EMBO/soda-nel huggingface dataset:
WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1. (2014)
Figure 7

Atg16L1 Mutants Unable to Bind WIPI2 Cannot Rescue LC3 Lipidation or LC3 Recruitment to Salmonella in Atg16L1Δ/Δ MEFs

(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.

(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn’s posttest. ∗p < 0.05. SEM for n = 3.

(C) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR) before analysis by immunoblotting.

(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey’s post hoc test. SEM for n = 3. ∗p < 0.05.

(E) HEK293A cells were treated with either RISC-free control or WIPI2 siRNA before infection with Salmonella (moi = 100) for 1 hr, labeled with anti-LC3, WIPI2, and p62 antibodies, and followed by confocal analysis. Scale bars, 5 μm.

(F) Statistical analysis for (E) was performed using an unpaired Student’s t test. ∗p < 0.05. SEM for n = 3.

(G) Atg16L1Δ/Δ MEFs were transiently transfected with FLAG-Atg16L1 WT, FLAG-Atg16L1 ERER, or FLAG-Atg16L2 DRDR 24 hr before being infected with Salmonella (moi = 25) for 1 hr, labeled with anti-LC3, anti-p62, and anti-FLAG antibodies, and followed by analysis by confocal microscopy. Scale bars, 5 μm.

(H) Statistical analysis of (G) was performed by one-way ANOVA with Dunn’s post hoc test. SEM for n = 4. ∗p < 0.05. See also Figure S7.
SUMOylation Coordinates BERosome Assembly in Active DNA Demethylation During Cell Differentiation (2018)
Figure 1

Figure 1. TDG and XRCC1 are SUMO-modified in mESCs during differentiation and oxidative stress.

Immunoblot analyses of TDG, XRCC1 and TDG-SUMO, XRCC1-SUMO, and TET1 isoforms in whole cell extracts of mESCs and MEFs.

(A) TDG and TDG-SUMO conjugates before (2i, 0 h) and during differentiation of TDG wildtype (TDGwt) and TDG deficient (TDGnull) mESC and MEFs (5 µM all-trans retinoic acid (RA, 24 h, 48 h). Percentages of TDG-SUMO relative to total TDG signal are indicated.

(B) TDG and TDG-SUMO before and after H2O2 treatment (10 mM, 15 min).

(C) Chromatin fractionation from mESCs expressing wildtype (ESC-TDGwt) or SUMOylation-deficient TDG (ESC-TDGsnm). Sol, chromatin unbound proteins; Chr1, chromatin associated proteins; Chr2, chromatin bound fraction; Histone H2B, stably chromatin bound marker.

(D) XRCC1 and XRCC1-SUMO conjugates before (2i, 0 h) and during RA-induced differentiation (24 h, 48 h) in mESCs and MEFs. Percentages of XRCC1-SUMO relative to total XRCC1 signal are indicated.

(E) XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease (SENP2364-489).

(F) XRCC1 SUMOylation before and after exposure to H2O2 (5 mM, 15 min).

(G) TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs.

(H) Fluorescence microscopy of GFP-TDGwt GFP-TDGpip in mESCs. Scale bar: 100µM.

(I) Quantitation of fluorescence signals of GFP-TDGwt and GFP-TDGpip in mESCs. median with interquartile range. >470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs. ***: P<0.0001. Non parametric, Mann-Whitney test.

(J) Immunoblot analysis of XRCC1 and XRCC1-SUMO in GFP-TDGwt and GFP-TDGpip mESCs.

Data information: In (A and D), membranes were sequentially probed with anti-TDG, anti-XRCC1 and anti β-actin antibodies. Quantitation of immunoblots in (A, D and J) was performed from exposed film using Fiji image processing package.
SUMOylation Coordinates BERosome Assembly in Active DNA Demethylation During Cell Differentiation (2018)
Figure 2

Figure 2. SUMOylation of XRCC1 facilitates TDG-BERosome assembly.

(A) "Far Western" blot analyses of BER protein interactions. Recombinant purified human TDG (10 µg), POLß (1 µg), APE1 (1 µg), and LIGIII (1 µg) probed with XRCC1 and XRCC1-SUMO (20 µg) and immune-stained for XRCC1.

(B) Immunoblot analysis of interactions of XRCC1 and XRCC1-SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1-SUMO was coupled to Ni-NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions.

C-H (C-F and H) Scheme of pulldown experiments and immunoblot analysis of purified 6hisTDG, 6hisXRCC1/XRCC1-SUMO and 6hisBER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. (C) 0.5 µM TDGwt, were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins. (D) Schematic of biotin-5caC DNA substrate pulldown experiment. 0.5 µM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1-SUMO (>50% SUMOylated), POLβ and LIGIII. (E) 1 µM biotinylated 5caC substrate (G•5caC DNA) and 10 µM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1-SUMO (50% SUMOylated) and BER proteins as indicated. (F) 0.5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate (G•5caC DNA), 0.5 µM SUMO and 10 µM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO. (G) "Far Western" blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1-SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1-SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated. (H) Schematic of biotin-5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG SUMO-binding mutant (TDGE310Q), 6hisXRCC1/XRCC1-SUMO and 6his BER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. 0.5 µM TDGwt or TDG SUMO-binding mutant (TDGE310Q) were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome.
SUMOylation Coordinates BERosome Assembly in Active DNA Demethylation During Cell Differentiation (2018)
Figure 3

Figure 3. SUMO transfer from XRCC1-SUMO to TDG within the TDG-BERosome.

(A) Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA.

(B) SUMOylation assay as in (A) with SUMO provided as XRCC1-SUMO instead of free SUMO.

(C) SUMO transfer as depicted in (B) with additional APE1, POLß, LIGIII, and TDG.

(D) Immunoblot analysis of XRCC1, TDG, TDG-SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null).

(E) Immunoblot analysis of TDG and TDG-SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H2O2 as indicated.

(F) Top: Schematic of the SUMO-mediated TDG-BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double-strands in presence of dynamic SUMO-conjugation/de-conjugation. Left panels lanes 3-14: BER reconstitution with XRCC1 or XRCC1-SUMO as indicated. Right panels lanes 15-21: BER reconstitution with free SUMO or XRCC1-SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1-2 CG/CG HpaII methylation sensitive restriction site, cleavable by HpaII; lane 3-6, 8-14, 15-17, 21 HpaII methylation sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII; lane 7 and 20 repaired Product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single stranded DNA.
SUMOylation Coordinates BERosome Assembly in Active DNA Demethylation During Cell Differentiation (2018)
Figure 4

Figure 4. SUMOylation of TDG is required for neural differentiation.

(A) Neural differentiation protocol.

(B) Bright field captures of TDGwt, TDGcat and TDGsnm mESCs at indicated stages of differentiation. DAPI, Alexa Fluor® 680 phalloidin actin staining at the bottom. Scale bars 25µM, 50µM, 500µM as indicated. Quantification of neural cells with neurites of total differentiating TDGwt, TDGcat and TDGsnm mESCs. n ≥ 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.

(C) Relative changes of lineage marker expression between NPCs and undifferentiated mESCs (ESCM LIF, 0 h), quantified with Gapdh as normalizer. n ≥ 3. Means with standard deviation (SD)). *: P<0.05; **: P<0.01 by non parametric Mann-Whitney test.

(D) LC-MS/MS analysis of global cytosine-modification levels in DNA from ground-state mESCs (2i) and EBs. Means with standard deviation (SD) of 5mC, 5hmC, 5fC and 5caC per 106 bases). n ≥ 3. *: P≤0.05, **: P<0.01, ***: P<0.0001 by one-way ANOVA, Dunnett's multiple comparisons test.

(E) Alkaline comet assay with pluripotent (0 h) and differentiating (24 h 5 µM RA) mESC as indicated. Olive Tail Moments and % DNA in tail. n ≥ 3. Medians with standard deviation (SD). *: P≤0.05; **: P≤0.01, ***: P≤0.0001 by one-way ANOVA, Sidak's multiple comparisons test.

(F) Cell proliferation assay. Relative viability of Talazoparib treated to untreated cells. n = 3. Means with standard deviation (SD), P≤0.05, **: P<0.01, by one-way ANOVA, Dunnett's multiple comparisons test.

(G) ChIP-qPCR analysis of TET1, TDG and XRCC1 at gene promoters in mESCs (24 h 5 µM RA). Relative enrichments to an unbound control region (Chr2neg). n ≥ 3. Means with standard deviation (SD). *: P≤0.05; **: P<0.001; ***: P≤0.0001, by two-tailed Mann-Whitney test.
SUMOylation Coordinates BERosome Assembly in Active DNA Demethylation During Cell Differentiation (2018)
Figure 5

Figure 5. XRCC1 is required for neural differentiation.

(A) Representative images of mESC neuronal differentiation as in Fig 4B. Shown are XRCC1wt and XRCC1null (XRCC1null1, XRCC1null2) mESCs in 2i medium supplemented with LIF (2i LIF) and in ESC medium supplemented with LIF (ESCM LIF), EBs in ESCM LIF and 5µM RA (EB 4d ESCM RA) and dissociated neuron-like cells in B27 (B27). Scale bars 25µM, 50µM, 500µM as indicated. Neurite grids were observed for XRCC1wt; heterogeneous cell populations formed with the XRCC1null (XRCC1null1, null2) clones. Quantification (below) of neural cells with neurites of total differentiating XRCC1wt, XRCC1null mESCs. n = 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.

(B) Quantification of mRNA expression of pluripotency and ectodermal/neurogenesis marker genes by RT-qPCR. n ≥ 3. Mean values and standard deviations (SD) of EBs relative to their mESC (ESCM LIF, 16h), normalised to Gapdh. Significance, *: P<0.05. **: P<0.01, ***: P≤0.0001, two-tailed Mann-Whitney test.

(C) SUMO coordinates BER in active DNA demethylation. SUMOylated XRCC1/BER-proteins are recruited to DNA-bound TDG to form the BERosome (grey oval) SUMO protease (SENP2) liberates SUMO from XRCC1 for attachment to TDG by the SUMO-conjugating enzymes SAE1/2 and UBC9. SUMOylated TDG dissociates from the DNA AP-site and the BERosome, facilitating completion of demethylation by BER, and neuronal differentiation.

Expanded View Figure Legends
The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy (2009)
Figure 1

Overall structure of the HsAtg4B–LC3 complex. (A) Ribbon diagram of the HsAtg4B–LC3(1–120) complex. HsAtg4B is coloured salmon red, and LC3 is coloured green. (B) Structural comparison between free and HsAtg4B‐bound LC3. Crystal structure of free LC3(1–120) (PDB code 1UGM) is superimposed on the HsAtg4B‐bound LC3. HsAtg4B‐bound LC3 is coloured green, and free LC3 is coloured grey. (C) Structural comparison between free and LC3‐bound HsAtg4B. Crystal structure of free HsAtg4B (PDB code 2CY7) is superimposed on the LC3‐bound HsAtg4B. LC3‐bound HsAtg4B is coloured salmon red, and free HsAtg4B is coloured grey. The regions with large conformational differences between free and LC3‐bound forms are coloured black (free) and red (LC3 bound). All the figures representing molecular structures were generated with PyMOL (DeLano, 2002).
The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy (2009)
Figure 2

Interaction between HsAtg4B and LC3. (A) Interaction between HsAtg4B and the ubiquitin core of LC3. HsAtg4B is coloured salmon red, and LC3 is coloured green. The side chains of the residues involved in the HsAtg4B–LC3 interaction are shown as stick models. Possible hydrophilic interactions are shown as broken lines. (B) Interaction between HsAtg4B and the C‐terminal tail of LC3. Trp142 and the regulatory loop of HsAtg4B are coloured blue, whereas the remainder is coloured as in (A). As the side chain of Cys74 has two conformations, both conformations are shown. Possible hydrophilic interactions are shown as broken lines. (C) In vitro proteolysis assay. Left, LC3 mutants fused to GST were incubated with HsAtg4B. Right, LC3–GST was incubated with HsAtg4B mutants. Procedures are described in detail in Materials and methods.
The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy (2009)
Figure 3

Structure of HsAtg4B complexed with a LC3 precursor. (A) Annealed Fo−Fc electron density map for the C‐terminal region (residues 119–122) of LC3(1–124). Left, LC3(1–124) bound to the HsAtg4B H280A mutant; right, LC3(1–124) bound to the HsAtg4B C74S mutant. Structure of the C‐terminal region of LC3 is also shown as a stick model, in which carbon, nitrogen and oxygen atoms are coloured green, blue and red, respectively. (B) Structure of the C‐terminal tail of LC3(1–124) and the catalytic site of HsAtg4B. Left, LC3(1–124) bound to HsAtg4B H280A mutant, right, LC3(1–124) bound to the HsAtg4B C74S mutant. HsAtg4B is shown as a ribbon model, whereas LC3 is shown as a stick model. The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model. Carbon, nitrogen, oxygen and sulphur atoms are coloured green, blue, red and yellow, respectively. Mutated catalytic residues are coloured cyan. (C) Structural comparison between the HsAtg4B–LC3 precursor and SENP2–SUMO‐3 precursor complexes. Left, structure of the HsAtg4B–LC3 precursor complex; right, structure of the SENP2–SUMO‐3 precursor complex. HsAtg4B and SENP2 are shown as a ribbon model, whereas LC3 and SUMO‐3 precursors are shown as a stick model. The side chains of the important residues at the catalytic site are also shown as a stick model. Atom colouring is the same as in (A, B).
The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy (2009)
Figure 4

Conformational change in HsAtg4B upon complex formation with LC3. (A) Surface model of free (left) and LC3(1–124)‐bound (right) HsAtg4B. W142 and the regulatory loop are coloured blue, and the N‐terminal tail is coloured cyan. The catalytic Cys74 is coloured red. LC3(1–124) is shown as a ribbon model. (B) In vitro proteolysis assay using N‐terminal tail‐deleted HsAtg4B. LC3–GST was incubated with either wild‐type or N‐terminally truncated HsAtg4B and was subjected to SDS–PAGE analysis. (C) Structural comparison between free and LC3(1–124)‐bound HsAtg4B and SENP2–SUMO‐3 precursor complexes. Left, free HsAtg4B; middle, the HsAtg4B–LC3 precursor complex; right, the SENP2–SUMO‐3 precursor complex. The three structures are shown in the same orientation. Model colouring is the same as in Figure 3. (D) Surface model of LC3(1–120) bound to HsAtg4B. Atom colouring is the same as in (A).
The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy (2009)
Figure 5

Interaction of the N‐terminal tail with the WXXL‐binding site of LC3. (A) Overall structure of HsAtg4B bound to both substrate and non‐substrate LC3. Substrate LC3 is designated as LC3(S) and coloured green, whereas non‐substrate LC3 is designated as LC3(N) and coloured blue. (B) Stereo‐view of the N‐terminal tail of HsAtg4B bound to the WXXL‐binding site of LC3. The side chains of the residues involved in the interaction between HsAtg4B and LC3 are labelled and shown with a stick model. Model colouring is the same as in (A). (C) Structural comparison of the N‐terminal tail–LC3 interaction with the p62–LC3 interaction. The structure of LC3 complexed with a p62 peptide is superimposed on that complexed with the N‐terminal tail peptide. As the structure of LC3 is almost identical to each other, only that bound to the N‐terminal tail peptide of HsAtg4B is shown. (D) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N‐terminal‐tail peptide. The combined 1H and 15N chemical shift differences, calculated using the equation Δp.p.m.=[(ΔδHN)2+(ΔδN/5)2]1/2 were plotted against residue numbers. (E) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B. The residues with Δp.p.m. >0.4 are shown in blue, 0.4>Δp.p.m.>0.3 in cyan and 0.3>Δp.p.m. >0.2 in green.
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. (2012)
Figure 1

Colocalization of HCV Proteins with organelle-specific markers in HCV infected cells.

Huh7 cells were infected with HCV (strain Jc1) using 30 TCID50/cell and 48 h later cells were fixed and processed for fluorescence microscopy. In case of samples shown in panels B–D, cells were first transfected with expression constructs specified in the left of each panel and 24 h later cells were infected as described above. Samples were analyzed with a Nikon TE2000-E inverted confocal microscope at 60× magnification. (A)–(D) Colocalization of HCV proteins specified in the top of each panel with protein disulphide isomerase (PDI; an ER marker), GFP-Rab21 (marker for early endosomes), GFP-Rab7 (marker for late endosomes) or βCOP-YFP (marker for COP I vesicles). The upper panels represent a low magnification overview; boxed areas are shown as enlargement in the corresponding panel below. The nucleus was stained with DAPI (blue). Scale bars represent 10 µm (top panels) and 2 µm (lower panels). The quantification of the degree of colocalization (Pearson's correlation coefficient) is given at the top of the enlarged pictures.
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. (2012)
Figure 2

Correlative light-electron microscopy of cells containing a GFP-tagged subgenomic replicon.

(A) Epifluorescence microscopy of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto carbon-patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) enlarged fluorescence image of the cell of interest; (c) merge of bright field and fluorescence images. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. White squares in a and c enclose the cell shown in b. (B) EM micrograph of the cell boxed in panel Ab overlapped with the fluorescence image. Areas marked with a green dotted line indicate regions of intense fluorescence. Note that the images do not match perfectly because the fluorescence image corresponds to the complete cell whereas the EM image represents one 60 nm ultrathin section of the same cell. (C) Higher magnification images of the two different regions, labeled 1 and 2 in panel B, corresponding to regions with intense fluorescence (1) or a region corresponding to the intersection of high to low fluorescence (2). Region 1 (top panel) corresponds to a DMV-containing area residing in close proximity of the ER; region 2 (bottom panel) corresponds to a LD-enriched area containing DMVs in very close proximity. Areas marked with white squares in the left images are magnified in the corresponding right panels. LD, lipid droplet; ER, endoplasmic reticulum; DMV, double membrane vesicle; m, mitochondrium; if, intermediate filaments.
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. (2012)
Figure 4

Immuno-EM localization of NS5A and NS3 in HCV-infected cells.

Huh7.5 cells were infected with 100 TCID50/cell of Jc1 and fixed 16 h post-infection. (A) Cells shown in panels b, c and e to g were post-fixed with 0.1% OsO4 providing best membrane preservation, especially in case of DMVs (f and g) and LDs (e). Both NS5A and NS3 localize predominantly to the ER (b and d) and 50–70 nm diameter single-membrane vesicles (SMVs; subpanel a, c and d). NS5A also localizes to lipid droplets (e). (B) Amount of gold particles per µm2 in Jc1-infected versus mock-infected cells after immunolabeling with NS3- and NS5A-specific primary antibodies. Note the higher immunolabeling with samples prepared without OsO4, but also the lower membrane preservation under this condition. (C) Relative labeling distribution of NS3 and NS5A. Thawed cryosections of cells post-fixed or not with OsO4 were labelled with NS3- or NS5A-specific antibodies by using two different blocks and 3 different labeling experiments. Per labeling experiment two grids were considered, counting ∼100–200 gold particles per grid and attributing the particles to the indicated structures. In the case of uninfected cells only background labeling in the cytoplasm, on mitochondria and undefined structures was seen. Numbers refer to the percent of total gold particles counted per sample. ER, endoplasmic reticulum; SV, small vesicles; Cyto, cytosol; Mito, mitochondria; n.d., non-defined structures; NE, nuclear envelope; EE/LE, early/late endosomes; PM, plasma membrane; DMV, double membrane vesicle; LD, lipid droplet.
Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. (2012)
Figure 5

Correlation between HCV RNA replication and appearance of double membrane vesicles.

(A) Time course of spread of HCV infection in Huh7.5 cells infected with 100 TCID50/cell of Jc1. Infected cells were detected by immunofluorescence using an NS5A-specific antiserum (upper panels). The graph shown below represents the result of counting ∼200 cells for each time point to determine the percentage of infected cells. Scale bars represent 50 µm. (B) Time course of accumulation of intracellular HCV RNA in infected Huh7.5 cells. The graph shows the result of two independent experiments (3 replicas each). Whiskers indicate the minimum and maximum values. (C) Colocalization of dsRNA and NS5A in cells infected with Jc1 (10 TCID50/cell). Cells were fixed at time points specified in the left of each panel row and NS5A and dsRNA were detected by indirect immunofluorescence microscopy. DNA was stained with DAPI (blue). Boxed areas in the left panels indicate areas that are shown as enlargements in the corresponding right panels. The quantification of the degree of colocalization (Pearson's correlation coefficient) is given in the enlarged pictures. Scale bars represent 10 µm and 2 µm (left and right panels, respectively). (D) Time course of accumulation of DMVs and MMVs. For each time point, 10 cellular profiles were counted. The Mann-Whitney (non-parametric) test was applied to determine statistical significance. Error bars refer to the standard deviation. Note the striking correlation between the increase of intracellular HCV RNA and DMV number between 16 and 24 hpi.