. ( D ) Ultrastructure of HCV - infected cells showing the formation of mitophagosome . Control naïve Huh7 cells ( uninfected ) , HCV FLR - JFH1 replicon cells , and Huh7 cells infected with HCVcc ( Jc1 ) were examined by electron microscopy . In the zoomed image , the formation of mitophagosome in HCV - infected cells is shown . Organelle mark : N , nucleus ; MF , mitophagosome formation . Scale bar = 0 . 2 µM ( uninfected ) ; 0 . 1 µM ( infected ) .

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. (D) Ultrastructure of HCV-infected cells showing the formation of mitophagosome. Control naïve Huh7 cells (uninfected), HCV FLR-JFH1 replicon cells, and Huh7 cells infected with HCVcc (Jc1) were examined by electron microscopy. In the zoomed image, the formation of mitophagosome in HCV-infected cells is shown. Organelle mark: N, nucleus; MF, mitophagosome formation. Scale bar = 0.2 µM (uninfected); 0.1 µM (infected).


( E ) Immunoelectron microscopic analysis showing the formation of mitophagosome in HCV - infected cells . Huh7 cells infected with HCVcc were stained by triple immunogold labeling method , as described in Materials and Methods . The immunogold particles of GFP - LC3 ( white arrow , 18 nm ) , HCV E2 ( blue arrow , 12 nm ) , and TOM20 ( red arrow , 6 nm ) are shown . Fluorescence image of HCV core identifies HCV - infected cells ( red ) . Nuclei were stained with DAPI ( blue ) . Scale bar = 0 . 2 µM .

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(E) Immunoelectron microscopic analysis showing the formation of mitophagosome in HCV-infected cells. Huh7 cells infected with HCVcc were stained by triple immunogold labeling method, as described in Materials and Methods. The immunogold particles of GFP-LC3 (white arrow, 18 nm), HCV E2 (blue arrow, 12 nm), and TOM20 (red arrow, 6 nm) are shown. Fluorescence image of HCV core identifies HCV-infected cells (red). Nuclei were stained with DAPI (blue). Scale bar = 0.2 µM.


Confocal microscopy showing the formation of mitophagolysosome in HCV - infected cells . ( A ) Huh7 cells transiently expressing GFP - LC3 protein were infected with HCVcc in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 12 h before fixation . At 3 days post - infection , cells prestained with LysoTracker ( red ) were immunostained with antibodies against TOM20 ( blue ) and HCV core ( cyan ) . Nuclei are demarcated with white dot circles . In the zoomed images , the arrows indicate the colocalization of GFP - LC3 puncta ( green ) , lysosome , and mitochondria in HCV - infected cells ( white puncta ) .

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Confocal microscopy showing the formation of mitophagolysosome in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 days post-infection, cells prestained with LysoTracker (red) were immunostained with antibodies against TOM20 (blue) and HCV core (cyan). Nuclei are demarcated with white dot circles. In the zoomed images, the arrows indicate the colocalization of GFP-LC3 puncta (green), lysosome, and mitochondria in HCV-infected cells (white puncta).


( B ) Ultrastructure of HCV - infected cells showing the formation of mitophagolysosome . HCV FLR - JFH1 replicon cells were examined by electron microscopy . In the zoomed image , the formation of mitophagolysosome in HCV - infected cells is shown . Organelle marker : ML , mitophagolysosome . Scale bar = 0 . 1 µM .

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(B) Ultrastructure of HCV-infected cells showing the formation of mitophagolysosome. HCV FLR-JFH1 replicon cells were examined by electron microscopy. In the zoomed image, the formation of mitophagolysosome in HCV-infected cells is shown. Organelle marker: ML, mitophagolysosome. Scale bar = 0.1 µM.


( C and D ) Quantitative analyses of the colocalization of GFP - LC3 containing lysosomes with mitochondria ( C ) or Parkin ( D ) in the panels ( A ) and Figure S5 ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 001 , * * p < 0 . 05 ) . ( E ) ImageJ quantitative analysis of the number of mitochondria ( mean ± SEM ; n ≥ 10 cells ; * p < 0 . 001 , * * p < 0 . 05 ) . P values were calculated by using an unpaired Student ' s t - test .

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(C and D) Quantitative analyses of the colocalization of GFP-LC3 containing lysosomes with mitochondria (C) or Parkin (D) in the panels (A) and Figure S5 (mean ± SEM; n≥10 cells; *p<0.001, **p<0.05). (E) ImageJ quantitative analysis of the number of mitochondria (mean ± SEM; n≥10 cells; *p<0.001, **p<0.05). P values were calculated by using an unpaired Student's t-test.


A and B ) Inhibitory effect of Parkin , PINKI , and ATG5 silencing on HCV replication . Huh7 cells transfected with Non - Targeting ( NT ) or gene - specific siRNA pools targeting Parkin , PINK1 , and ATG5 genes , respectively , were infected with HCVcc . At day 3 post - infection , the levels of HCV RNA ( A ) and targeted gene

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A and B) Inhibitory effect of Parkin, PINKI, and ATG5 silencing on HCV replication. Huh7 cells transfected with Non-Targeting (NT) or gene-specific siRNA pools targeting Parkin, PINK1, and ATG5 genes, respectively, were infected with HCVcc. At day 3 post-infection, the levels of HCV RNA (A) and targeted gene mRNA


A ( B ) were analyzed by real - time qRT - PCR , as described in Materials and Methods ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . GAPDH was used to normalize changes in Parkin , PINK1 , and ATG5 gene expression .

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A (B) were analyzed by real-time qRT-PCR, as described in Materials and Methods (mean ± SD; n = 3; *p<0.01). GAPDH was used to normalize changes in Parkin, PINK1, and ATG5 gene expression.


( C and D ) Rescue of HCV RNA replication by Parkin overexpression . Huh7 cells stably expressing mock vector ( M - KD ) , Non - target - shRNA ( NT - KD ) , and Parkin - shRNA ( P - KD ) , respectively , were infected with HCVcc . P - KD cells were further transfected with two different concentration of the plasmid DNA encoding wild - type Parkin for 2 days before harvest . At 3 days post - infection , intracellular HCV RNA levels were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in HCV RNA expression ( C ) ( mean ± SD ; n = 3 ; * p < 0 . 01 , * * p < 0 . 05 ) .

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(C and D) Rescue of HCV RNA replication by Parkin overexpression. Huh7 cells stably expressing mock vector (M-KD), Non-target-shRNA (NT-KD), and Parkin-shRNA (P-KD), respectively, were infected with HCVcc. P-KD cells were further transfected with two different concentration of the plasmid DNA encoding wild-type Parkin for 2 days before harvest. At 3 days post-infection, intracellular HCV RNA levels were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in HCV RNA expression (C) (mean ± SD; n = 3; *p<0.01, **p<0.05).


( D ) The expression levels of Parkin and HCV core protein were analyzed by immunoblotting with anti - Parkin and HCV core antibodies . Ectopic expression level of HA - tagged wild - type Parkin was detected by immunoblotting with anti - HA antibody . β - actin was used as an internal loading control . P values were calculated by using an unpaired Student ' s t - test . The relative intensity of HCV core expression normalized to β - actin was analyzed by ImageJ .

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(D) The expression levels of Parkin and HCV core protein were analyzed by immunoblotting with anti-Parkin and HCV core antibodies. Ectopic expression level of HA-tagged wild-type Parkin was detected by immunoblotting with anti-HA antibody. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test. The relative intensity of HCV core expression normalized to β-actin was analyzed by ImageJ.


( A ) Rescue effect of 3 - MA and BafA1 on reduction of mitochondrial complex I enzyme activity caused by HCV infection . Huh7 cells infected with HCVcc were treated with 3 - MA ( 10 mM ) or BafA1 ( 100 nM ) for 12 h before harvest . At 3 days post - infection , the activity of mitochondrial complex I enzyme was measured according to manufacturer ' s instructions ( mean ± SD ; n = 3 ; * p < 0 . 01 ) .

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

(A) Rescue effect of 3-MA and BafA1 on reduction of mitochondrial complex I enzyme activity caused by HCV infection. Huh7 cells infected with HCVcc were treated with 3-MA (10 mM) or BafA1 (100 nM) for 12 h before harvest. At 3 days post-infection, the activity of mitochondrial complex I enzyme was measured according to manufacturer's instructions (mean ± SD; n = 3; *p<0.01).


. ( B ) Rescue effect of Parkin knockdown on reduction of mitochondrial complex I enzyme activity caused by HCV infection . NT - KD and P - KD cells infected with HCVcc were harvested on day 3 post - infection and used for analysis of the activity of mitochondrial complex I enzyme ( mean ± SD ; n = 3 ; * p < 0 . 05 ) .

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. (B) Rescue effect of Parkin knockdown on reduction of mitochondrial complex I enzyme activity caused by HCV infection. NT-KD and P-KD cells infected with HCVcc were harvested on day 3 post-infection and used for analysis of the activity of mitochondrial complex I enzyme (mean ± SD; n = 3; *p<0.05).


( C ) Western blot analysis of mitochondrial respiratory chain complex enzyme expression . NT - KD and P - KD cells were infected with HCVcc and at 3 days post - infection , the expression levels of complex I and IV enzyme were analyzed by immunoblotting with anti - Hu total OxPhos complex antibody . Huh7 cells treated with CCCP ( 10 µM ) for 12 h were also analyzed as a control . β - actin was used as an internal loading control . P values were calculated by using an unpaired Student ' s t - test .

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(C) Western blot analysis of mitochondrial respiratory chain complex enzyme expression. NT-KD and P-KD cells were infected with HCVcc and at 3 days post-infection, the expression levels of complex I and IV enzyme were analyzed by immunoblotting with anti-Hu total OxPhos complex antibody. Huh7 cells treated with CCCP (10 µM) for 12 h were also analyzed as a control. β-actin was used as an internal loading control. P values were calculated by using an unpaired Student's t-test.


( D ) Effect of Parkin silencing on depletion of mitochondrial DNA caused by HCV infection . Huh7 cells transfected with NT or Parkin - specific siRNA pools were infected with HCVcc . At day 3 post - infection , mitochondrial DNA levels of ND - 2 and COX - 2 were analyzed by real - time qPCR . GAPDH was used to normalize changes in ND - 2 and COX - 2 gene expression ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . ( A , B , and D ) P values were calculated by using an unpaired Student ' s t - test .

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(D) Effect of Parkin silencing on depletion of mitochondrial DNA caused by HCV infection. Huh7 cells transfected with NT or Parkin-specific siRNA pools were infected with HCVcc. At day 3 post-infection, mitochondrial DNA levels of ND-2 and COX-2 were analyzed by real-time qPCR. GAPDH was used to normalize changes in ND-2 and COX-2 gene expression (mean ± SD; n = 3; *p<0.01). (A, B, and D) P values were calculated by using an unpaired Student's t-test.


Real - time qRT - PCR analysis of C / EBPβ mRNA in CD4 + naïve T cells inactivated ( naive ) or cultured for 24 hr under conditions as indicated with DMSO or ATRA ( all - trans retinoic acid ) . Data are representative of two independent experiments with consistent results and normalized with β - actin ( mean and s . e . m . of quadruplicates )

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Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid). Data are representative of two independent experiments with consistent results and normalized with β-actin (mean and s.e.m. of quadruplicates)


Immunoblot analysis of C / EBPβ in the nuclear fraction of naïve CD4 + T cells cultured for 2 d under conditions as indicated . The immunoblot was quantified using Image

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Immunoblot analysis of C/EBPβ in the nuclear fraction of naïve CD4+ T cells cultured for 2 d under conditions as indicated. The immunoblot was quantified using Image J


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean value from 8 ( C are shown on the right Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean value from 8(C are shown on the right Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean values fro 6 ( D are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro 6(D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean values fro E 7 ( F are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro E 7(F are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the upper panel and pooled data from 4 ( A are shown in the bottom . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gate . Error bars represent mean ± s . e .

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Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data from 4 (A are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.m


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the upper panel and pooled data fro ) , 3 ( B are shown in the bottom . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gate . Error bars represent mean ± s . e .

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Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data fro ), 3 (B are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.m


Real - time qRT - PCR analysis of FACS - sorted GFP + cells from CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d in the conditions as indicated . Data are representative of two independent experiments with consistent results and normalized with β - actin ( mean ± s . e . m . of triplicates )

Real-time qRT-PCR analysis of FACS-sorted GFP+ cells from CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d in the conditions as indicated. Data are representative of two independent experiments with consistent results and normalized with β-actin (mean ± s.e.m. of triplicates)


Body weight of RAG - 2 - / - mice after transfer with 5 x 105 CD4 + CD45RBhigh cells alone ( circles ) or with 2 x 105 FACS - sorted CD4 + GFP + cells transduced with control retrovirus ( MigRI ) ( squares ) or retrovirus encoding C / EBPβ ( triangles ) and cultured for 2 d in the presence of TGF - β and culture supernatant . Each time point contains three ( circles ) or four ( squares and triangles ) mice in each group . Error bars represent mean ± s .

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Body weight of RAG-2-/- mice after transfer with 5 x 105 CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD4+GFP+ cells transduced with control retrovirus (MigRI) (squares) or retrovirus encoding C/EBPβ (triangles) and cultured for 2 d in the presence of TGF-β and culture supernatant. Each time point contains three (circles) or four (squares and triangles) mice in each group. Error bars represent mean ± s.d


Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm Histological scores Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

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Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm Histological scores Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


Comparison of the spleen weight Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

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Comparison of the spleen weight Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb / c mice that received RAG2 - / - DO11 . 10 CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPβ . ( A ) The recipients were immunized via intravenous injection of OVA323 - 339 peptide ( 20 ug ) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization . ( B ) The recipients were fed 1 % OVA solution in drinking water for 5 consecutive days . Representative experiments are shown in the left panel and pooled data from 4 ( A ) or 3 ( B ) independent experiments with mean values are shown on the right . Dot plots are gated for CD4 + KJ . 1 . 26 + GFP + ( top ) or CD4 + KJ . 1 . 26 + GFP - ( bottom ) and numbers indicate percent Foxp3 + cells in the gate . Statistical analysis was performed using one - way ANOVA . ( * p < 0 . 05 , * * p < 0 . 01 ; ns , not

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Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb/c mice that received RAG2-/-DO11.10 CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPβ. (A) The recipients were immunized via intravenous injection of OVA323-339 peptide (20 ug) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization. (B) The recipients were fed 1% OVA solution in drinking water for 5 consecutive days. Representative experiments are shown in the left panel and pooled data from 4 (A) or 3 (B) independent experiments with mean values are shown on the right. Dot plots are gated for CD4+KJ.1.26+GFP+ (top) or CD4+KJ.1.26+GFP- (bottom) and numbers indicate percent Foxp3+ cells in the gate. Statistical analysis was performed using one-way ANOVA. (*p<0.05, **p<0.01; ns, not significant


EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl - or methyl - CRE sequence in the

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EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl- or methyl-CRE sequence in the TSDR


ChIP - qPCR assay of C / EBPβ on the Foxp3 TSDR in naïve CD4 + T cells cultured for 2 d under conditions as indicated . Data are representative of two independent experiments with consistent results ( mean ± s . e . m . of triplicates )

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ChIP-qPCR assay of C/EBPβ on the Foxp3 TSDR in naïve CD4+ T cells cultured for 2 d under conditions as indicated. Data are representative of two independent experiments with consistent results (mean ± s.e.m. of triplicates)


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data from 4 ( C with mean values are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data from 4 (C with mean values are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data fro 5 ( D are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

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

Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data fro 5 (D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


CD4 + naïve T cells from Foxp3EGFP mice were transduced with control retrovirus ( MIN ) or retrovirus encoding C / EBPβ ( MIN - C / EBPβ ) and stimulated with anti - CD3 and anti - CD28 for 2 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . GFP + ( Foxp3 + ) hNGFR + T cells were sorted to high purity ( 0 hr ) and restimulated with anti - CD3 and anti - CD28 for 2 d in the presence of IL - 4 , IL - 6 , IL - 12 or none . Foxp3 expression was assessed based on GFP expression . Data are representative of two independent experiments with consistent

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

CD4+ naïve T cells from Foxp3EGFP mice were transduced with control retrovirus (MIN) or retrovirus encoding C/EBPβ (MIN-C/EBPβ) and stimulated with anti-CD3 and anti-CD28 for 2 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. GFP+(Foxp3+) hNGFR+ T cells were sorted to high purity (0 hr) and restimulated with anti-CD3 and anti-CD28 for 2 d in the presence of IL-4, IL-6, IL-12 or none. Foxp3 expression was assessed based on GFP expression. Data are representative of two independent experiments with consistent results


Flow cytometry of CFSE - labeled CD45 . 1 + CD4 + T cells stimulated 3 d alone or together with CD45 . 2 + GFP ( Foxp3 ) + NGFR + - sorted CD4 + naïve T cells from Foxp3EGFP mice transduced with control retrovirus ( Min ) or retrovirus encoding C / EBPβ and cultured for 3 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . Histograms are gated for CD45 . 1 + . The ratios shown are responder to suppressor . Data are representative of two independent experiments with consistent

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

Flow cytometry of CFSE-labeled CD45.1+CD4+ T cells stimulated 3 d alone or together with CD45.2+GFP(Foxp3)+NGFR+-sorted CD4+ naïve T cells from Foxp3EGFP mice transduced with control retrovirus (Min) or retrovirus encoding C/EBPβ and cultured for 3 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Histograms are gated for CD45.1+. The ratios shown are responder to suppressor. Data are representative of two independent experiments with consistent results


EAE disease course of RAG2 - / - mice that received CD45 . 1 + 2D2CD4 + naive T cells alone or together with CD4 + GFP / Foxp3 + iTreg cells from CD45 . 1 - 2D2CD4 + GFP ( Foxp3 ) + naïve T cells transduced with either control retrovirus ( MIN ) or retrovirus encoding C / EBPβ , cultured in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs and sorted based on GFP / Foxp3 expression . The recipients were immunized with myelin oligodendrocyte glycoprotein peptide ( 100 ug ) 1 d after transfer . Data show mean ± s . e . m of the EAE clinical score of 9 ( day 1 to day 11 ) , 5 ( day 12 to day 16 ) or 4 ( day 17 to day 20 ) mice of each

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

EAE disease course of RAG2-/- mice that received CD45.1+2D2CD4+ naive T cells alone or together with CD4+GFP/Foxp3+ iTreg cells from CD45.1-2D2CD4+GFP(Foxp3)+ naïve T cells transduced with either control retrovirus (MIN) or retrovirus encoding C/EBPβ, cultured in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs and sorted based on GFP/Foxp3 expression. The recipients were immunized with myelin oligodendrocyte glycoprotein peptide (100 ug) 1 d after transfer. Data show mean ± s.e.m of the EAE clinical score of 9 (day 1 to day 11), 5 (day 12 to day 16) or 4 (day 17 to day 20) mice of each group


The percentage of IL - 17 - producing cells in CD45 . 1 + Vβ11 + CD4 + T cells in the spinal cords 11 days after transfer Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . d )

The percentage of IL-17-producing cells in CD45.1+Vβ11+ CD4+ T cells in the spinal cords 11 days after transfer Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)


The absolute number of IL - 17A + CD45 . 1 + Vβ11 + CD4 + T cells in the spinal cords 11 days after transfer . Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . d )

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

The absolute number of IL-17A+CD45.1+Vβ11+CD4+ T cells in the spinal cords 11 days after transfer. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)


Flow cytometry of Foxp3 staining of splenic cells . Histograms are gated for CD45 . 1 - CD4 + Vβ11 + . Data are representative of two independent experiments with consistent

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

Flow cytometry of Foxp3 staining of splenic cells. Histograms are gated for CD45.1-CD4+Vβ11+. Data are representative of two independent experiments with consistent results


Body weight of RAG - 2 - / - mice after transfer with 5 x 105 CD45 . 1 + CD4 + CD45RBhigh cells alone ( circles ) or with 2 x 105 FACS - sorted CD45 . 1 - CD4 + GFP ( Foxp3 ) + NGFR + cells transduced with control retrovirus ( Min ) ( filled rectangles ) or retrovirus encoding C / EBPβ ( empty rectangles ) and cultured for 2 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . Each time point contains three mice in each group . Error bars represent mean ± s . e .

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

Body weight of RAG-2-/- mice after transfer with 5 x 105 CD45.1+CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD45.1-CD4+GFP(Foxp3)+NGFR+ cells transduced with control retrovirus (Min)(filled rectangles) or retrovirus encoding C/EBPβ (empty rectangles) and cultured for 2 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Each time point contains three mice in each group. Error bars represent mean ± s.e.m


Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm )

Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm)


Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm ) Histological scores . Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

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

Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm) Histological scores. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


The percentage of Foxp3 + cells in CD45 . 1 - CD4 + T cells in colonic lamina propria 11 weeks after

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

The percentage of Foxp3+ cells in CD45.1-CD4+ T cells in colonic lamina propria 11 weeks after transfer


Flow cytometry of intracellular FOXP3 staining ( right ) or isotype control ( left ) in human CD4 + CD25 - CD45RA + naïve T cells transduced with control retrovirus ( Min ) or retrovirus encoding hC / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean ± s . e . m are shown on the right . Histograms are gated for CD4 + NGFR + . Statistical analysis was performed using unpaired two - tailed t test . ( * p < 0 . 05 ; ns , not significant )

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

Flow cytometry of intracellular FOXP3 staining (right) or isotype control (left) in human CD4+CD25-CD45RA+ naïve T cells transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean±s.e.m are shown on the right. Histograms are gated for CD4+NGFR+. Statistical analysis was performed using unpaired two-tailed t test. (*p<0.05; ns, not significant)


Flow cytometry of CFSE - labeled human CD4 + CD25 - T cells ( responder ) stimulated 4 d alone or together with NGFR + - sorted human CD4 + CD25 - CD45RA + naïve T cells ( suppressor ) transduced with control retrovirus ( Min ) or retrovirus encoding hC / EBPβ and cultured for 5 d in the presence of hTGF - β , anti - hIFN - γ and anti - hIL - 4 Abs . Histograms are gated for CFSE + . The ratio of responder to suppressor is 1 : 0 . 2 ( top ) or 1 : 0 . 4 ( bottom ) . Data are representative of two independent experiments with consistent

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

Flow cytometry of CFSE-labeled human CD4+CD25- T cells (responder) stimulated 4 d alone or together with NGFR+-sorted human CD4+CD25-CD45RA+ naïve T cells (suppressor) transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 5 d in the presence of hTGF-β, anti-hIFN-γ and anti-hIL-4 Abs. Histograms are gated for CFSE+. The ratio of responder to suppressor is 1:0.2 (top) or 1:0.4(bottom). Data are representative of two independent experiments with consistent results


( E , F ) Kymograph and individual images of time - lapse observation of Mal3 - GFP ( green ) and Dli1 - 3mCherry ( magenta ) along the longitudinal axis . Yellow arrowheads indicate temporary accumulation of growing plus tips during the SPB is turning . Blue asterisks indicate dynein accumulating at the distal cell end cortex in the direction of the SPB movement . Individual images indicated by ( a - d ) are shown in ( F ) . Data information : Scale 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 O O 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, F) Kymograph and individual images of time-lapse observation of Mal3-GFP (green) and Dli1-3mCherry (magenta) along the longitudinal axis. Yellow arrowheads indicate temporary accumulation of growing plus tips during the SPB is turning. Blue asterisks indicate dynein accumulating at the distal cell end cortex in the direction of the SPB movement. Individual images indicated by (a-d) are shown in (F). Data information: Scale bars, 5 µm.


( I ) Kymographs of the SPB ( Sfi1 - mCherry , yellow ) and histone H2B ( Htb1 - CFP , blue ) in dhc1 ∆ mutant . At 20 min ( white solid lines ) , dimethyl sulfoxide ( DMSO , as a negative control ) or MBC ( final concentration of 20 µg / mL ) were added . Dashed lines indicate cell shape . Data information : Scale 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 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

(I) Kymographs of the SPB (Sfi1-mCherry, yellow) and histone H2B (Htb1-CFP, blue) in dhc1∆ mutant. At 20 min (white solid lines), dimethyl sulfoxide (DMSO, as a negative control) or MBC (final concentration of 20 µg/mL) were added. Dashed lines indicate cell shape. Data information: Scale bars, 5 µm.


( E - H ) Spectrograms indicating changes in the power spectral density for variable parameters in the pulling - only model . The gray scale ( black to white ) corresponds to the values of the power spectral density ( low to high ) . The default parameters are indicated by the arrowheads on the horizontal axes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) Spectrograms indicating changes in the power spectral density for variable parameters in the pulling-only model. The gray scale (black to white) corresponds to the values of the power spectral density (low to high). The default parameters are indicated by the arrowheads on the horizontal axes.


( D ) Scatter plot of cell length versus peak frequency during the azygotic oscillation . n = 45 cells .

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

(D) Scatter plot of cell length versus peak frequency during the azygotic oscillation. n = 45 cells.


( C ) Tracks of the SPB along the longitudinal ( x , blue line ) and lateral ( y , red line ) axes . The 64 - minute period that gives the fastest movement of the SPB is indicated by the green bar and analyzed in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tracks of the SPB along the longitudinal (x, blue line) and lateral (y, red line) axes. The 64-minute period that gives the fastest movement of the SPB is indicated by the green bar and analyzed in (D).


( A ) Time - lapse observation of microtubules ( GFP - Atb2 , green ) , dynein ( Dli1 - 3mCherry , red ) and the SPB ( Sad1 - CFP , blue ) in wild - type and klp6 ∆ cells . Arrowheads indicate the SPB . The arrow shows an extensively elongated microtubule along the cell cortex in a klp6 ∆ cell . Data information : Scale 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 O O O O O O O O O

(A) Time-lapse observation of microtubules (GFP-Atb2, green), dynein (Dli1-3mCherry, red) and the SPB (Sad1-CFP, blue) in wild-type and klp6∆ cells. Arrowheads indicate the SPB. The arrow shows an extensively elongated microtubule along the cell cortex in a klp6∆ cell. Data information: Scale bars, 5 µm.


( B and C ) Time - lapse observation of Klp6 ( Klp6 - GFP , green ) , dynein ( Dli1 - 3mCherry , red ) and microtubules ( CFP - Atb2 , blue ) . A magnified view of the square area is shown in ( C ) . Arrowheads indicate accumulation of Klp6 on microtubules extending along the cell cortex . Data information : Scale 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 O O O O O O O

(B and C) Time-lapse observation of Klp6 (Klp6-GFP, green), dynein (Dli1-3mCherry, red) and microtubules (CFP-Atb2, blue). A magnified view of the square area is shown in (C). Arrowheads indicate accumulation of Klp6 on microtubules extending along the cell cortex. Data information: Scale bars, 5 µm.


( A ) Representative immunofluorescence images of Sec31A marked ERES in HeLa cells cultured on crossbow shaped micropatterned surface of small ( upper panel ) or large ( lower panel ) size for 4 hours . Cells were treated with DMSO ( Ctrl ) or 50 µM NSC23766 ( Rac1 inhibitor ) for 4 hours prior to fixation and immunostaining . The number of ERES per cell for each pattern size in HeLa ( B ) The number of ERES was quantified from at least 74 cells per condition for HeLa ( B ) Each dot represents an individual cell .

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

(A) Representative immunofluorescence images of Sec31A marked ERES in HeLa cells cultured on crossbow shaped micropatterned surface of small (upper panel) or large (lower panel) size for 4 hours. Cells were treated with DMSO (Ctrl) or 50 µM NSC23766 (Rac1 inhibitor) for 4 hours prior to fixation and immunostaining. The number of ERES per cell for each pattern size in HeLa (B) The number of ERES was quantified from at least 74 cells per condition for HeLa (B) Each dot represents an individual cell.


( A ) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries . Cells were transfected with 10 nM non - targeting control ( siCtrl ) or co - transfected with siRNAs targeting Sar1A and Sar1B . After 48 h , cells were trypsinized and 80 , 000 cells were seeded on micropatterned chips . Cells were allowed to attach for 3 h , stained with CellMask for 5 min and processed for microscopy . Scale bars : 5 µm ( B ) Quantification graph showing the percentage of cells failing to entirely cover the different geometries ( described in A ) . Data derived from at least 100 cells from a set of three independent experiments . Error bars represent standard deviation . Asterisks ( * ) denote statistical significance ( p - value < 0 . 05 ; chi - squared test ) .

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

(A) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were transfected with 10 nM non-targeting control (siCtrl) or co-transfected with siRNAs targeting Sar1A and Sar1B. After 48 h, cells were trypsinized and 80,000 cells were seeded on micropatterned chips. Cells were allowed to attach for 3 h, stained with CellMask for 5 min and processed for microscopy. Scale bars: 5 µm (B) Quantification graph showing the percentage of cells failing to entirely cover the different geometries (described in A). Data derived from at least 100 cells from a set of three independent experiments. Error bars represent standard deviation. Asterisks (*) denote statistical significance (p-value < 0.05; chi-squared test).


( D ) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries . Cells were treated with 50 µM of the Rac1 inhibitor NSC23766 for 4 hours prior to staining with CellMask and processing for immunofluorescence microscopy . ( E ) Quantification of ( D ) . Percentage of cells failing to cover entire micropatterned surface is shown for control ( ctrl ) and the Rac1 inhibitor NSC23766 . Between 113 - 117 cells were counted in total from a set of three independent experiments .

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

(D) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were treated with 50 µM of the Rac1 inhibitor NSC23766 for 4 hours prior to staining with CellMask and processing for immunofluorescence microscopy. (E) Quantification of (D). Percentage of cells failing to cover entire micropatterned surface is shown for control (ctrl) and the Rac1 inhibitor NSC23766. Between 113 - 117 cells were counted in total from a set of three independent experiments.


( A ) HeLa cells were transfected with 10 nM non - targeting control siRNA ( siCtrl ) or siRNAs targeting Rac1 ( siRac1 ) . After 72 h , cells were fixed and processed for immunostaining against Sec31A to label ERES . Representative confocal microscopy images are shown . ( B ) Quantification graph shows the number of ERES / cell in cells transfected with control siRNA ( siCtrl ) or siRNA against Rac1 ( two siRNAs # 1 and # 2 ) . Values are expressed as % of siCtrl . From three independent experiments , with > 30 cells per condition were counted . ( C ) Representative immunofluorescence images showing ERES in HeLa cells treated with DMSO ( Ctrl ) , Rac1 inhibitor NSC23766 ( 50 µM , 4 h ) , or NSC23766 washout ( 50 µM for 4h , then washout for 2h ) . ( D ) Graph shows the number of ERES per cell ( displayed as % of Ctrl ) derived from at least 30 cells per condition from three experiments .

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

(A) HeLa cells were transfected with 10 nM non-targeting control siRNA (siCtrl) or siRNAs targeting Rac1 (siRac1). After 72 h, cells were fixed and processed for immunostaining against Sec31A to label ERES. Representative confocal microscopy images are shown. (B) Quantification graph shows the number of ERES/cell in cells transfected with control siRNA (siCtrl) or siRNA against Rac1 (two siRNAs #1 and #2). Values are expressed as % of siCtrl. From three independent experiments, with >30 cells per condition were counted. (C) Representative immunofluorescence images showing ERES in HeLa cells treated with DMSO (Ctrl), Rac1 inhibitor NSC23766 (50 µM, 4 h), or NSC23766 washout (50 µM for 4h, then washout for 2h). (D) Graph shows the number of ERES per cell (displayed as % of Ctrl) derived from at least 30 cells per condition from three experiments.


( E ) Fluorescence recovery after photobleaching ( FRAP ) of GFP - Sec16A marked ERES in HeLa cells . Images show an individual ERES before ( - 1 ) , immediately after ( 0 ) and 28 sec after photobleaching . The graph illustrates FRAP analysis of individual ERES from 23 ( for control ) and 24 ( for NSC23766 ) regions in 3 experiments . Line connects individual time points . Data are ± SD . ( F ) Quantification of the mobile fraction of GFP - Sec16A in control and Rac1 inhibited cells . Mobile fraction is derived from a total of 23 ( Ctrl ) and 24 ( NSC23766 ) individual ERES from three independent experiments .

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

(E) Fluorescence recovery after photobleaching (FRAP) of GFP-Sec16A marked ERES in HeLa cells. Images show an individual ERES before (-1), immediately after (0) and 28 sec after photobleaching. The graph illustrates FRAP analysis of individual ERES from 23 (for control) and 24 (for NSC23766) regions in 3 experiments. Line connects individual time points. Data are ± SD. (F) Quantification of the mobile fraction of GFP-Sec16A in control and Rac1 inhibited cells. Mobile fraction is derived from a total of 23 (Ctrl) and 24 (NSC23766) individual ERES from three independent experiments.


( G ) The rate of ER - export was monitored using GFP - ManII - RUSH in HeLa cells after perturbation of Rac1 with siRNA ( siRac1 ) , or Rac1 inhibitor NSC23766 , or a combination of both . Representative images show GFP - ManII - RUSH distribution in HeLa cells at indicated time points . ( H ) Quantification shows ratio of ManII fluorescence intensity within Golgi to outside Golgi region after addition of biotin at indicated time points . Between 76 - 104 cells were used for measurement of ManII intensity in 3 experiments .

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

(G) The rate of ER-export was monitored using GFP-ManII-RUSH in HeLa cells after perturbation of Rac1 with siRNA (siRac1), or Rac1 inhibitor NSC23766, or a combination of both. Representative images show GFP-ManII-RUSH distribution in HeLa cells at indicated time points. (H) Quantification shows ratio of ManII fluorescence intensity within Golgi to outside Golgi region after addition of biotin at indicated time points. Between 76 - 104 cells were used for measurement of ManII intensity in 3 experiments.


( A ) Co - occurrence of mCherry - Rac1 and GFP - Sec61β at the endoplasmic reticulum ( ER ) in HeLa cells stably expressing mCherry - Rac1 and transiently expressing GFP - Sec61β as monitored by confocal microscopy . SRRF imaging mode was used in Andor DragonFly spinning disk confocal microscope for obtaining the image . Scale bar : 5 µm and 1 µm ( inset ) . See also movieEV3 . ( B ) Line profile of two marked spots from ( A ) showing the overlap between mCherry - Rac1 and GFP - Sec61β signal at the ER .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-occurrence of mCherry-Rac1 and GFP-Sec61β at the endoplasmic reticulum (ER) in HeLa cells stably expressing mCherry-Rac1 and transiently expressing GFP-Sec61β as monitored by confocal microscopy. SRRF imaging mode was used in Andor DragonFly spinning disk confocal microscope for obtaining the image. Scale bar: 5 µm and 1 µm (inset). See also movieEV3. (B) Line profile of two marked spots from (A) showing the overlap between mCherry-Rac1 and GFP-Sec61β signal at the ER.


( E ) Representative images showing Sec31A marked ERES in HeLa cells transfected with indicated plasmids . HeLa cells were transfected with the plasmids 16 h before immunolabelling with Sec31A . Scale bar : 5 µm . ( F ) Quantification of ERES number in HeLa cells from the experiment described in C . Between 90 - 100 cells were quantified from 4 independent experiments . Average ERES per cell ± standard deviation is shown . Each dot represents average ERES / cell from an experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images showing Sec31A marked ERES in HeLa cells transfected with indicated plasmids. HeLa cells were transfected with the plasmids 16 h before immunolabelling with Sec31A. Scale bar: 5 µm. (F) Quantification of ERES number in HeLa cells from the experiment described in C. Between 90-100 cells were quantified from 4 independent experiments. Average ERES per cell ± standard deviation is shown. Each dot represents average ERES/cell from an experiment.


( G ) Images represent ERES in HeLa cells that were transfected with the indicated plasmids and cultured on the crossbow micropattern for 4 hours . Cells were transfected with the plasmids 16 h before they were trypsinized and seeded on the micropatterns for ERES staining . ( H ) The dot plot shows ERES count in individual cells from ( G ) . Mean ± standard deviation is shown for each condition . At least 58 cells were used for counting ERES number . Independent experiments were performed 3 times .

(G) Images represent ERES in HeLa cells that were transfected with the indicated plasmids and cultured on the crossbow micropattern for 4 hours. Cells were transfected with the plasmids 16 h before they were trypsinized and seeded on the micropatterns for ERES staining. (H) The dot plot shows ERES count in individual cells from (G). Mean ± standard deviation is shown for each condition. At least 58 cells were used for counting ERES number. Independent experiments were performed 3 times.


( I ) Merged confocal images show distribution of Rac1 specific GEF TIAM ( magenta ) in HeLa cells before ( 0 ) , and after optogenetic recruitment at indicated time points . CIBN - Sec61β ( green ) was used to recruit Cry2 - TIAM to the ER with 488 nm laser . ( J ) Graph shows the number of SNAP - Sec16A marked ERES in HeLa cells before ( 0 ) and after ( 30 , 60 and 90 seconds ) recruitment of TIAM to the ER from 2 independent experiments . Whiskers indicate min - max range and central band indicates the median of all data . Number of cells = 9 .

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(I) Merged confocal images show distribution of Rac1 specific GEF TIAM (magenta) in HeLa cells before (0), and after optogenetic recruitment at indicated time points. CIBN-Sec61β (green) was used to recruit Cry2-TIAM to the ER with 488 nm laser. (J) Graph shows the number of SNAP-Sec16A marked ERES in HeLa cells before (0) and after (30, 60 and 90 seconds) recruitment of TIAM to the ER from 2 independent experiments. Whiskers indicate min-max range and central band indicates the median of all data. Number of cells = 9.


( A and B ) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO ( Ctrl ) , 0 . 5 µM cytochalasin D ( CytoD ) and 0 . 5 µM latrunculin A ( LatA ) for 15 and 30 min ( A ) . Scale bar : 5 µm . The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation ( 90 - 100 cells from 3 experiments ) ( B ) .

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(A and B) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO (Ctrl), 0.5 µM cytochalasin D (CytoD) and 0.5 µM latrunculin A (LatA) for 15 and 30 min (A). Scale bar: 5 µm. The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation (90 -100 cells from 3 experiments) (B).


( C ) Distribution of ManII - RUSH in HeLa cells . Cells were pre - treated with DMSO ( Ctrl ) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII - trafficking . Golgi arrival for ManII - RUSH was monitored for 30 min .

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(C) Distribution of ManII-RUSH in HeLa cells. Cells were pre-treated with DMSO (Ctrl) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII-trafficking. Golgi arrival for ManII-RUSH was monitored for 30 min.


( D - E ) Graph showing ratio of ManII within Golgi to outside Golgi . Values were derived from HeLa cells treated with LatA or CytoD as described in C . At least 30 cells per condition were used for quantification in each experiment ( n = 3 ) .

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(D-E) Graph showing ratio of ManII within Golgi to outside Golgi. Values were derived from HeLa cells treated with LatA or CytoD as described in C. At least 30 cells per condition were used for quantification in each experiment (n = 3).


( F ) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D ( CytoD ) and ERES were labelled with anti - Sec31A antibody before ( no strain ) or after 15 % equibiaxial strain ( 15 % strain ) . Anti - phalloidin staining was performed to visualize actin in cells . Representative images are shown . ( G ) Quantification of experiment described in ( F ) . The graph shows average ERES / Cell . Data were derived from a total of 90 - 115 cells from 3 experiments . Each dot represents several cells and error bars show standard deviation .

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(F) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D (CytoD) and ERES were labelled with anti-Sec31A antibody before (no strain) or after 15% equibiaxial strain (15% strain). Anti-phalloidin staining was performed to visualize actin in cells. Representative images are shown. (G) Quantification of experiment described in (F). The graph shows average ERES/Cell. Data were derived from a total of 90 - 115 cells from 3 experiments. Each dot represents several cells and error bars show standard deviation.


( H and I ) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect . HeLa cells were transfected with non - targeting control siRNA ( siCtrl ) or siRNA against INF2 ( siINF2 ) for 72 h before processing cells immunostaining with Sec31A to quantify ERES . Representative images showing Sec31A - labelled ERES in HeLa cells ( H ) , and quantification of ERES per cell presented as % of control ( I ) . In total 137 cells ( siCtrl ) and 113 cells ( siINF2 ) were used for ERES quantification from three independent experiments .

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(H and I) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect. HeLa cells were transfected with non-targeting control siRNA (siCtrl) or siRNA against INF2 (siINF2) for 72 h before processing cells immunostaining with Sec31A to quantify ERES. Representative images showing Sec31A-labelled ERES in HeLa cells (H), and quantification of ERES per cell presented as % of control (I). In total 137 cells (siCtrl) and 113 cells (siINF2) were used for ERES quantification from three independent experiments.


( J and K ) Knockdown of INF2 does not alter the kinetics of ER - to - Golgi transport . HeLa cells stably expressing ManII - RUSH were transfected with siRNAs as described in E , and the ER - to - Golgi transport of ManII - RUSH was followed by confocal microscopy ( J ) . Representative images for indicated time points are shown . Scale bars , 5 µm . The ratio of ManII - RUSH within the Golgi to outside the Golgi was calculated for the indicated time points ( K ) . Three independent experiments were performed and a total of 82 ( siCtrl , 0 min ) , 72 ( siINF2 , 0 min ) and 93 ( siCtrl and siINF2 , 20 min ) were used for quantification .

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(J and K) Knockdown of INF2 does not alter the kinetics of ER-to-Golgi transport. HeLa cells stably expressing ManII-RUSH were transfected with siRNAs as described in E, and the ER-to-Golgi transport of ManII-RUSH was followed by confocal microscopy (J). Representative images for indicated time points are shown. Scale bars, 5 µm. The ratio of ManII-RUSH within the Golgi to outside the Golgi was calculated for the indicated time points (K). Three independent experiments were performed and a total of 82 (siCtrl, 0 min), 72 (siINF2, 0 min) and 93 (siCtrl and siINF2, 20 min) were used for quantification.


( A ) Co - localization of mCherry - Rac1 and GFP - Sec16A in HeLa cells as monitored by SRRF live cell imaging . See also movieS4 .

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(A) Co-localization of mCherry-Rac1 and GFP-Sec16A in HeLa cells as monitored by SRRF live cell imaging. See also movieS4.


( B ) Representative immunofluorescence images of HeLa cells co - transfected with YFP ( N ) - Rac1 and Sar1 - YFP ( C ) and immunostained with Sec31A . Rac1 and Sar1 form complexes at ERES in HeLa cells , which partially overlap with Sec31A . ( C ) Box plot showing the fraction of ERES that overlap with the Rac1 - Sar1 complex ( number of cells = 24 , number of experiments = 4 ) . Individual data points shown in the graph represent number of images . Whiskers indicate min - max range and central band indicates the median of all data .

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(B) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and immunostained with Sec31A. Rac1 and Sar1 form complexes at ERES in HeLa cells, which partially overlap with Sec31A. (C) Box plot showing the fraction of ERES that overlap with the Rac1-Sar1 complex (number of cells = 24, number of experiments = 4). Individual data points shown in the graph represent number of images. Whiskers indicate min-max range and central band indicates the median of all data.


( D ) GFP - tagged Rac1 was immunoprecipitated by GFP - trap and the fraction was immunoblotted for endogenous Sar1 . Representative immunoblot showing co - immunoprecipitation of Sar1 by Rac1 .

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(D) GFP-tagged Rac1 was immunoprecipitated by GFP-trap and the fraction was immunoblotted for endogenous Sar1. Representative immunoblot showing co-immunoprecipitation of Sar1 by Rac1.


( E ) Representative immunofluorescence images of HeLa cells co - transfected with YFP ( N ) - Rac1 and Sar1 - YFP ( C ) and cultured on crossbow micropatterns of indicated size . Cells were co - transfected with the indicated DNA constructs and incubated overnight prior to trypsinization and seeding on the micropatterned coverslip . Cells were allowed to grow for 3h , fixed and processed for microscopy . ( F ) Quantification of Rac1 - Sar1 puncta described in ( E ) ( number of experiments = 3 , number of cells = 28 ( small ) and 27 ( large ) ) . In the quantification graph , error bars represent standard deviation and asterisk ( * ) denotes statistical significance at p - value < 0 . 05 .

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(E) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and cultured on crossbow micropatterns of indicated size. Cells were co-transfected with the indicated DNA constructs and incubated overnight prior to trypsinization and seeding on the micropatterned coverslip. Cells were allowed to grow for 3h, fixed and processed for microscopy. (F) Quantification of Rac1-Sar1 puncta described in (E) (number of experiments = 3, number of cells = 28 (small) and 27 (large)). In the quantification graph, error bars represent standard deviation and asterisk (*) denotes statistical significance at p-value < 0.05.


( E ) Cells expressing GFP - Rac1 were lysed followed by immunoprecipitation against GFP and immunoblotting against endogenous Sar1 and Sec12 . Blotting against GFP was performed to determine efficiency of the IP .

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(E) Cells expressing GFP-Rac1 were lysed followed by immunoprecipitation against GFP and immunoblotting against endogenous Sar1 and Sec12. Blotting against GFP was performed to determine efficiency of the IP.


( F ) HeLa cells were co - transfected with the indicated plasmids and cells were incubated overnight prior to cell lysis and immunoprecipitation against GFP . Immunoblotting against anti - mCherry was performed to detect Sar1A - mCherry . The IP efficiency was determined using anti - GFP . Different exposure times are shown for anti - mCherry blot .

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(F) HeLa cells were co-transfected with the indicated plasmids and cells were incubated overnight prior to cell lysis and immunoprecipitation against GFP. Immunoblotting against anti-mCherry was performed to detect Sar1A-mCherry. The IP efficiency was determined using anti-GFP. Different exposure times are shown for anti-mCherry blot.


( A ) Microsomes isolated from HeLa cells were incubated as indicated . Active Sar1 pulldown was then carried out , followed by immunoblotting against Sar1 .

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(A) Microsomes isolated from HeLa cells were incubated as indicated. Active Sar1 pulldown was then carried out, followed by immunoblotting against Sar1.


( B ) Microsomes ( M ) from HeLa cells stably expressing mCherry - ManII - RUSH were incubated with ATP and an ATP - regenerating system , GTP , biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25 ° C . COPII vesicles ( V ) were pelleted by ultracentrifugation ( 100 , 000 xg ) and immunoblotted for anti - mCherry to detect ManII - RUSH and anti - calnexin ( ER marker ) . ( C ) Quantification data of two independent vesicle budding assays . All values were normalized to 4 ℃ Ctrl condition .

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(B) Microsomes (M) from HeLa cells stably expressing mCherry-ManII-RUSH were incubated with ATP and an ATP-regenerating system, GTP, biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25°C. COPII vesicles (V) were pelleted by ultracentrifugation (100,000 xg) and immunoblotted for anti-mCherry to detect ManII-RUSH and anti-calnexin (ER marker). (C) Quantification data of two independent vesicle budding assays. All values were normalized to 4 ℃ Ctrl condition.


Purified NK cells were activated with IL - 2 or IL - 15 in the presence or absence of PGE2 ( 1µM ) and tested for ( A ) proliferation by flow cytometry for Ki - 67 staining after four days of culture ( n = 6 , biological replicates ) ,

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (A) proliferation by flow cytometry for Ki-67 staining after four days of culture (n=6, biological replicates),


Purified NK cells were activated with IL - 2 or IL - 15 in the presence or absence of PGE2 ( 1µM ) and tested for , ( B ) ability to kill K562 cells after two days of culture in a 4 - hour chromium - assay at an E : T ratio of 5 : 1 ( n = 4 , biological replicates ) .

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for , (B) ability to kill K562 cells after two days of culture in a 4-hour chromium-assay at an E:T ratio of 5:1 (n=4, biological replicates).


Purified NK cells were activated with IL - 2 or IL - 15 in the presence or absence of PGE2 ( 1µM ) and tested for ( C ) Viability by flow cytometry using fixable live / dead staining after two days of culture ( n = 7 , biological replicates ) .

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Purified NK cells were activated with IL-2 or IL-15 in the presence or absence of PGE2 (1µM) and tested for (C) Viability by flow cytometry using fixable live/dead staining after two days of culture (n=7, biological replicates).


D ) Intracellular cAMP detected in two - day IL - 2 or IL - 15 activated NK cells before and 5 minutes after the addition of PGE2 ( 1 µM ) ( n = 3 , biological replicates )

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D) Intracellular cAMP detected in two-day IL-2 or IL-15 activated NK cells before and 5 minutes after the addition of PGE2 (1 µM) (n=3, biological replicates)


E ) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells ( n = 3 - 4 , biological replicates ) .

E) mRNA levels of selected PGE2 response elements as measured by qPCR in NK cells (n=3-4, biological replicates).


F ) Fold change comparing flow cytometry mean fluorescence intensity ( MFI ) values of PDE4A expression between IL - 2 and IL - 15 activated NK cells after two days activation with cytokines ( n = 5 , biological replicates ) .

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F) Fold change comparing flow cytometry mean fluorescence intensity (MFI) values of PDE4A expression between IL-2 and IL-15 activated NK cells after two days activation with cytokines (n=5, biological replicates).


G ) Representative images showing PDE4A expression in NK cells activated by IL - 2 or IL - 15 for two days . Scale bar : 10 μm .

G) Representative images showing PDE4A expression in NK cells activated by IL-2 or IL-15 for two days. Scale bar: 10 μm.


A - B ) NK cells were activated with IL - 2 or IL - 15 for two days in the presence or absence of PGE2 ( 1µM ) and analyzed for A ) the levels of phosphorylated S6 ( pS235 / pS236 ) , STAT3 ( S727 ) , STAT5 ( Y694 ) , Akt ( S473 ) ( n = 4 , biological replicates ) and B ) 16 categories of phosphorylation array showed . Red box indicates category 1 ( pS6 + , pSTAT3 + , pSTAT5 + , pAKT + ) .

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A-B) NK cells were activated with IL-2 or IL-15 for two days in the presence or absence of PGE2 (1µM) and analyzed for A) the levels of phosphorylated S6 (pS235/pS236), STAT3 (S727), STAT5 (Y694), Akt (S473) (n=4, biological replicates) and B)16 categories of phosphorylation array showed. Red box indicates category 1 (pS6+, pSTAT3+, pSTAT5+, pAKT+).


C ) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 ( n = 4 , biological replicates ) .

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C) Relative changes in frequency of NK cells expressing pS6 in the presence and absence of PGE2 (n=4, biological replicates).


D ) Flow cytometry analysis of PDE4A in the presence or absence of Torin - 1 ( 1µM ) ( left , n = 7 , biological replicates ) with representative histograms ( right ) .

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D) Flow cytometry analysis of PDE4A in the presence or absence of Torin-1 (1µM) (left, n=7, biological replicates) with representative histograms (right).


E ) NK cell - mediated cytotoxicity of K562 cells by 4 - hour chromium - release assay of two - day cytokine activated NK cells in the presence or absence of Torin - 1 ( 1µM ) and PGE2 ( 1µM ) ( n = 6 , biological replicates ) . E : T ratio = 5 : 1 .

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E) NK cell-mediated cytotoxicity of K562 cells by 4-hour chromium-release assay of two-day cytokine activated NK cells in the presence or absence of Torin-1 (1µM) and PGE2 (1µM) (n=6, biological replicates). E:T ratio=5:1.


F , G ) ( F ) Flow cytometry analysis of pS6 and ( G ) 4 - hour chromium - release assay of PGE2 - treated NK cells against K562 measured after 48 hours treatment with Roflumilast ( PDE4i , 1µM ) ( n = 3 , biological replicates ) E : T ratio = 5 : 1 .

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F, G) (F) Flow cytometry analysis of pS6 and (G) 4-hour chromium-release assay of PGE2-treated NK cells against K562 measured after 48 hours treatment with Roflumilast (PDE4i, 1µM) (n=3, biological replicates) E:T ratio=5:1.


B ) Volcano plot showing genes compared after two - days cytokine activated NK cells . Genes with significant changes ( p value < 0 . 05 ) and > 1 . 5 log2 fold change between IL - 2 and IL - 15 activated NK cells are labeled in red . Genes belonging to cell surface ( GO : 0009986 ) are annotated with gene name .

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B) Volcano plot showing genes compared after two-days cytokine activated NK cells. Genes with significant changes (p value<0.05) and >1.5 log2 fold change between IL-2 and IL-15 activated NK cells are labeled in red. Genes belonging to cell surface (GO:0009986) are annotated with gene name.


C ) Quantification of CD25 + CD54 + population changes after two days treatment ( n = 4 , biological replicates ) .

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C) Quantification of CD25+ CD54+ population changes after two days treatment (n=4, biological replicates).


D ) Representative flow cytometry graphs for CD25 and CD54 staining in IL - 2 and IL - 15 NK cells in the presence or absence of PGE2 .

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D) Representative flow cytometry graphs for CD25 and CD54 staining in IL-2 and IL-15 NK cells in the presence or absence of PGE2.


E ) Quantification of the number of cell clusters . NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of < 0 . 8 ) . ( n = 4 , biological replicates ) .

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E) Quantification of the number of cell clusters. NK cell clusters were defined as cell aggregates occupying an area at least 2000 μm2 and an eccentricity of <0.8). (n=4, biological replicates).


F ) Representative phase contrast image of IL - 2 and IL - 15 activated NK cells in the presence or absence of PGE2 . Scale bar 50 µm .

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F) Representative phase contrast image of IL-2 and IL-15 activated NK cells in the presence or absence of PGE2. Scale bar 50 µm.


G ) Chromium - release cytotoxicity assay by two - day cytokine activated NK cells in the presence or absence of PGE2 ( 1µM ) and anti - CD54 ( 20 µg / ml ) ( n = 4 , biological replicates ) , E : T ratio = 5 : 1 .

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G) Chromium-release cytotoxicity assay by two-day cytokine activated NK cells in the presence or absence of PGE2 (1µM) and anti-CD54 (20 µg/ml) (n=4, biological replicates), E:T ratio=5:1.


A ) NK cells were stimulated with either IL - 2 or IL - 15 in the presence or absence of PGE2 for two days and thereafter purified as CD25 + / CD54 + and CD25 - / CD54 - and tested in a 51Cr - release assay against K562 . ( E : T = 5 : 1 , n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - NK cells for each condition .

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A) NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of PGE2 for two days and thereafter purified as CD25+/CD54+ and CD25-/CD54- and tested in a 51Cr-release assay against K562. (E: T=5:1, n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54- NK cells for each condition.


B - F ) NK cells were stimulated with IL - 15 and purified as CD25 + / CD54 + and CD25 - / CD54 - and co - cultures with K562 cells ( 5 : 1 ratio ) for six hours and analyzed for the expression of B ) CD107a , C ) IFNγ , D ) Perforin , E ) TRAIL and F ) Granzyme B were by flow cytometry ( n = 4 , biological replicates ) .

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B-F) NK cells were stimulated with IL-15 and purified as CD25+/CD54+ and CD25-/CD54- and co-cultures with K562 cells (5:1 ratio) for six hours and analyzed for the expression of B) CD107a, C) IFNγ, D) Perforin, E) TRAIL and F) Granzyme B were by flow cytometry (n=4, biological replicates).


G ) Flow cytometry analysis of levels ( Left = % frequency , right = fold change of mean fluorescence intensity ) of phosphorylated S6 ( S235 / 236 ) in CD25 + / CD54 + and CD25 - / CD54 - IL - 15 activated NK cells ( n = 4 , biological replicates ) . H ) Flow cytometry analysis of PDE4A expression in CD25 + / CD54 + and CD25 - / CD54 - IL - 15 activated NK cells ( n = 4 , biological replicates ) .

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G) Flow cytometry analysis of levels (Left=% frequency, right=fold change of mean fluorescence intensity) of phosphorylated S6 (S235/236) in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates). H) Flow cytometry analysis of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells (n=4, biological replicates).


I ) Representative image of PDE4A expression in CD25 + / CD54 + and CD25 - / CD54 - IL - 15 activated NK cells . Scale bar : 10 µm .

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I) Representative image of PDE4A expression in CD25+/CD54+ and CD25-/CD54- IL-15 activated NK cells. Scale bar: 10 µm.


J ) NK cells were first stimulated with IL - 15 for two days in the absence of PGE2 , then purified as CD25 + / CD54 + or CD25 - / CD54 - NK cells and tested in a 51Cr - release cytotoxicity assay against K562 cells at an E : T ratio of 5 : 1 in the presence ( filled bars ) or absence ( open bars ) of PGE2 ( 1 µM ) ( n = 3 , biological replicates ) .

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J) NK cells were first stimulated with IL-15 for two days in the absence of PGE2, then purified as CD25+/CD54+ or CD25-/CD54- NK cells and tested in a 51Cr-release cytotoxicity assay against K562 cells at an E:T ratio of 5:1 in the presence (filled bars) or absence (open bars) of PGE2 (1 µM) (n=3, biological replicates).


K ) Mitochondria mass from purified CD25 + / CD54 + and CD25 - / CD54 - NK cells measured by flow cytometry ( n = 5 , biological replicates ) .

K) Mitochondria mass from purified CD25+/CD54+ and CD25-/CD54- NK cells measured by flow cytometry (n=5, biological replicates).


L ) Representative image of Mitotracker Red FM in CD25 + / CD54 + NK cells and CD25 - / CD54 - NK cells . Scale bar : 20 μm .

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L) Representative image of Mitotracker Red FM in CD25+/CD54+ NK cells and CD25-/CD54- NK cells. Scale bar: 20 μm.


A ) Expression of PTGES in normal and tumor tissue .

A) Expression of PTGES in normal and tumor tissue.


B ) Kaplan - Meier analysis of overall ( left ) and progression - free ( right ) survival in patients with lung adenocarcinoma with high PTGES ( top 10 % ) and low PTGES ( bottom 90 % ) expression .

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B) Kaplan-Meier analysis of overall (left) and progression-free (right) survival in patients with lung adenocarcinoma with high PTGES (top 10%) and low PTGES (bottom 90%) expression.


C ) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma .

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C) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma.


D ) IL15 gene expression analysis in high and low NK gene signature ( NK GS ) .

D) IL15 gene expression analysis in high and low NK gene signature (NK GS).


E ) Overall survival in patients with stage I ( n = 269 ) lung adenocarcinoma with high ( top 50 % ) and low ( bottom 50 % ) NK cell gene expression signature expression .

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E) Overall survival in patients with stage I (n=269) lung adenocarcinoma with high (top 50%) and low (bottom 50%) NK cell gene expression signature expression.


F ) Overall survival in patients with high ( top 50 % ) PTGES expression in relation to high ( top 50 % ) and low ( bottom 50 % ) NK cell gene expression signature .

F) Overall survival in patients with high (top 50%) PTGES expression in relation to high (top 50%) and low (bottom 50%) NK cell gene expression signature.


A ) Real time image - based killing of A549 cells by NK cells isolated from two individual healthy donors at an E : T ratio of 10 : 1 .

A) Real time image-based killing of A549 cells by NK cells isolated from two individual healthy donors at an E:T ratio of 10:1.


B ) NK cells were stimulated with IL - 2 or IL - 15 in the presence or absence of PGE2 and then purified as CD25 + / CD54 + or CD25 - / CD54 - cells and tested for cytotoxicity against A549 tumor cell in a 51Cr - release assay . E : T ratio = 5 : 1 ( n = 4 , biological replicates ) . Stars indicate significant p - values comparing CD25 + / CD54 + vs . CD25 - / CD54 - .

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B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-.


C ) Representative image of NK cell infiltration ( red ) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells , scale bar : 400 μm .

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C) Representative image of NK cell infiltration (red) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells, scale bar: 400 μm.


D , E ) Incucyte quantification of D ) total and E ) tumor - infiltrating CD25 + / CD54 + and CD25 - / CD54 - NK cells . RCU , Red Calibrated Unit , ( n = 4 , biological replicates ) .

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D, E) Incucyte quantification of D) total and E) tumor-infiltrating CD25+/CD54+ and CD25-/CD54- NK cells. RCU, Red Calibrated Unit, (n=4, biological replicates).


F , G ) Flow cytometry quantification of ( F ) tumor - infiltrating and ( G ) non - infiltrating NK cells . Results are depicted as total NK cells among CD45 positive live cells . ( n = 4 , biological replicates ) .

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F, G) Flow cytometry quantification of (F) tumor-infiltrating and (G) non-infiltrating NK cells. Results are depicted as total NK cells among CD45 positive live cells. (n=4, biological replicates).


H ) 48 hours dpi zebrafish injected with A549 , A549 + CD25 + / CD54 + , or A549 + CD25 - / CD54 - NK cells after 24 hours . Fluorescent image was taken by Leica epi - fluorescent stereomicroscope . Scale bar : 500 µm . The tumor injection area ( perivitelline space ) is depicted with a dashed line .

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H) 48 hours dpi zebrafish injected with A549, A549+ CD25+/CD54+, or A549+ CD25-/CD54- NK cells after 24 hours. Fluorescent image was taken by Leica epi-fluorescent stereomicroscope. Scale bar: 500 µm. The tumor injection area (perivitelline space) is depicted with a dashed line.


I ) 3D view of tumor injection area 24 hours posts tumor inoculation ( left ) . Scale bar = 50 µm . The tumor injection area ( perivitelline space ) is depicted with a dashed line . Magnified view for tumor area ( right ) . The A549 - td tumor cells are annotated with a purple translucent mask and NK cells are color - coded based on the distance to the tumor area ( near - far : blue - red ) . Scale bar = 10 µm .

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I) 3D view of tumor injection area 24 hours posts tumor inoculation (left). Scale bar=50 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. Magnified view for tumor area (right). The A549-td tumor cells are annotated with a purple translucent mask and NK cells are color-coded based on the distance to the tumor area (near-far: blue-red). Scale bar=10 µm.


J ) The distance between NK cells and tumor area in CD25 - / CD54 - and CD25 + / CD54 + NK cell treated fish ( n = 5 , biological replicates ) . K ) Frequency of tumor infiltrating NK cells in in CD25 - / CD54 - and CD25 + / CD54 + NK cell treated fish ( n = 5 , biological replicates ) .

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J) The distance between NK cells and tumor area in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates). K) Frequency of tumor infiltrating NK cells in in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates).


L , M ) Quantification of ( L ) tumor cell and ( M ) NK cell counts 24 hours after injection of NK cells by Imaris ( n = 5 , biological replicates ) .

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L, M) Quantification of (L) tumor cell and (M) NK cell counts 24 hours after injection of NK cells by Imaris (n=5, biological replicates).


N ) Frequency of tissue - infiltrating total NK cells ( CD56 + / CD3 - ) ( left ) and CD54 positive NK cells ( right ) among CD45 positive live cells ( n = 10 , biological replicates ) .

N) Frequency of tissue-infiltrating total NK cells (CD56+/CD3-) (left) and CD54 positive NK cells (right) among CD45 positive live cells (n=10, biological replicates).


( A ) . Cell growth assessment of ΔIndLon ( upper plot ) and ΔIndFtsH ( lower plot ) mutants grown under inducing ( blue ) or depleting conditions ( red ) . Growth was monitored by measuring DNA and protein biomass over time . The average from two independent biological replicates is shown .

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(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.


( D ) . Role of Lon and FtsH under heat shock stress conditions . ΔIndLon and ΔIndFtsH mutants were grown under inducing ( Lon + or FtsH + ) or depleting conditions ( Lon + or FtsH + , 60h of depletion for both , Lon and FtsH ) , and then exposed at 45 ° C during 0 , 5 , 10 , 15 or 20 min . Then , growth after the heat treatment was monitored over time under inducing conditions by the 430 / 560 absorbance rate index that shows pH changes in the medium . The average from two independent biological replicates is shown for each condition .

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(D). Role of Lon and FtsH under heat shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon+ or FtsH+, 60h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15 or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition.


( C ) In vivo degradation assays of FtsA , FtsZ and DnaB . ΔIndLon mutants expressing the different N - terminal FLAG - tagged derivatives were grown in depleting conditions for 36h . Then , Lon expression was transiently induced for 3h before translation was blocked with gentamicin ( Gm ) . Samples were taken at the indicated time points after gentamicin treatment , and processed for immunoblot analysis using anti - FLAG antibodies . LC , loading control . As controls , non - induced cells were also treated with gentamicin and processed at the same time points . Immunoblots are representative of two independent experiments .

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(C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.


( E and F ) Similar to panel B ( E ) and C ( F ) but for a C - terminal FLAG - tagged derivative of MPN201 as a representative of an unstable HsdS subunit .

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(E and F) Similar to panel B (E) and C (F) but for a C-terminal FLAG-tagged derivative of MPN201 as a representative of an unstable HsdS subunit.


( H ) Similar to panel B but for C - terminal FLAG - tagged derivatives of FtsH candidate substrates . Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti - FtsH and anti - FLAG antibodies . LC , loading control .

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(H) Similar to panel B but for C-terminal FLAG-tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti-FtsH and anti-FLAG antibodies. LC, loading control.


( I ) Similar to panel C , but for FtsH candidate substrates . In this case , C - terminal FLAG - tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment .

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(I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.


( B ) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons . N - terminal FLAG - tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant . Protein levels were then determined by immunoblot using anti - Lon and anti - FLAG antibodies comparing inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

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(B) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons. N-terminal FLAG-tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant. Protein levels were then determined by immunoblot using anti-Lon and anti-FLAG antibodies comparing inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


( B ) Lon surveillance of truncated protein variants . As an example , an N - terminal FLAG - tagged derivative of MPN304 , which encodes a truncated variant of ArcA ( ArcA - Nt ) , was expressed in the ΔIndLon mutant , and its accumulation monitored over time under non - inducing conditions using anti - FLAG antibodies . Anti - Lon antibodies were used to monitor Lon depletion along the time course experiment ( western blot figure on left ) . MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants ( 36h of depletion ) , in which protein expression was blocked with gentamicin ( Gm ) during the indicated time points after transient induction of Lon for 3h . As control , samples after Gm treatment were also taken from non - induced samples . LC , loading control ( western blot figure on right ) . Immunoblots are representative of two independent experiments . A schematic representation of the arginine deiminase pathway , which is inactive in M . pneumoniae , and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing ( + ) or depleting ( - ) conditions .

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(B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions.