( g ) Clonogenicity of Bax / Bak − / − MEFs after exposure to etoposide staurosporine . Bax / Bak − / − MEFs that were transfected with indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h . After collection , 2 , 000 were seeded in normal medium . After one week , colonies were counted .

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(g) Clonogenicity assay of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine. Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h. After collection, 2,000 cells were seeded in the normal medium. After one week, colonies were counted.


h , i ) Inhibition of APG5 - APG12 induction by silencing of Bcl - x or Beclin 1 . Bax / Bak − / − MEFs were treated with indicated siRNAs ( 10 μg ) for 24 h then incubated with 20 μM etoposide for indicated times . Expression of APG5 - APG12 , Beclin 1 GAPDH / VDAC ( loading control ) was analysed by Western blot .

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h, i) Inhibition of APG5-APG12 induction by silencing of Bcl-x or Beclin 1. Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex, Beclin 1 and GAPDH/VDAC (loading control) was analysed by Western blot analysis.


Figure 1 . Ret - eGFP is Expressed in Multiple Sensory Neuron Subsets ( A − L ) Ret - positive neurons largely overlap with markers for myelinated neurons ( NF200 ) , non - peptidergic nociceptors ( IB4 ) C - fiber low - threshold mechanoreceptors ( TH ) . However some Ret positive neurons are negative for these markers ( indicated by arrows in D , H L ) suggesting existence of a further subset of Ret + neurons . ( M ) of proportion of Ret - eGFP positive neurons in DRG . ( N ) of Ret - eGFP co - expression with other markers . ( n = 2278 from 3 mice ) Scale bars , 50 μm .

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Figure 1. Ret-eGFP is Expressed in Multiple Sensory Neuron Subsets(A−L) Ret-positive neurons largely overlap with markers for myelinated neurons (NF200), non-peptidergic nociceptors (IB4) and C-fiber low-threshold mechanoreceptors (TH). However some Ret positive neurons are negative for these markers (indicated by the arrows in D,H and L) suggesting the existence of a further subset of Ret+ neurons. (M) Quantification of the proportion of Ret-eGFP positive neurons in DRG . (N) Quantification of Ret-eGFP co-expression with other markers. (n=2278 cells from 3 mice) Scale bars, 50 μm.


( A ) Flow cytometric of dissociated sensory neurons plotted according to their level of endogenous Ret - eGFP expression IB4 binding ( also indicated in Figure EV1 ) . There are 5 well - defined subsets , 2 of which do not bind to IB4 but display a differential level of eGFP ( termed Ret - eGFPLo : IB4Neg , A , Ret - eGFPHi : IB4Neg , B , respectively ) . other 3 subsets bind IB4 display a range of eGFP intensities , termed Ret - eGFPLo : IB4Lo , C , Ret - eGFPHi : IB4Lo , D , Ret - eGFPHi : IB4Hi , E . of total events are indicated in each box .

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(A) Flow cytometric analysis of dissociated sensory neurons plotted according to their level of endogenous Ret-eGFP expression and IB4 binding (also indicated in Figure EV1). There are 5 well-defined subsets, 2 of which do not bind to IB4 but display a differential level of eGFP intensity (termed Ret-eGFPLo:IB4Neg, A, and Ret-eGFPHi:IB4Neg ,B, respectively). The other 3 subsets bind IB4 and display a range of eGFP intensities, termed Ret-eGFPLo:IB4Lo, C, Ret-eGFPHi:IB4Lo, D, and Ret-eGFPHi:IB4Hi, E. Percent of total events are indicated in each box.


( B ) No correlation between median of different Ret + subsets eGFP or IB4 binding . graph shows forward scatter values plotted against normalized number of , displayed as of Max . ( C − D ) Variations in endogenous eGFP are clearly visible with microscopy .

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(B) No correlation between the median cell size of different Ret+ subsets and eGFP intensity or IB4 binding. The graph shows the forward scatter values plotted against the normalized number of cells, displayed as the percent of Max. (C−D) Variations in endogenous eGFP intensity are clearly visible with fluorescent microscopy.


( C ) Cultured neurons ( D ) DRG section from Avil - Cre : : Ret + / eGFPmice displaying native eGFPfluorescence stained with IB4 . Different of eGFP ( high - indicated by arrows low - indicated by arrowheads ) are detected across IB4 + IB4 - . Scale bar , 50 μm .

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(C) Cultured neurons and (D) DRG section from Avil-Cre::Ret+/eGFPmice displaying native eGFPfluorescence and stained with IB4. Different levels of eGFP intensity (high -indicated by arrows and low -indicated by arrowheads) are detected across IB4+ and IB4- cells. Scale bar, 50 μm.


( A ) Ret - eGFPHi : IB4Neg Ret - eGFPLo : IB4Negneurons display distinct expression profiles . A volcano plot of fold change expression in Ret - eGFPLo : IB4Neg versus Ret - eGFPHi : IB4Neg against probability . Ret has higher expression in Ret - eGFPHi : IB4Neg subset , which also shows an up regulation of Ret co - receptor Gfra2 Fam38b , encoding for mechanosensitive ion channel Piezo2 . Ret - eGFPLo : IB4Neg subset , displays an array of molecules previously associated with itch ( marked in red ) .

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(A) Ret-eGFPHi:IB4Neg and Ret-eGFPLo:IB4Negneurons display distinct expression profiles. A volcano plot of fold change expression in Ret-eGFPLo:IB4Neg versus Ret-eGFPHi:IB4Neg against probability. Ret has higher expression in the Ret-eGFPHi:IB4Neg subset, which also shows an up regulation of the Ret co-receptor Gfra2 and Fam38b, encoding for the mechanosensitive ion channel Piezo2. The Ret-eGFPLo:IB4Neg subset, displays an array of molecules previously associated with itch (marked in red).


( B ) Itch - associated transcripts are enriched in Ret - eGFPLo : IB4Neg population compared to DRGneurons . Volcano plot of fold change expression in Ret - eGFPLo : IB4Negneurons versus sorted neurons from Avil - Cre : : R26tdRFPmice against probability . Molecules linked to itch perception are significantly upregulated in Ret - eGFPLo : IB4Neg subset .

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(B) Itch-associated transcripts are enriched in the Ret-eGFPLo:IB4Neg population compared to all DRGneurons. Volcano plot of fold change expression in Ret-eGFPLo:IB4Negneurons versus sorted neurons from Avil-Cre::R26tdRFPmice against probability. Molecules linked to itch perception are significantly upregulated in the Ret-eGFPLo:IB4Neg subset.


( C ) Gene expression within Ret - eGFPLo : IB4Neg population is confirmed by triple comparison between Ret - eGFPLo : IB4Neg , Ret - eGFPHi : IB4Neg Avil - Cre : : R26tdRFPdatasets .

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(C) Gene expression within the Ret-eGFPLo:IB4Neg population is confirmed by triple comparison between Ret-eGFPLo:IB4Neg, Ret-eGFPHi:IB4Neg and Avil-Cre::R26tdRFPdatasets.


( D ) Validation of differential microarray screening by quantitative RT - PCR . Transcripts encoding ion channels , receptors , neuropeptides , signaling molecules transcription factors were selected for quantitative RT - PCR . Differential expression between Ret - eGFPLo : IB4Neg Ret - eGFPHi : IB4Neg populations correlates with microarray ( n = 3 ) .

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(D) Validation of differential microarray screening by quantitative RT-PCR. Transcripts encoding ion channels, receptors, neuropeptides, signaling molecules and transcription factors were selected for quantitative RT-PCR. Differential expression between Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg populations correlates with microarray analysis (n=3).


( A − H ) SstCre mediated recombination of Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co - express NF200 or TH . Triple immunostaining of DRG from Sst - Cre : : ReteGFP / + mice with RetGFP ( A ) , IB4 ( B ) NF200 ( C ) , RetGFP ( E ) , IB4 ( F ) TH ( G ) .

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(A−H) SstCre mediated recombination of the Ret locus drives eGFP expression in sensory neurons that do not bind to IB4 or co-express NF200 or TH. Triple immunostaining of DRG from Sst-Cre::ReteGFP/+mice with RetGFP (A), IB4 (B) and NF200 (C), and RetGFP (E), IB4 (F) and TH (G).


( I ) of SstCre : : Ret - eGFP expression in DRG . ( J ) of co - expression of SstCre : : Ret - eGFP with neuronal markers ( n = 8827 from 3 mice ) .

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(I) Quantification of SstCre::Ret-eGFP expression in DRG. (J) Quantification of co-expression of SstCre::Ret-eGFP with neuronal markers (n=8827 cells from 3 mice).


( K − M ) Peripheral projections of sensory neurons from Sst - Cre : : Rosa26SnapCaaXmice terminate in hairy skin as free nerve endings . Non - innervated hair follicles are indicated by arrows . Double labelling using TMRStar to label SNAP - tag ( K ) DAPI ( L ) . Scale bars , 50 μm .

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(K−M) Peripheral projections of sensory neurons from Sst-Cre::Rosa26SnapCaaXmice terminate in the hairy skin as free nerve endings. Non-innervated hair follicles are indicated by arrows. Double labelling using TMRStar to label SNAP-tag (K) and DAPI (L). Scale bars, 50 μm.


( A - B ) Contour diagram of dissociated sensory neurons from Sst - Cre : : ReteGFP / + ( A ) Avil - Cre : : ReteGFP / + neurons ( B ) , plotted according to their native eGFP expression IB4 binding . Only one subset of Ret - eGFP can be defined within Sst - Cre : : ReteGFP / + dissociated DRGneurons . This displays same range of eGFPfluorescence as Ret - eGFPLo : IB4Neg population doesn ' t bind to IB4 .

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(A-B) Contour diagram of dissociated sensory neurons from Sst-Cre::ReteGFP/+ (A) and Avil-Cre::ReteGFP/+neurons (B), plotted according to their native eGFP expression and IB4 binding. Only one subset of Ret-eGFP cells can be defined within the Sst-Cre::ReteGFP/+ dissociated DRGneurons. This displays the same range of eGFPfluorescence as the Ret-eGFPLo:IB4Neg population and doesn't bind to IB4.


( C ) Quantitative RT - PCR of selected transcripts in Sst - Cre : : ReteGFP ( darker colors ) Ret - eGFPHi : IB4Neg ( lighter colors ) subsets displayed as differential expression compared to Ret - eGFPHi : IB4Neg subset . Almost genes that are up - regulated in Ret - eGFPLo : IB4Neg compared to Ret - eGFPHi : IB4Neg subset are also enriched in Sst - Cre : : ReteGFP population ( n = 3 )

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(C) Quantitative RT-PCR analysis of selected transcripts in the Sst-Cre::ReteGFP (darker colors) and Ret-eGFPHi:IB4Neg (lighter colors) subsets displayed as differential expression compared to the Ret-eGFPHi:IB4Neg subset. Almost all the genes that are up-regulated in the Ret-eGFPLo:IB4Neg compared to the Ret-eGFPHi:IB4Neg subset are also enriched in the Sst-Cre::ReteGFP population (n=3)


( A - G ) Ablation of Sst - Cre : : AviliDTR positive sensory neurons by injection of diphtheria toxin ( DTX ) . Immunostaining of DRG from Sst - Cre : : AviliDTRmice with an antibody against diphtheria toxin receptor IB4 in absence of diphtheria toxin ( A - C ) after systemic injection of diphtheria toxin ( D - F ) .

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(A-G) Ablation of Sst-Cre::AviliDTR positive sensory neurons by injection of diphtheria toxin (DTX). Immunostaining of DRG from Sst-Cre::AviliDTRmice with an antibody against the diphtheria toxin receptor and IB4 in the absence of diphtheria toxin (A-C) and after systemic injection of diphtheria toxin (D-F).


( G ) of diphtheria toxin receptor positive neurons before after injection of diphtheria toxin .

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(G) Quantification of diphtheria toxin receptor positive neurons before and after injection of diphtheria toxin.


( H - K ) Examples of calcium flux in dissociated DRG upon application of histamine ( H ) , chloroquine ( I ) , IL -

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(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31


( H - K ) Examples of calcium flux in dissociated DRG upon application of histamine ( H ) , chloroquine ( I ) , IL - 31 ( J ) 5HT1F agonist Ly344864 ( K ) .

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(H-K) Examples of calcium flux in dissociated DRG upon application of histamine (H), chloroquine (I), IL-31 (J) and 5HT1F agonist Ly344864 (K).


( L ) of responding neurons to indicated pruritogens before or after systemic injection of diphtheria toxin in Sst - Cre : : AviliDTRmice . Scale bars , 50 μm , asterisk denotes p < 0 . 05 , n = 4 - 6 mice , Student ' s t - , error bars indicate .

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(L) Quantification of percent responding neurons to the indicated pruritogens before or after systemic injection of diphtheria toxin in Sst-Cre::AviliDTRmice. Scale bars, 50 μm, asterisk denotes p<0.05, n=4-6 mice, Student's t-test, error bars indicate SEM.


( A - B ) Nociceptive withdrawal thresholds in control ( AviliDTR without Cre ) Sst - Cre : : AviliDTRmice after systemic diphtheria toxin injection . No significant differences in thermal ( A ) or mechanical ( B ) withdrawal reflexes after ablation of SstCre positive neurons .

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(A-B) Nociceptive withdrawal thresholds in control (AviliDTR without the Cre) and Sst-Cre::AviliDTRmice after systemic diphtheria toxin injection. No significant differences in thermal (A) or mechanical (B) withdrawal reflexes after ablation of SstCre positive neurons.


( C - F ) Scratching behavior after a single intradermal injection of indicated pruritogen observed for 1 hour . compounds evoked a significant increase in number of scratching bouts compared to vehicle alone in control ( AviliDTR ) or Sst - Cre : : AviliDTRmice . Diphtheria toxin injection in control mice ( AviliDTR ) had no effect on scratching behavior evoked by histamine ( C ) , chloroquine ( D ) , IL - 31 ( E ) or 5HT1F agonist Ly344864 ( F ) but significantly reduced responses to IL31 , or Ly344864 in Sst - Cre : : AviliDTRmice . n - numbers indicated in brackets , asterisk denotes p < 0 . 05 , two way RM ANOVA , Holm - Sidak multiple comparison . Error bars indicate .

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(C-F) Scratching behavior after a single intradermal injection of the indicated pruritogen observed for 1 hour. All compounds evoked a significant increase in the number of scratching bouts compared to vehicle alone in control (AviliDTR) or Sst-Cre::AviliDTRmice. Diphtheria toxin injection in control mice (AviliDTR) had no effect on scratching behavior evoked by histamine (C), chloroquine (D), IL-31 (E) or 5HT1F agonist Ly344864 (F) but significantly reduced responses to IL31, or Ly344864 in Sst-Cre::AviliDTRmice. n-numbers indicated in brackets, asterisk denotes p<0.05, two way RM ANOVA, Holm-Sidak multiple comparison. Error bars indicate SEM.


A ) Representative confocal showing perinuclear clustering of mitochondria Parkin on mitochondria in Huh7 infected with HCVcc . At 3 days post - infection , prestained with MitoTracker ( Mito , red ) were immunostained with anti - Parkin ( green ) HCV ( orange ) antibodies . Nuclei were stained with DAPI ( blue ) . Infected ( + ) uninfected ( − ) are marked . illustrated display typical distribution of mitochondria in uninfected altered distribution of mitochondria in perinuclear region of infected ( gray ) . In zoomed , arrows indicate accumulation of endogenous Parkin recruited to mitochondria in HCV - infected ( yellow ) .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-ORGANISM O O O O O O

A) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc. At 3 days post-infection, cells prestained with MitoTracker (Mito, red) were immunostained with anti-Parkin (green) and HCV core (orange) antibodies. Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked. The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells (gray). In the zoomed images, the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV-infected cells (yellow).


( B ) Ultrastructure of HCV - infected showing perinuclear clustering of damaged mitochondria . Control naïve Huh7 ( left ) stable harboring HCV full - replicon FLR - JFH1 ( right ) were examined by electron microscopy . In zoomed , typical ultrastructure of mitochondria in naïve ultrastructural abnormalities of mitochondria in HCV replicon are shown . Organelle mark : N , nucleus ; M arrow , mitochondria . Scale bar = 1 µM . ( below ) indicate expression of HCV in HCV replicon . were immunostained with anti - HCV antibody ( red ) . Nuclei were stained with DAPI ( blue ) .

O O O O O B-ORGANISM O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-CELL_LINE O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O

(B) Ultrastructure of HCV-infected cells showing perinuclear clustering of damaged mitochondria. Control naïve Huh7 cells (left) and stable cells harboring HCV full-length replicon FLR-JFH1 (right) were examined by electron microscopy. In the zoomed images, typical ultrastructure of mitochondria in naïve cells and ultrastructural abnormalities of mitochondria in HCV replicon cells are shown. Organelle mark: N, nucleus; M and white arrow, mitochondria. Scale bar = 1 µM. Fluorescent images (below) indicate the expression of HCV core protein in HCV replicon cells. Cells were immunostained with anti-HCV core antibody (red). Nuclei were stained with DAPI (blue).


( C ) of number of mitochondria in perinuclear region ( mean ± ; n ≥ 5 ; * p < 0 . 05 ) .

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(C) Quantification of the number of mitochondria in the perinuclear region (mean ± SEM; n≥5 cells; *p<0.05).


( D ) of of Parkin on mitochondria in CCCP - treated ( see Figure S1 ) or HCV - infected ( A ) those treated with 3 - MA or BafA1 , respectively ( see Figure 4A ) ( mean ± ; n = 10 ; * p < 0 . 01 ) . P values were calculated by using an unpaired Student ' s t - .

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(D) Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in CCCP-treated (see Figure S1) or HCV-infected cells (A) and those treated with 3-MA or BafA1, respectively (see Figure 4A) (mean ± SEM; n = 10 cells; *p<0.01). P values were calculated by using an unpaired Student's t-test.


( E ) Western blot showing endogenous Parkin recruitment to mitochondria in HCV - infected . Huh7 were infected with HCVcc . At 5 days post - infection , pure cytoplasm mitochondria fractions were isolated by ultracentrifugation as described in Materials Methods . fractions of HCV - infected were analyzed by immunoblotting using antibodies specific for indicated . Fractions : whole lysates , WCL ; purified cytoplasm , pCyto ; purified mitochondria , pMito . Organelle markers : VDAC1 , mitochondria ; Calreticulin , ER ; GAPDH , cytoplasm .

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(E) Western blot analysis showing endogenous Parkin recruitment to mitochondria in HCV-infected cells. Huh7 cells were infected with HCVcc. At 5 days post-infection, pure cytoplasm and mitochondria fractions were isolated by ultracentrifugation as described in Materials and Methods. Cellular fractions of HCV-infected cells were analyzed by immunoblotting using antibodies specific for the indicated proteins. Fractions: whole cell lysates, WCL; purified cytoplasm, pCyto; purified mitochondria, pMito. Organelle markers: VDAC1, mitochondria; Calreticulin, ER; GAPDH, cytoplasm.


( A ) Parkin ubiquitination in purified mitochondria isolated from HCV - infected . Parkin in pCyto pMito fractions , respectively , was immunoprecipitated by anti - Parkin antibody , followed by immunoblotting with anti - ubiquitin ( Ub ) antibody . Normal rabbit IgG was used as a negative control for immunoprecipitation ( IP ) .

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(A) Parkin ubiquitination in the purified mitochondria isolated from HCV-infected cells. Parkin protein in pCyto and pMito fractions, respectively, was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a negative control for immunoprecipitation (IP).


( B , E , F ) At indicated time points post - infection , WCL were extracted from HCV - infected Huh7 . ( B ) Parkin ubiquitination in WCL extracted from HCV - infected . ubiquitinated were immunoprecipitated with anti - Ub antibody analyzed by immunoblotting with anti - Parkin antibody . HCV infection was verified by immunoblotting with anti - HCV antibody . β - actin was used as an internal loading control .

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(B, E, and F) At the indicated time points post-infection, WCL were extracted from HCV-infected Huh7 cells. (B) Parkin ubiquitination in WCL extracted from HCV-infected cells. All ubiquitinated proteins were immunoprecipitated with anti-Ub antibody and analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( C ) Confocal microscopy showing Parkin ubiquitination in HCV - infected . HCV - infected prestained with MitoTracker ( Mito ) were immunostained with anti - Parkin ubiquitin ( Ub ) antibodies . In zoomed , arrows ( spots ) indicate merge of Mito ( green ) , Parkin ( red ) , Ub ( blue ) . Nuclei are demarcated with dot circles . image of HCV E2 verifies HCV - infected ( gray ) .

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(C) Confocal microscopy showing Parkin ubiquitination in HCV-infected cells. HCV-infected cells prestained with MitoTracker (Mito) were immunostained with anti-Parkin and ubiquitin (Ub) antibodies. In the zoomed images, the arrows (white spots) indicate the merge of Mito (green), Parkin (red), and Ub (blue). Nuclei are demarcated with white dot circles. Fluorescence image of HCV E2 verifies HCV-infected cells (light gray).


( D ) Quantitative of ubiquitination of endogenous Parkin colocalized with mitochondria in panels ( C ) ( mean ± ; n ≥ 10 ; * p < 0 . 05 ) .

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(D) Quantitative analysis of the ubiquitination of endogenous Parkin colocalized with mitochondria in the panels (C) (mean ± SEM; n≥10 cells; *p<0.05).


( E ) Western blot analyses of Mfn2 VDAC1 expression , mitochondrial substrate of Parkin , in HCV - infected . Intracellular HCV were analyzed by real - time qRT - PCR , as described in Materials Methods ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . WCL extracted from HCV - infected were analyzed by immunoblotting with anti - Parkin , Mfn2 , VDAC1 antibodies , respectively . HCV infection was verified by immunoblotting with anti - HCV antibody . β - actin was used as an internal loading control .

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(E) Western blot analyses of Mfn2 and VDAC1 expression, mitochondrial substrate of Parkin, in HCV-infected cells. Intracellular HCV RNA levels were analyzed by real-time qRT-PCR, as described in Materials and Methods (mean ± SD; n = 3; *p<0.01). WCL extracted from HCV-infected cells were analyzed by immunoblotting with anti-Parkin, Mfn2, and VDAC1 antibodies, respectively. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( F ) Western blot of p62 expression , autophagy - associated factor , in HCV - infected . WCL extracted from HCV - infected were analyzed by immunoblotting with anti - p62 antibody . β - actin was used as an internal loading control . ( E F ) relative of Mfn2 , VDAC1 , p62 expression normalized to β - actin was analyzed by ImageJ .

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(F) Western blot analysis of p62 expression, the autophagy-associated factor, in HCV-infected cells. WCL extracted from HCV-infected cells were analyzed by immunoblotting with anti-p62 antibody. β-actin was used as an internal loading control. (E and F) The relative intensity of Mfn2, VDAC1, and p62 expression normalized to β-actin was analyzed by ImageJ.


( G ) Parkin - mediated ubiquitination of Mfn2 VDAC1 in HCV - infected . ubiquitinated Mfn2 VDAC1 were analyzed by immunoprecipitation with anti - Mfn2 VDAC1 antibodies , respectively , followed by immunoblotting with anti - Ub antibody . expression of Parkin were analyzed by immunoblotting with anti - Parkin antibody . HCV infection was verified by immunoblotting with anti - HCV antibody . β - actin was used as an internal loading control . Normal mouse IgG was used as a negative control for immunoprecipitation ( IP ) . ( D E ) P values were calculated by using an unpaired Student ' s t - .

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(G) Parkin-mediated ubiquitination of Mfn2 and VDAC1 in HCV-infected cells. The ubiquitinated Mfn2 and VDAC1 proteins were analyzed by immunoprecipitation with anti-Mfn2 and VDAC1 antibodies, respectively, followed by immunoblotting with anti-Ub antibody. The protein expression levels of Parkin were analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control. Normal mouse IgG was used as a negative control for immunoprecipitation (IP). (D and E) P values were calculated by using an unpaired Student's t-test.


( A B ) Quantitative analyses of ATF4 , Parkin , PINK1 gene expression in HCV - infected . Huh7 infected with HCVcc for 5 days or treated with CCCP ( 10 or 20 µM ) for 24 h were used for of ATF4 , Parkin , PINK1 gene expression . ( A ) Intracellular mRNA of ATF4 , Parkin , PINK1 were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in Parkin PINK1 gene expression ( mean ± SD ; n = 3 ; * p < 0 . 01 , * * p < 0 . 05 ) . P values were calculated by using an unpaired Student ' s t - .

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(A and B) Quantitative analyses of ATF4, Parkin, and PINK1 gene expression in HCV-infected cells. Huh7 cells infected with HCVcc for 5 days or treated with CCCP (10 or 20 µM) for 24 h were used for analysis of ATF4, Parkin, and PINK1 gene expression. (A) Intracellular mRNA levels of ATF4, Parkin, and PINK1 were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin and PINK1 gene expression (mean ± SD; n = 3; *p<0.01, **p<0.05). P values were calculated by using an unpaired Student's t-test.


B ) expression of Parkin PINK1 were analyzed by immunoblotting with anti - Parkin PINK1 antibodies . expression of HCV was analyzed by immunoblotting with anti - HCV antibody . β - actin was used as an internal loading control .

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B) The protein expression levels of Parkin and PINK1 were analyzed by immunoblotting with anti-Parkin and PINK1 antibodies. The expression of HCV core protein was analyzed by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.


( C ) Western blot of Parkin expression in liver biopsies of chronic hepatitis C . Samples 1 2 represent liver biopsies of HCV - negative samples 3 - 7 represent HCV - positive chronic hepatitis . ( B C ) relative of Parkin PINK1 expression normalized to β - actin analyzed by ImageJ .

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(C) Western blot analysis of Parkin expression in liver biopsies of chronic hepatitis C patients. Samples 1 and 2 represent liver biopsies of HCV-negative patients and samples 3-7 represent HCV-positive chronic hepatitis patients. (B and C) The relative intensity of Parkin and PINK1 expression normalized to β-actin analyzed by ImageJ.


( D ) Confocal microscopy showing Parkin overexpression in HCV - infected . Huh7 infected with HCVcc were immunostained with anti - Parkin antibody ( red ) . image of HCV E2 verifies HCV - infected ( gray ) . Nuclei were stained with DAPI ( blue ) . Infected ( + ) uninfected ( − ) are marked .

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(D) Confocal microscopy showing Parkin overexpression in HCV-infected cells. Huh7 cells infected with HCVcc were immunostained with anti-Parkin antibody (red). Fluorescence image of HCV E2 verifies HCV-infected cells (light gray). Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked.


Confocal microscopy showing mitophagosome formation associated with Parkin in HCV - infected . ( A ) Huh7 transiently expressing GFP - LC3 were infected with HCVcc in absence or presence of 3 - MA ( 10 mM ) BafA1 ( 100 nM ) , respectively , for 12 h before fixation . At 3 day post - infection , were immunostained with antibodies against HCV E2 ( orange ) , TOM20 ( blue ) , Parkin ( red ) . In zoom , arrows ( ) indicate GFP - LC3 ( green ) colocalized with TOM20 Parkin . Nuclei are demarcated with dot circles . (

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Confocal microscopy showing mitophagosome formation associated with Parkin 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 day post-infection, cells were immunostained with antibodies against HCV E2 (orange), TOM20 (blue), and Parkin (red). In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. Nuclei are demarcated with white dot circles. (


( B ) of number of GFP - LC3 colocalized with TOM20 in panel ( A ) ( mean ± ; n ≥ 10 ; * p < 0 . 01 ) .

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(B) Quantification of the number of GFP-LC3 puncta colocalized with TOM20 in the panel (A) (mean ± SEM; n≥10 cells; *p<0.01).


( C ) Quantitative of of GFP - LC3 ( ) representing merge of GFP - LC3 , TOM20 , Parkin in panel ( A ) ( mean ± ; n ≥ 10 ; * p < 0 . 001 ) . ( B C ) P values were calculated by using an unpaired Student ' s t - .

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(C) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A) (mean ± SEM; n≥10 cells; *p<0.001). (B and C) P values were calculated by using an unpaired Student's t-test.


. ( D ) Ultrastructure of HCV - infected showing formation of mitophagosome . Control naïve Huh7 ( uninfected ) , HCV FLR - JFH1 replicon , Huh7 infected with HCVcc ( Jc1 ) were examined by electron microscopy . In zoomed image , formation of mitophagosome in HCV - infected 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 showing formation of mitophagosome in HCV - infected . Huh7 infected with HCVcc were stained by triple immunogold labeling method , as described in Materials Methods . immunogold particles of GFP - LC3 ( arrow , 18 nm ) , HCV E2 ( blue arrow , 12 nm ) , TOM20 ( red arrow , 6 nm ) are shown . image of HCV identifies HCV - infected ( 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 formation of mitophagolysosome in HCV - infected . ( A ) Huh7 transiently expressing GFP - LC3 were infected with HCVcc in absence or presence of 3 - MA ( 10 mM ) BafA1 ( 100 nM ) , respectively , for 12 h before fixation . At 3 days post - infection , prestained with LysoTracker ( red ) were immunostained with antibodies against TOM20 ( blue ) HCV ( cyan ) . Nuclei are demarcated with dot circles . In zoomed , arrows indicate colocalization of GFP - LC3 ( green ) , lysosome , mitochondria in HCV - infected ( ) .

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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 showing formation of mitophagolysosome . HCV FLR - JFH1 replicon were examined by electron microscopy . In zoomed image , formation of mitophagolysosome in HCV - infected 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 D ) Quantitative analyses of colocalization of GFP - LC3 containing lysosomes with mitochondria ( C ) or Parkin ( D ) in panels ( A ) Figure S5 ( mean ± ; n ≥ 10 ; * p < 0 . 001 , * * p < 0 . 05 ) . ( E ) ImageJ quantitative of number of mitochondria ( mean ± ; n ≥ 10 ; * p < 0 . 001 , * * p < 0 . 05 ) . P values were calculated by using an unpaired Student ' s t - .

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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 B ) Inhibitory effect of Parkin , PINKI , ATG5 silencing on HCV replication . Huh7 transfected with Non - Targeting ( NT ) or gene - specific siRNA pools targeting Parkin , PINK1 , ATG5 genes , respectively , were infected with HCVcc . At day 3 post - infection , of HCV ( A ) 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 Methods ( mean ± SD ; n = 3 ; * p < 0 . 01 ) . GAPDH was used to normalize changes in Parkin , PINK1 , 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 D ) Rescue of HCV replication by Parkin overexpression . Huh7 stably expressing mock vector ( M - KD ) , Non - target - shRNA ( NT - KD ) , Parkin - shRNA ( P - KD ) , respectively , were infected with HCVcc . P - KD were further transfected with two different of plasmid encoding wild - type Parkin for 2 days before harvest . At 3 days post - infection , intracellular HCV were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in HCV 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 ) expression of Parkin HCV were analyzed by immunoblotting with anti - Parkin HCV 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 - . relative of HCV expression normalized to β - actin was analyzed by ImageJ .

O O O O O B-GENEPROD B-ORGANISM O O O B-EXP_ASSAY O O O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD 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-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

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

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O B-CELL_LINE O O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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 I enzyme activity caused by HCV infection . NT - KD P - KD infected with HCVcc were harvested on day 3 post - infection used for of activity of mitochondrial I enzyme ( mean ± SD ; n = 3 ; * p < 0 . 05 ) .

O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O 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) 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 of mitochondrial respiratory chain enzyme expression . NT - KD P - KD were infected with HCVcc at 3 days post - infection , expression of I IV enzyme were analyzed by immunoblotting with anti - Hu total OxPhos antibody . Huh7 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 - .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-ORGANISM O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O 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) 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 of C / EBPβ mRNA in CD4 + naïve T inactivated ( naive ) or cultured for 24 hr under conditions as indicated with DMSO or ATRA ( - trans retinoic acid ) . Data are representative of two independent experiments with consistent results normalized with β - actin ( mean s . e . m . of quadruplicates )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

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

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 in CD4 + naïve T transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ cultured for 2 d under conditions as indicated . Representative experiments are shown in left panel pooled data with mean values fro 6 ( D are shown on right . Dot plots are gated for CD4 + GFP + numbers indicate Foxp3 + in

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 in CD4 + naïve T transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ cultured for 2 d under conditions as indicated . Representative experiments are shown in left panel pooled data with mean values fro E 7 ( F are shown on right . Dot plots are gated for CD4 + GFP + numbers indicate Foxp3 + in

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 in CD4 + naïve T transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ cultured for 2 d under conditions as indicated . Representative experiments are shown in upper panel pooled data from 4 ( A are shown in bottom . Dot plots are gated for CD4 + GFP + numbers indicate Foxp3 + in gate . Error bars represent mean ± s . e .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 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 in CD4 + naïve T transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ cultured for 2 d under conditions as indicated . Representative experiments are shown in upper panel pooled data fro ) , 3 ( B are shown in bottom . Dot plots are gated for CD4 + GFP + numbers indicate Foxp3 + in gate . Error bars represent mean ± s . e .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 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 of FACS - sorted GFP + from CD4 + naïve T transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ cultured for 2 d in conditions as indicated . Data are representative of two independent experiments with consistent results normalized with β - actin ( mean ± s . e . m . of triplicates )

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

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)


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

O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

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

B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O B-ORGANISM 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)


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

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O O B-ORGANISM O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O 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 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 extracts of Jurkat transfected with indicated expression vectors assessed with a probe covering putative unmethyl - or methyl - CRE in

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

B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 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 in CD4 + naïve T transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ cultured for 2 d under conditions as indicated . Representative experiments are shown in left panel pooled data fro 5 ( D are shown on right . Dot plots are gated for CD4 + GFP + numbers indicate Foxp3 + in

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

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

B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD B-ORGANISM O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD 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 alone or together with CD4 + GFP / Foxp3 + iTreg from CD45 . 1 - 2D2CD4 + GFP ( Foxp3 ) + naïve T transduced with either control retrovirus ( MIN ) or retrovirus encoding C / EBPβ , cultured in presence of TGF - β , anti - IFN - γ anti - IL - 4 Abs sorted on GFP / Foxp3 expression . recipients were immunized with myelin oligodendrocyte glycoprotein peptide ( 100 ug ) 1 d after transfer . Data show mean ± s . e . m of 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

B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O 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-ORGANISM 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


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

O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

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)


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

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM 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 of splenic . Histograms are gated for CD45 . 1 - CD4 + Vβ11 + . Data are representative of two independent experiments with consistent

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD 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


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

O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM 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 eosin of distal colons of mice ( Scale bars , 200μm )

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


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

B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM 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)


of Foxp3 + in CD45 . 1 - CD4 + T in colonic lamina propria 11 after

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


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

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

B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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 of time - lapse observation of Mal3 - GFP ( green ) Dli1 - 3mCherry ( magenta ) along longitudinal axis . Yellow arrowheads indicate temporary accumulation of growing plus tips during SPB is turning . Blue asterisks indicate dynein accumulating at distal end cortex in direction of SPB movement . indicated by ( a - d ) are shown in ( F ) . Data information : Scale bars , 5 µm .

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

O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O 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 power spectral density for variable parameters in pulling - only model . gray scale ( black to ) corresponds to values of power spectral density ( low to high ) . default parameters are indicated by arrowheads on horizontal axes .

O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 versus peak frequency during azygotic oscillation . n = 45 .

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


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

O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR 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 ) SPB ( Sad1 - CFP , blue ) in wild - type klp6 ∆ . Arrowheads indicate SPB . arrow shows an extensively elongated microtubule along cortex in a klp6 ∆ . Data information : Scale bars , 5 µm .

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

O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O B-SUBCELLULAR 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 of Sec31A marked ERES in HeLa cultured on crossbow shaped micropatterned surface of small ( upper panel ) or large ( lower panel ) for 4 hours . were treated with DMSO ( Ctrl ) or 50 µM NSC23766 ( Rac1 inhibitor ) for 4 hours prior to fixation immunostaining . number of ERES per for each pattern in HeLa ( B ) number of ERES was quantified from at least 74 per condition for HeLa ( B ) Each dot represents an .

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

O O O O B-EXP_ASSAY O B-CELL_LINE O O O 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-GENEPROD I-GENEPROD 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-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of HeLa cultured on micropatterned surface of multiple geometries . were treated with 50 µM of Rac1 inhibitor NSC23766 for 4 hours prior to with CellMask processing for immunofluorescence microscopy . ( E ) of ( D ) . of failing to cover entire micropatterned surface is shown for control ( ctrl ) Rac1 inhibitor NSC23766 . Between 113 - 117 were counted in total from a set of three independent experiments .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 were transfected with 10 nM non - targeting control siRNA ( siCtrl ) or siRNAs targeting Rac1 ( siRac1 ) . After 72 h , were fixed processed for immunostaining against Sec31A to label ERES . Representative confocal microscopy are shown . ( B ) graph shows number of ERES / in transfected with control siRNA ( siCtrl ) or siRNA against Rac1 ( two siRNAs # 1 # 2 ) . Values are expressed as % of siCtrl . From three independent experiments , with > 30 per condition were counted . ( C ) Representative immunofluorescence showing ERES in HeLa 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 number of ERES per ( displayed as % of Ctrl ) derived from at least 30 per condition from three experiments .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O B-CELL_LINE O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 ) recovery after photobleaching ( FRAP ) of GFP - Sec16A marked ERES in HeLa . show an ERES before ( - 1 ) , immediately after ( 0 ) 28 sec after photobleaching . graph illustrates FRAP of ERES from 23 ( for control ) 24 ( for NSC23766 ) regions in 3 experiments . connects time points . Data are ± SD . ( F ) of mobile fraction of GFP - Sec16A in control Rac1 inhibited . Mobile fraction is derived from a total of 23 ( Ctrl ) 24 ( NSC23766 ) ERES from three independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR 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 ) rate of ER - export was monitored using GFP - ManII - RUSH in HeLa after perturbation of Rac1 with siRNA ( siRac1 ) , or Rac1 inhibitor NSC23766 , or a combination of both . Representative show GFP - ManII - RUSH distribution in HeLa at indicated time points . ( H ) shows of ManII within Golgi to outside Golgi region after addition of biotin at indicated time points . Between 76 - 104 were used for measurement of ManII in 3 experiments .

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

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

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

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(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.