EMBO/soda-nel huggingface dataset:
Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme (2022)
Figure 5

Figure 5: Both slit3 and s1pr2 mutants show loss of focal adhesion markers and sensitivity to Fibronectin levels

A-B: Immunofluorescent staining of 48hpf tail fins in WT sqet37Et (Top row), slit3sq49/sq49; sqet37Et (middle row), and s1pr2te273/te273; sqet37Et (bottom row) transgenic larvae, stained for phospho-FAK (magenta; A, A'), phospho-Non Muscle myosin II (magenta; B, B') and eGFP (green; A', B'). Mutant mesenchymal cells show significantly reduced p-FAK and p-NM myosin II (magenta; A, B) signals in slit3 and s1pr2 homozygous mutants compared to the WT fin mesenchyme. Arrowheads indicate fin mesenchymal cells.

C-E: Lateral Nomarski images of 48hpf larval fins which are WT (C) or slit3+/sq49 heterozygotes (D) and are injected with 300µM fn1a morpholino. Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes (quantified in E).

F-G: Lateral Nomarski images of 48hpf WT larval fins injected with 200µM gna13b alone (F) or with 300µM fn1a morpholino (G).

H: Quantification of the proportion of larvae with fin blisters when low amounts of gna13 morpholinos (125µM each gna13a and gna13b MO combined or 200µm gna13b MO alone) are injected into WT, fn1a morphants (300µM MO), fn1a+/- heterozygotes and fn1a-/- mutants. Loss of a single or both copies of fn1a exacerbates reduced gna13 levels, as does knockdown of fn1a.

Data information: *** p<0.001, Chi-squared tests used in (E) and (H). Scale bar A-B: 20µm; C-G: 50µm.
RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules. (2022)
Figure 1

Figure 1. The number of P-bodies is reduced in cells with active RNase L.

A. GAPDH smFISH and IF analysis using anti-G3BP antibody (G3BP) and anti-Dcp1b antibody (DCP1) in A549 cells (RL-WT) or A549 RL-KO cells either mock transfected (mock) or transfected with poly(I:C) (PIC) for 5 hours. Scale bar 10 microns.

B. Graph of the fraction of G3BP foci with different volumes in PIC treated RL-WT and RL-KO cells.

C. Number of DCP1 foci per cell in PIC treated RL-WT and RL-KO cells relative to the number in mock treated cells. Wilcoxon Signed Rank test, ns=non significant

D. Average volume of individual Dcp1 foci in mock and PIC treated RL-WT and RL-KO cells. One way ANOVA with Sidak's multiple comparisons test, ns=non significant

Data information: C-D. Bar graphs show mean + SD for N=4 independent experiments.
RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules. (2022)
Figure 2

Figure 2. Nuclear-localized RNase L degrades nuclear RNA in response to poly(I:C).

A. Western analysis of nuclear (N) or cytoplasmic (C) fractions from whole cell lysates (W) from A549 cells (RL WT), RNase L knock out A549 cells (RL KO), or A549 cells with either wild-type RNase L (NLS-RL-WT) or RNase L-R667A (NLS-RL-CM) fused to a nuclear localization signal sequence. Short and long exposure with anti-RL antibody. GAPDH protein used as cytoplasmic marker. Histone H3 protein used as nuclear marker.

B. Graph depicting the fraction of RNase L found in the nucleus or cytoplasm. Mean and SEM of two experiments with individual experiment values plotted.

C. FISH with probes to GAPDH mRNA or oligo(dT) to detect poly(A)+ RNA in A549 cells without or with either NLS-RL-WT or NLS-RL-CM either mock transfected or transfected with poly(I:C) for 4 hours. Scale bar 5 micron.

D. Graph depicting median and the value of the intensity of oligo(dT) fluorescence in individual nuclei divided by nuclear volume in the indicated cell types either mock transfected or transfected with poly(I:C) for 4 hours. At least 43 nuclei analyzed per condition. Kruskal-Wallis test and Dunn's multiple compartisons test. **** P value <0.0001, *** P value 0.0001, ns not significant.
RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules. (2022)
Figure 3

Figure 3. The structure of nucleoli, nuclear speckles and Cajal bodies is dependent on nuclear RNA.

Analysis of nuclear RNA granules in A549 cells with RNase L targeted to the nucleus (NLS-RL-WT) either mock transfected or treated with poly(I:C) (PIC) for 4 to 5 hours.

A. FISH analysis to detect poly(A)+ RNA and nucleolar-localized snoRD3A RNA. IF analysis to detect nucleolar granular component protein nucleophosmin (NPM1).

B. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 17 nuclei mock treated cells, 31 nuclei PIC treated cells.

C. Fraction of nuclei with NPM1 protein enriched in ring structures classified as granular component assemblies or dispersed in nucleoplasm. 17 nuclei mock treated cells, 31 nuclei PIC treated cells.

D. IF analysis to detect dense fibrillar component protein, ribosome processing factor 1 (RPF1).

E. Graph of the mean and value of the average volume of individual snoRD3A foci in nuclei. 43 nuclei mock treated cells, 69 nuclei PIC treated cells.

F. Graph of the mean and value of the average volume of RPF1 foci in 43 mock treated cells and 69 nuclei PIC treated cells.

G. FISH analysis to detect poly(A)+ RNA and nuclear speckle-localized MALAT1 RNA and IF analysis with anti-sc35 antibody to detect nuclear speckle protein SRRM2.

H. Graph of the mean and value of the intensity of MALAT1 signal divided by the nuclear volume of individual nuclei. 51 nuclei mock treated cells, 56 nuclei PIC treated cells.

I. Graph of the median and value of the volume of individual SRRM2 foci in nuclei. Mock 1526 SRRM2 foci in 51 nuclei. PIC 136 SRRM2 foci in 20 nuclei that contain <10 SRRM2 foci.

J. FISH analysis to detect poly(A)+ RNA and IF analysis to detect Cajal body protein coilin.

K. Graph depicting the fraction of nuclei in mock and PIC treated cells with decreased oligo(dT) with different distributions of coilin protein. 90 nuclei in mock treated cells, 192 nuclei in PIC treated cells.

Data information: All images scale bar 5 microns. All graphs with significance testing. Mann-Whitney two-tailed test. **** P value <0.0001
RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules. (2022)
Figure 5

Figure 5. Analysis of the effect of nuclear RNA degradation on super-enhancer condensates and FUS assembly.

Analysis of A549 cells with NLS-RL-WT or NLS-RL-CM either mock transfected or treated with poly(I:C) (PIC) for 5 hours.

A. FISH analysis to detect poly(A)+ RNA and IF analysis against FUS protein. Images are a single 0.2 micron Z section.

B. Graph depicting the fraction of nuclei with FUS dispersed in the nucleoplasm or in foci in cells with reduced nuclear oligo(dT) signal. All the nuclei (total) in which nuclear RNA was degraded (N=64) or the same nuclei classified as to whether or not they contained residual poly(A) foci. Nuclei with poly(A) foci (N=14). Nuclei without poly(A) foci (N=50).

C. FISH analysis to detect poly(A)+ RNA and IF analysis against mediator complex protein, MED1, to detect super-enhancer condensates.

D. Number of MED1 foci in mock and PIC treated NLS-RL-WT cells.

E. Average volume of individual MED1 foci in mock and PIC treated NLS-RL-WT cells.

F. FISH analysis to detect poly(A)+ RNA and IF analysis against BRD4, to detect super-enhancer condensates.

G. Number of BRD4 foci in mock and PIC treated NLS-RL-WT cells.

H. Average volume of individual BRD4 foci in mock and PIC treated NLS-RL-WT cells.

Data information: Scale bar 5 microns. D-E. Mean and SEM of 3 independent experiments with at least 10 cells of each cell type and condition per experiment. G-H. Mean and SEM of 3 independent experiments with 8-13 cells of each cell type and condition per experiment. D, E, G and H. Unpaired two-tailed t test, *P value=0.02, ns non-significant.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 1

Figure 1. Chm7 localizes to a focus on the NE.

A. Schematic of the predicted domain architecture of Chm7 and truncations. Green circle is a Chm7-specific insertion (relative to Vps25; see Appendix Fig S1). MIM is microtubule interacting motif. Red stripes denote putative autoinhibitory helix. Numbers are amino acid residues.

B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm.

C. Scatter plot of the mean fluorescence intensity with SD of individual NE foci (horizontal line is mean). >50 foci were measured for each GFP fusion; p values from un-paired student’s T-test. *** is p ≤ 0.001; **** is p ≤ 0.0001.

D. Deconvolved fluorescence micrographs of Chm7-GFP and Mps3-mCherry with merge of green and red images (right). Scale bar is 5 μm.

E. 10-fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid (-) or those expressing the indicated Chm7 constructs (pRS416-HA-CHM7, pRS416-HA-chm7-NTD, pRS416-HA-chm7-CTD). See Appendix Figure S2A for protein levels.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 2

Figure 2. Heh1 is required for Chm7 recruitment to the NE subdomain.

A. Deconvolved inverted fluorescence micrographs Chm7-GFP and truncations in the indicated null strains. Scale bar is 5 μm.

B. Plot of the percentage of cells with SD of Chm7-GFP (and truncations) NE foci from A. Data are from 3 independent replicates where >200 cells were counted for each strain. p values from un-paired student’s T-test. ** is a p value ≤ 0.01; ****, p ≤ 0.0001.

C. Deconvolved inverted fluorescence micrographs of Chm7-GFP in a heh1Δ strain containing either an empty pRS426 plasmid (-) or those expressing HEH1 (pRS426-HEH1) or HEH2 (pRS426-HEH2).

D. Plots of the percentage of cells from C with Chm7-GFP NE-foci. Data are from 3 independent replicates where >200 cells were counted for each strain. Error bars are SD. p values from T-test. **, p ≤ 0.01; ****, p ≤ 0.0001.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 3

Figure 3. Heh2 directly binds Chm7 and Snf7.

A, B. Schematics of the domain organization and secondary structure of Heh2, Snf7 and truncations with amino acid numbers. LEM is LAP2-Emerin-MAN1 (green), TM is transmembrane (black), MCHD is MAN1 C-terminal Homology Domain (brown), MIM is microtubule interacting motif (purple). The striped α-helix 5 represents the autoinhibitory helix.

C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers.

D. In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead-bound GST, GST-snf7OPEN or GST-Snf7 before washing, elution and detection of bound proteins by autoradiography (top panel) or coomassie staining (bottom).

E. As in C except with GST-Chm7 constructs. Upper panel shows western blot (WB) with anti-His6 antibody. *chm7-NTD is also expressed with a His6 tag. Bottom panel is coomassie stained.

F. As in E except with MBP-Snf7.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 4

Figure 4. Heh1-Chm7 interactions occur at the NE subdomain.

A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm.

B. Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1-VN and Snf7-VC with either Chm7-mCherry or Mps3-mCherry and merge of both channels. Scale bar is 5 µm.

C. Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains. Cell borders outlined. Scale bar is 5 μm.

D,E. Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds. Data are from three independent replicates where > 225 cells per strain per replicate were quantified. Error bars are SD from the mean of each replicate. p values from un-paired student’s T-test where ns is p > 0.05. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

F. Deconvolved inverted fluorescence micrographs of Heh1-VN Snf7-VC BiFC signal in a chm7Δ strain with either an empty plasmid (“-”; pRS416) or one expressing CHM7 (pRS416-HA-CHM7). Cell borders outlined. Scale bar is 5 μm.

G. Plot of the percentage of cells with BiFC signal from F. Error bars are SD from the mean of each replicate. p values from un-paired student’s T-test where ns is p > 0.05. ***, p ≤ 0.001.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 5

Figure 5. Chm7 accumulates in the SINC.

A. Deconvolved inverted fluorescence micrographs of Chm7-GFP in the indicated strains. Scale bar is 5 µm. The percentage of cells ± SD with Chm7-GFP foci is indicated below each panel.

B. Deconvolved fluorescence images of SINC-containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7-GFP and Nup170-mCherry (green, red and merged images are shown). Scale bar is 1 μm.

C. Plot of the proportion of nup clusters with Chm7-GFP enrichment in the indicated genetic backgrounds. Error bars are SD of the mean from 3 independent replicates.

D. Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7-GFP and clustered Nup170-mCherry in a nup133Δ strain. Scale bar is 1 μm.

E. Plot of the total fluorescence/integrated density (in arbitrary units (A.U.)) of Chm7-GFP within (and outside of) the SINC. Error bars represent SD deviation of the mean from > 50 SINC or non-SINC accumulations pooled from 3 independent replicates. p values from un-paired student’s T-tests with Welch’s correction. **** is p ≤ 0.0001.

F. Correlation of the total fluorescence intensity/integrated density of SINC-enriched Chm7-GFP and Nup170-mCherry within individual SINCs. Linear regression calculated from 150 SINCs pooled from 3 independent replicates; r is the linear correlation (Pearson’s) coefficient.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 6

Figure 6. CHM7 is required for SINC formation.

A.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging.

B. Schematic of Sna3-mCherry localization. Sna3 accumulates in the vacuole (V) or class E compartment (E) in cells with, or lacking, ESCRT function, respectively.

C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm.

D. Deconvolved fluorescence micrographs (maximum intensity projections of a z-series of images) of GFP-Nup49 in the indicated yeast strains (arrows indicate SINCs).

E. Plot of proportion of cells in the indicated strains with SINCs. Error bars are the SD from the mean from 3 independent replicates quantifying >300 cells for each strain. p values from un-paired student’s T-tests where ns is p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 7

Figure 7. Perturbation of NPC assembly leads to NE accumulation of Chm7.

A. Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7-GFP (percentages ± SD of the proportion of cells with Chm7-GFP foci is at bottom of each panel, see Figure EV5B). Scale bar is 5 μm.

B. Plot of the percentage of cells with the indicated number of Chm7-GFP foci. Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns represents p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

C. Deconvolved inverted fluorescence micrographs of strains expressing Chm7-GFP after 5 h at 23°C (top) or 37°C (bottom). Scale bar is 5 μm. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).

D. Plots of percentage of cells from C with the indicated number of Chm7-GFP foci at 23°C (left) and 37°C (right). Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns is p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 8

Figure 8. nup116Δ cells require CHM7 for growth and accumulate Chm7 and NE herniations.

A. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student’s T-test where *** is p ≤ 0.001.

B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).

C. Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37°C. N denotes nuclear interior. Scale bars are 100 nm.

D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.
Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing (2016)
Figure 9

Figure 9. Chm7 protects nuclear compartmentalization.

A. Deconvolved fluorescence micrographs of NLS-GFP in the indicated yeast strains at either 30°C or after 2 h at 37°C. Scale bar is 5 μm.

B. Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS-GFP from cells represented in A. Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain. p values from un-paired student’s T-test where ns is p > 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Only chm7Δ and chm7Δapq12Δ cells have N:C ratios below 1.2 (dotted red line).

C. Re-plotting of data from B showing the percentage of cells with a N:C NLS-GFP fluorescence ratio <1.2. Error bars are the SD from the mean from 3 independent replicates of 150 cells/strain. p values from student’s T-test where *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.

D. Model of a proposed stepwise recruitment and activation of Chm7 and other ESCRTs at defective NPCs (bottom, red), or NE holes.
Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis. (2013)
Figure 1

HBV/HBx promotes mitochondrial fission via Drp1 stimulation and activation.

(A and B) Confocal microscopy images showing mitochondrial fragmentation in HBV- and HBx-expressing cells, respectively. Huh7 cells were transiently transfected with HBV (A) or the HBx-flag construct (B). At 2 days post-transfection, cells prestained with MitoTracker (Mito, white) were immunostained with anti-HBsAg (A, green) and anti-flag (B, green) antibodies, respectively. In the zoomed images, typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown. (C) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody. (D–F) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV- and HBx-expressing cells, respectively. Huh7 cells transfected with HBV (D and E) or HBx-flag construct (F) were immunostained with antibodies specific to TOM20 (red), Drp1 (D, green), and phospho-Drp1 (Ser616) (E and F, green), respectively. HBV and HBx gene expression (white) was verified using anti-HBsAg (D and E) and anti-flag (F) antibodies, respectively. In the zoomed images, the yellow color indicates the merge of Drp1 or phospho-Drp1 (Ser616) with mitochondria. (A, B, D, E, and F) Transfected (+) and untransfected (−) cells are marked. Nuclei were stained with DAPI (blue).
Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis. (2013)
Figure 2

HBV induces mitochondrial translocation of Parkin and stimulates mitophagy-related genes.

(A) Huh7 cells transfected with HBV construct were prestained with MitoTracker (Mito, red) and subsequently, immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria. Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV-expressing cells. (B) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm. (C) Parkin protein in whole cell lysates of HBV-expressing cells was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a control for immunoprecipitation (IP). Western blot analysis for β-actin indicates equivalent amount of cell lysates for IP (C and D). (D) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non-targeting (NT) and Parkin (P) siRNA, respectively, for 48 h was immunoprecipitated by anti-Mfn2 antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal mouse IgG was used as a control for immunoprecipitation (IP). The protein expression was analyzed by Western blotting with antibodies specific to Mfn2, Parkin, and β-actin proteins. (E and F) HepAD38 and HepG2 cells were grown in the absence or presence of tetracycline (1 µg/ml) for 12 h. (E) Intracellular mRNA levels of Parkin, PINK1, and LC3B were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin, PINK1, and LC3B gene expression. (F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading control (F and G). The relative intensity of ATF4, Parkin, PINK1, and LC3B protein expression normalized to β-actin was analyzed by ImageJ software. (G) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins.
Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis. (2013)
Figure 3

HBV induces mitophagosomes.

(A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies against HBsAg (white), TOM20 (blue), and Parkin (red). Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. (B and C) Quantification of the number of total GFP-LC3 puncta (B) and GFP-LC3 puncta colocalized with TOM20 (C) in the panel (A). (D) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A).