EMBO/soda-nel huggingface dataset:
Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis. (2013)
Figure 4

HBV/HBx induces mitophagolysosomes.

(A) Novel system for monitoring mitophagic flux using dual fluorescence p-mito-mRFP-EGFP reporter (pAT016). Lysosomal delivery of the tandem fusion protein Mito-mRFP-EGFP along with entire mitochondria results in differential quenching and degradation of the two individual fluorochromes, thus allowing for visual analysis of mitophagic flux. (B) Huh7 cells transiently expressing Mito-mRFP-EGFP were transfected with HBV, HBx-flag, and HBV-ΔX constructs, respectively, for 48 h. Cells were immunostained with antibodies specific to HBsAg and flag (white), respectively. Nuclei were stained with DAPI (blue). Transfected (+) and untransfected (−) cells are marked. In the zoomed images, fluorescence signals indicate the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. (C) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel (B). (D) GFP-LC3-expressing Huh7 cells 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 prestained with LysoTracker (Lyso, red) were immunostained with antibodies against HBsAg (white) and TOM20 (blue). Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (−) cells are marked. In the zoom images, the arrows denoting white puncta indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (E) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (D).
Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis. (2013)
Figure 5

Attenuation of mitochondrial apoptosis by HBV.

(A–C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C). (D) A model elucidating HBV-induced aberrant mitochondrial dynamics in mitochondrial apoptosis. HBV induces immoderate stimulation in mitochondrial fission and mitophagy through mitochondrial translocation of Drp1 and Parkin, respectively, eventually causing disruption in mitochondrial dynamics. Inhibition of mitophagy by Parkin silencing can lead to apoptotic cell death in HBV-infected host cells via accumulation of apoptotic signal (red).
Trans-generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy (2020)
Figure 1

Figure 1 - Celf2a inactivation through CRISPR/Cas9 in UP∆44 myocytes induces skipping of exon 45.

A GS∆44 myoblasts were transfected with an empty vector (CTRL) or with a CELF2a overexpressing construct (OE CELF2A) (Martone et al, 2016). Cells were collected after 10 days in differentiation medium (DM) and the RNA was analysed by RT-PCR for CELF2a expression and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).

B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UP∆44 (UP∆44#3) and ∆44 edited clones (UP∆44#3.1 and UP∆44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm.

C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.

D RNA extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).

E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP∆44#3 to reach a total amount of 40 μg. Representative results are shown (n=3).
Trans-generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy (2020)
Figure 2

Figure 2 - Celf2a repression in GS∆44 is due to epigenetic silencing.

A iPSCs obtained from a control (WT#1; (Lenzi et al, 2015), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo) and RNA was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown (n=3).

B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm.

C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.

D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).

E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).
Trans-generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy (2020)
Figure 3

Figure 3 - Chromatin accessibility and epigenetic signature of control and GS∆44 myoblasts.

A DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).

B Pie charts showing ATAC-seq peaks' genomic distribution (blue: promoter region, grey: gene bodies, white: intergenic regions).

C Pie charts showing genomic distribution of ChIP-seq peaks (H3K4me3, H3K27ac and H3K27me3) (blue: promoter region, grey: gene bodies, white: intergenic regions).

D Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.
Trans-generational epigenetic regulation associated with the amelioration of Duchenne Muscular Dystrophy (2020)
Figure 4

Figure 4 - A trans-acting lncRNA controls Celf2a expression.

A Schematic representation of the exon/intron organization of Celf2 genomic locus. Arrows indicate TSS sites.

B RT-PCR analyses of indicated RNAs in control (WT), ∆44 and in GS∆44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown (n=3).

C RT-PCR analyses for CELF2a and DUXAP8 expression in WT and GS∆44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown (n=3).

D RT-PCR analysis of DUXAP8 expression in RNA extracted from GS∆44, GS∆44's mother (GSM) and GS∆44's father (F) fibroblasts. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).

E RT-PCR analysis for DUXAP8 expression in RNA extracted from WT and GS∆44 myocytes and from WT (WT#1), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).

F RT-PCR for the indicated RNAs on extracts from GS∆44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected two days after transfection. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).

G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96h. Relative mRNA levels were calculated with the delta delta Ct method. qPCR reactions were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown (n=3).
Iron loss triggers mitophagy through induction of mitochondrial ferritin (2020)
Figure 1

Figure 1. Induction of mitophagy by iron chelators (DFP, DFX or DFO).

(A) Immunoblots for LC3 using whole cell lysates of Huh7 cells before and after treatment with iron chelators (DFP, DFX or DFO). β-actin was used as loading control. Bafilomycin (-): without Bafilomycin, Bafilomycin (+): with 100 mM of Bafilomycin.

(B) Dual excitation (550/488 nm) of mt-mKeima transfected-Huh7 cells shows red fluorescence indicating mitophagy with DFP treatment at 24 h and green fluorescence without DFP treatment. Scale bar: 10 μm.

(C) The high ratio signal cells were quantified by flow cytometry with dual excitation of mt-mKeima at 633/488 nm for untreated (left panel) and DFP-treated (24 h, right panel) Huh7 cells.

(D) Untreated Huh7 cells or those treated with DFP (10 μM), DFX (10 μM) or DFO (10 μM) were quantified for mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates). A high signal (633/488) area/ cell area indicates the proportion of cells undergoing mitophagy. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.

(E) Electron microscopic images of untreated (left panel) and DFP-treated (right panel) Huh7 cells. Arrows indicate mitophagosome-like structures. Scale bar: 500 nm. The number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) between cells treated with DFP (1.0 mM) and those without DFP. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.

(F) Quantification of mitophagy using mt-mKeima transfection and FACS analysis for Huh7 cells treated with DFP (0, 0.01. 0.5 or 1.0 mM as indicated) in the absence or presence of bafilomycin (n=4, biological replicates). The central horizontal bar and the error bards indicate mean ± SD. *: P<0.05, **: P<0.01.

(G) Immunoblots for Parkin, PINK1, phosphorylated p-62 (p-p62), HSP60, LC3, p62/SQSTM, and Atg5 using the mitochondrial fraction and whole cell lysates of Huh7 cells. HSP60 and β-actin were used as loading controls for mitochondria and whole cell lysates, respectively.
Iron loss triggers mitophagy through induction of mitochondrial ferritin (2020)
Figure 3

Figure 3. Mitochondrial ferritin is required for iron loss-induced mitophagy.

(A) Immunoblots for mitochondrial ferritin (FTMT) with HSP60 as the loading control using the mitochondrial fraction of Huh7 cells and HepG2 cells (upper panels) and the quantification of mitophagy using mt-mKeima transfection and FACS analysis (n=4, biological replicates) (lower panels) before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. NC: negative control siRNA, **: P<0.01.

(B) Immunofluorescence staining for LC3 (red), the mitochondrial marker Tom20 (green) and FTMT (blue) in Huh7 cells before and after DFP (1.0 mM) treatment with or without FTMT knockdown by siRNA. FTMT colocalize with LC3 at the mitochondria (white puncta indicated by arrows). Boxed areas are enlarged below. Scale bar: 5 μm

(C) Immunoblots for mitochondrial ferritin (FTMT) following FTMT knockdown in the absence or presence of DFP using the mitochondrial fraction of Huh7 cells and those transfected with FTMT siRNA-resistant FTMT (left panel). HSP60 as the loading control. Those cells were subjected to the quantification of mitophagy using mt-mKeima transfection and FACS analysis before and after DFP (1.0 mM) treatment (n=5, biological replicates) (right panel). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. ***: P<0.001.

(D) Super resolution images of immunofluorescence staining for LC3 (red), FTMT (green) and Tom20 (blue) in FTMT knockdown-Huh7 cells transfected with FTMT-GFP or FTMT siRNA-resistant FTMT-GFP before and after DFP (1.0 mM) treatment. White arrows indicate colocalization of LC3, FTMT, and Tom20.
Iron loss triggers mitophagy through induction of mitochondrial ferritin (2020)
Figure 4

Figure 4. Interaction of mitochondrial ferritin with nuclear receptor coactivator 4.

(A) Extracted proteins from Huh7 cells that were untreated or treated with DFP were mixed with anti-FTMT-coupled magnetic beads and immunoblotted with the indicated antibodies (anti-NCOA4 or anti-LC3).

(B) GST pulldown assay using recombinant GST-NCOA4 mixed with recombinant His-FTMT. Bound FTMT was analyzed by immunoblotting using either anti-FTMT or anti-His antibodies.

(C) Immunofluorescence staining for Tom20 (red), NCOA4 (green) and FTMT (blue) in Huh7 cells before and after DFP (1 mM) treatment. Boxed areas are enlarged on the right. The white puncta indicated by white arrows show the colocalization of FTMT, NCOA4 and Tom20. Scale bar: 10 μm and 5 μm in enlarged images.

(D) Super-resolution images of immunofluorescence staining for Tom20 (blue), NCOA4 (red) and FTMT (green) in Huh7 cells after DFP (1 mM) treatment, using the single-molecule fluorescence microscope. The white arrows indicate colocalization of these proteins.

(E) Immunofluorescence staining for Tom20 (blue), FTMT-GFP (green), recombinant FTMTR82A-GFP (green), NCOA4-mCherry (red) and NCOA4I489A/W497A-mCherry (red) in Huh7 cells. Boxed areas are enlarged on the right. The white puncta pointed by white arrows in the overlaid picture indicate the colocalization of Tom20, FTMT-GFP and NCOA4-mCherry. The yellow puncta shown by white arrows in the overlaid picture indicate the colocalization of FTMT-GFP and NCOA4-mCherry. Scale bar: 10 μm and 5 μm in enlarged images.

(F) Extracted proteins from Huh7 cells expressing the recombinant proteins as indicated were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies. Co-immunoprecipitation revealed a specific interaction of NCOA4-mCherry with FTMT-GFP or FTMTD57A-GFP, which has a substitution of alanine (A) for aspartic acid (D) at position 57 in the FTMT mitochondrial leader sequence (lanes 1 and 3).

(G) Following DFP (1.0 mM) or ammonium iron sulfate (FAS) treatment as indicated, extracted proteins from Huh7 cells expressing FTMT-GFP and NCOA4-mCherry were mixed with anti-GFP-coupled magnetic beads and immunoblotted with anti-mCherry and anti-GFP antibodies.
Iron loss triggers mitophagy through induction of mitochondrial ferritin (2020)
Figure 5

Figure 5. Induction of mitochondrial damage by deferiprone and localization of mitochondrial ferritin precursor in the outer mitochondrial membrane.

(A) Flowcytometric analysis of the mitochondrial membrane potential of the isolated mitochondria. The mitochondria isolated from Huh7 cells with CCCP treatment and with or without DFP treatment were incubated with JC-1 for 1 h at 30°C and subjected to flow cytometric analysis (n=4, biological replicates) by excitation with a blue laser (488 nm) with simultaneous emission assessment using a 585/42 detector for JC-1 red and a 530/30 for JC-1 green. The trapezoid areas show the population of depolarized mitochondria. Quantification of the proportion of depolarized mitochondria in Huh7 cells with or without DFP treatment is shown in the lower panel. The central horizontal bar and the error bards indicate mean ± SD. Two sample t test was used for statistical analysis. **: P<0.01.

(B) The oxygen consumption rate was measured using a Seahorse XF24 Extracellular Flux Analyzer (n=4, technical replicates). Huh7 cells were incubated with 1 mM of DFP for different lengths of time (0, 3, 6, 18, and 24 h). Oxygen consumption was measured, and the respiration rate was analyzed by injecting of 1μM oligomycin, 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 10 μM rotenone/antimycin A. OCR: oxygen consumption rate. The central point and the error bards indicate mean ± SD.

(C) Immunofluorescence staining for FTMT-GFP (green) and LC3 (red) in Huh7 cells transfected with FTMT-GFP. The proportion of colocalization was quantified for four randomly selected areas (technical replicates) using ImageJ software version 1.46 (NIH, Bethesda, MD). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. **: P<0.01.

(D) Immunofluorescence staining for Ferritin-GFP heavy chain (FTH1) (green) and LC3 (red) in Huh7 cells transfected with FTH1-GFP. The proportion of colocalization was quantified as described in (C). *: P<0.05, **: P<0.01.

(E) Immunoblots for the precursor form and processed form of FTMT-GFP, endogenous FTMT, NCOA4, LC3, and Tom20 using the mitochondrial fractions of Huh7 cells expressing FTMT-GFP with or without digestion with proteinase K in the presence or absence of DFP. mtHSP70 was used as the loading control.

(F) Immunoblots for FTMT, VDAC, and mtHSP70 using the mitochondrial membrane from Huh7 cells with or without DFP treatment. The mitochondrial membrane was fractionated from Huh7 cells by means of sodium carbonate treatment. mtHSP70 was used as the non-membrane-integral protein control and VDAC as the membrane-integral protein control. mtHSP70 at the right-hand side in the third lane indicates the positive control obtained from the mitochondrial fraction of Huh7 cells without DFP treatment.
Iron loss triggers mitophagy through induction of mitochondrial ferritin (2020)
Figure 6

Figure 6. Selective induction of iron loss-induced mitophagy for damaged mitochondria and suppression of liver tumor, fibrosis and reactive oxygen species production by deferiprone.

(A) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), and LC3 (green) in Huh7 cells untreated or treated with DFP (1 mM) for 24 h, or with CCCP (30 μM) for 6 h. Boxed areas are enlarged on the right. Scale bar: 10 μm, and 5 μm in enlarged images.

(B) Immunofluorescence staining for the polarized mitochondria (red), Tom20 (blue), LC3 (blue), and FTMTD57A (green) using Huh7 cells expressing FTMTD57A. Boxed areas are enlarged on the right. The white puncta in overlaid pictures indicate the colocalization of the polarized mitochondria, Tom20 and FTMTD57A or the colocalization of the polarized mitochondria, FTMTD57A, and LC3. Scale bar: 10 μm, and 5 μm in enlarged images.

(C) STAM mice were fed a high-fat diet and orally administered 0.0375 mg/g body weight of DFP, 0.075 mg/g body weight of DFP or distilled water from 4 weeks of age to 16 weeks of age (n=6). Arrows indicate liver tumors. Scale bar: 1.0 cm. The number and maximum size of liver tumors were compared among the three groups. The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05, **: P<0.01.

(D) Histology of the nontumorous liver (H&E and Sirius red stain, original magnification x40) and superoxide anion production in the livers of STAM mice fed a high-fat diet with or without DFP (0.075 mg/g) administration. Frozen liver sections of mice were stained with dihydroethidium for assessment of superoxide anion. Scale bar: 100 μm.

(E) Histology of the hepatocellular carcinoma using H&E staining in STAM mice fed a high-fat diet. The boxed area is enlarged on the right (x40 in left panel and x400 in right panel). The tumor has increased cell density, an increased ratio of nucleus to cytoplasm, and thickened trabeculae Scale bar: 100 μm.

(F) Electron microscopic images of livers in control (C57BL/6) and DMBA + HFD mice that were orally administered 0.075 mg/g of DFP or distilled water. Scale bars: 2.0 μm. The boxed areas (x 1,500) are enlarged below (x6,000 on left and x10,000 on right). The white arrow indicates mitophagosome-like structures. The mean number of mitophagosome-like structures/100 μm2 was compared for four randomly selected areas (technical replicates) among the four groups (control, DMBA + HFD mice treated with 0, 0.0375 or 0.075 mg/g of DFP) (n=6). The central horizontal bar and the error bards indicate mean ± SD. The Tukey's honestly significant test was used for statistical analysis. *: P<0.05.