EMBO/soda-nel huggingface dataset:
Histone acetyltransferase 1 is a succinyltransferase for histones and non-histones and promotes tumorigenesis (2020)
Figure 5

Figure 5. HAT1 succinylates PGAM1 to enhance glycolysis

A. PGAM1 was immunoprecipitated from HepG2, HAT1 KO Clone 1 HepG2, and HAT1 KO Clone 1 HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). The levels of succinylation, PGAM1, HAT1, and β-actin were analyzed by Western blot analysis in the cells.

B. Relative PGAM1 activity was measured by PGAM1 activity assays in the indicated tumor cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

C. The decrease of 2-PG and increase of 3-PG were measured by ELISA assays in HepG2, HAT1 KO HepG2, and HAT1 KO HepG2 cells reconstituted with either wildtype HAT1 or mutant HAT1 (T188A). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

D. HAT1-catalyzed PGAM1 succinylation was analyzed by mixing purified HAT1, PGAM1, and succinyl-CoA/acetyl-CoA in the in vitro succinylation assays. Western blot analysis was performed with indicated antibodies.

E. Purified HAT1, PGAM1 or PGAM1 mutant, and succinyl-CoA were mixed in the in vitro succinylation assays and relative PGAM1 activity was measured by PGAM1 activity assays in vitro. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

F. The decrease of glucose and increase of lactate in the culture medium and the relative amounts of 2-PG and 3-PG were analyzed by ELISA assays in HepG2 cells depleted endogenous PGAM1 and reconstituted expression of PGAM1 or PGAM1 mutant (K99R). N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.
Histone acetyltransferase 1 is a succinyltransferase for histones and non-histones and promotes tumorigenesis (2020)
Figure 6

Figure 6. HAT1-mediated succinylation displays critical roles in tumor growth

A. HAT1 levels were assessed by immunohistochemical staining in clinical tissues of liver cancer patients, pancreatic cancer patients, and cholangiocarcinoma patients. Scale bar = 100 μm

B. The effect of HAT1-mediated succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

C. The effect of PGAM1 succinylation on cancer cell proliferation was measured by MTS assays in the indicated cells. N = 3 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.

D-H. HepG2 cells, HAT1 KO HepG2 cells, and HAT1 KO HepG2 cells reconstituted with either wild type HAT1 or mutant HAT1 (T188A) were subcutaneously injected into athymic nude mice (N=6). (D, E) Photographs showing the tumors from the nude mice. Tumor volumes and average tumor weight were calculated. N = 6 biological replicates. Scale bar = 10 mm. In the boxplots, boxes extend from the 25th to 75th percentiles (inter‐quartile range (IQR)), central band represents the median, whiskers indicate the lowest and highest data within 1.5 × IQR from the lower and upper quartiles, respectively. Student's t-test, ***P < 0.001 (F) Immunohistochemical analysis of the indicated tumor sections was performed with an anti-Ki67 antibody. The images represent the results of six tissue slides. Scale bar = 100 μm. (G) Western blot analysis was performed with the indicated antibodies. Representative images of triplicate experiments are shown. (H) The relative amounts of 3-PG and 2-PG were determined by ELISA assays in the tumor tissues. N = 6 biological replicates. Data are presented as mean ± SD. Student's t-test, **P < 0.01.
Recruitment of TRIM33 to cell-context specific PML nuclear bodies regulates Nodal signaling in mESCs (2022)
Figure 1

Figure 1. TRIM33 forms puncta in the nucleus of mESCs but not in the differentiated cells and interacts stronger with PML in mESCs than in differentiated cells.

(A) Confocal microscopy of GFP-TRIM33-overexpressing mESCs and differentiated cells. Scale bar = 5 µm.

(B) Line scans of GFP-TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software.

(C) FRAP assays showing recovery time of normalized fluorescence signals for GFP-TRIM33 puncta in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of three biological experiments.

(D) Sphericity analysis and 3D imaging by NIS-Elements 3D tools of TRIM33 puncta in live mESCs. Panels show the xz, xy, and yz planes, from left to right. Scale bar = 1 µm.

(E) Table of top ranking GFP-TRIM33 interacting proteins. GFP-trap-based affinity purification of extracts prepared from both mESCs and differentiated cells were carried out to identify factor(s) that associate with GFP-TRIM33. Mass spectrometry (MS) analysis was performed following the affinity purification.

(F) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co-transfected with plasmids encoding Flag-PML and GFP-TRIM33. GFP-TRIM33 was immunoprecipitated with anti-GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP. Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates. (n = 3).

(G) Lysates from mESCs and differentiated cells were immunoprecipitated with anti-PML antibody, and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML. (n = 3).

(H) Lysates from HEK293T cells co-transfected with plasmids encoding either full length Flag-TRIM33 or the Flag-TRIM33-RBCC domain alone and full-length HA-PML or the HA-PML-RBCC domain alone immunoprecipitated with anti-Flag affinity beads. Protein complexes were detected using antibodies against Flag and HA. The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates. (n = 3).

(I) Schematic diagram of TRIM33 and PML interaction domains.

Data information: All data are from three biological replicates.
Recruitment of TRIM33 to cell-context specific PML nuclear bodies regulates Nodal signaling in mESCs (2022)
Figure 2

Figure 2. TRIM33 puncta formation is dependent on proper assembly of PML NBs and TRIM33 puncta colocalize with DAPI in the absence of PML.

(A) Representative images of immunofluorescence staining of PML in WT and Pml null mESCs. Scale bar = 5 µm.

(B) Representative confocal microscopy images of mESCs overexpressing mCherry-PML or GFP-PML (top). Representative confocal microscopy images of mESCs overexpressing mCherry-EV or GFP-EV (bottom). Scale bar = 5 µm.

(C) FRAP assays quantifying normalized fluorescence recovery of GFP-PML signal in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of four biological experiments.

(D-L) (D, G, I, K) Representative images of co-localization assays with mCherry-PML and GFP-TRIM33 in mESCs (D), differentiated cells (G), NaAsO2-treated mESCs (200 µM for 2 hr) (I) and Pml null (K). Scale bar = 5 µm. (E, H, J, L) Line scans of GFP-TRIM33 and mCherry-PML fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (F) Column graph of the numbers of GFP-TRIM33 puncta and mCherry-PML NBs in mESCs.

(M) Column graph of the numbers of GFP-TRIM33 puncta and DAPI in mESCs.

(N) Scatter plot of the numbers of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20).

(O) Scatter plot of the volume of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20).

(P) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs. Scale bar = 10 µm.

(Q) Scatter plot of the numbers of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20).

(R) Scatter plot of the proportion of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20).

Data information: In panels (N, O, Q and R), 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001).
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 1

HCV infection induces perinuclear clustering of mitochondria and Parkin translocation to mitochondria.

(A) Representative confocal images showing perinuclear clustering of mitochondria and Parkin aggregates on the mitochondria in Huh7 cells infected with HCVcc. At 3 days post-infection, cells prestained with MitoTracker (Mito, red) were immunostained with anti-Parkin (green) and HCV core (orange) antibodies. Nuclei were stained with DAPI (blue). Infected (+) and uninfected cells (−) are marked. The illustrated images display typical distribution of mitochondria in uninfected cells and altered distribution of mitochondria in the perinuclear region of infected cells (gray). In the zoomed images, the white arrows indicate accumulation of endogenous Parkin recruited to the mitochondria in HCV-infected cells (yellow). (B) Ultrastructure of HCV-infected 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.
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 2

HCV infection induces the ubiquitination of Parkin and its mitochondrial substrates Mfn2 and VDAC1, and autophagy-associated factor p62.

(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.
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 3

HCV infection stimulates ATF4, Parkin, and PINK1 gene expression.

(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.
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 4

HCV induces Parkin-mediated mitophagosome formation.

Confocal microscopy showing mitophagosome formation associated with Parkin in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 day post-infection, cells were immunostained with antibodies against HCV E2 (orange), TOM20 (blue), and Parkin (red). In the zoom images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. Nuclei are demarcated with white dot circles. (B) 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.
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 5

HCV induces complete mitophagolysosome formation.

Confocal microscopy showing the formation of mitophagolysosome in HCV-infected cells. (A) Huh7 cells transiently expressing GFP-LC3 protein were infected with HCVcc in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 12 h before fixation. At 3 days post-infection, cells prestained with LysoTracker (red) were immunostained with antibodies against TOM20 (blue) and HCV core (cyan). Nuclei are demarcated with white dot circles. In the zoomed images, the arrows indicate the colocalization of GFP-LC3 puncta (green), lysosome, and mitochondria in HCV-infected cells (white puncta). (B) Ultrastructure of HCV-infected 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.
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 6

Parkin and PINK1 affects HCV replication.

(A and B) Inhibitory effect of Parkin, PINKI, and ATG5 silencing on HCV replication. Huh7 cells transfected with Non-Targeting (NT) or gene-specific siRNA pools targeting Parkin, PINK1, and ATG5 genes, respectively, were infected with HCVcc. At day 3 post-infection, the levels of HCV RNA (A) and targeted gene mRNA (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.
Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. (2013)
Figure 7

Mitochondrial functions altered by HCV infection are associated with Parkin-mediated mitophagy.

(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.