EMBO/SourceData huggingface dataset:
Figure 2(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 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 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.
Figure 3(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 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 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 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).
Figure 4(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) 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 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.
Figure 5(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 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)
Figure 6(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) 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-31(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) 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.
Figure 7(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 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.
Figure 1A) 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 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) Quantification of the number of mitochondria in the perinuclear region (mean ± SEM; n≥5 cells; *p<0.05).(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 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.
Figure 2(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, 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 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 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 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 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 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.
Figure 3(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) 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 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 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.
Figure 4Confocal 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) 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 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 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.
Figure 5Confocal 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.(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.
Figure 6A 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 mRNAA (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).(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.
Figure 7(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).(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.
Figure 1Real-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 JFlow 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 gateFlow 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 gateFlow 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
Figure 2Flow 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.mFlow 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.mReal-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.dHematoxylin 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)
Figure 3Flow 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