeiss ) .

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(C) Combined detection of eIF4EHP by immunofluorescence (green) and polyA mRNA by oligo-dT FISH (red) in normoxic and hypoxic WT S2 cells. nuclei were counterstained with DAPI (blue). Red and green signal intensities were quantified with the Zen software (Zeiss).


( D ) Combined detection of GFP - Rox8 or GFP - Dcp1 ( green ) and endogenous eIF4EHP ( red ) in transfected S2 cells cultivated in normoxia ( 21 % O2 ) , in hypoxia ( 1 % O2 for 24h ) or after oxidative stress ( 250μM arsenite for 1h ) . Representative images of confocal imaging ( Z - stack maximum intensity projection ) and zooms are shown . , nuclei were counterstained with DAPI ( blue ) . Red and green signal intensities were quantified with the Zen software ( Zeiss ) .

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(D) Combined detection of GFP-Rox8 or GFP-Dcp1 (green) and endogenous eIF4EHP (red) in transfected S2 cells cultivated in normoxia (21% O2), in hypoxia (1% O2 for 24h) or after oxidative stress (250μM arsenite for 1h). Representative images of confocal imaging (Z-stack maximum intensity projection) and zooms are shown. , nuclei were counterstained with DAPI (blue). Red and green signal intensities were quantified with the Zen software (Zeiss).


( A ) Cell extracts from normoxic or hypoxic S2 cells were ultracentrifuged as described in fig . 1B , protein extracts from pooled fractions corresponding to the same ribosomal content were analysed by western blot with anti - eIF4EHP and RpS23 antibodies .

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(A) Cell extracts from normoxic or hypoxic S2 cells were ultracentrifuged as described in fig.1B, protein extracts from pooled fractions corresponding to the same ribosomal content were analysed by western blot with anti-eIF4EHP and RpS23 antibodies.


( B ) Adult Canton S flies were kept at 21 % or 1 % O2 for 24h . Total protein extracts were prepared as described in the method section and fractionated by ultracentrifugation on sucrose gradients ( 15 - 50 % ) . OD at 260 nm was measured in line during fractionation ( upper panels ) . EIF4EHP was detected by western blot in representative fractions of the gradients .

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(B) Adult Canton S flies were kept at 21% or 1% O2 for 24h. Total protein extracts were prepared as described in the method section and fractionated by ultracentrifugation on sucrose gradients (15-50%). OD at 260 nm was measured in line during fractionation (upper panels). EIF4EHP was detected by western blot in representative fractions of the gradients.