EMBO/soda-nel huggingface dataset:
PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice (2021)
Figure 3

Figure 3

PLK1 phosphorylates the EBNA2 residue S457 and T465 within the C-terminal transactivation domain

(A) Doxycycline (Dox) induction of EBNA2 in DG75Dox HA-EBNA2 cells treated for 24 hours. Co-immunoprecipitates of HA-EBNA2 and endogenous PLK1 are visualized by Western blotting.

(B) EBNA2/PLK1 co-precipitates were submitted to kinase reactions using [γ-32P] ATP in the absence (control) or presence of 50 ng recombinant active PLK1.

(C) EBNA2 co-precipitates were submitted to kinase reactions in the absence (control) or the presence of the PLK1 inhibitor Volasertib (40 nM).

(D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting.

(E) Immunoprecipitates were submitted to kinase reactions as in B, but samples were treated with Volasertib (40 nM) (+) or treated with solvent only (-).

(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate (Yuan et al, 2002), was used as a positive control.

(G) Schematic presentation of EBNA2 phosphorylation sites by CDK1 and PLK1.

(H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity. Data were presented as the mean ± S.E.M. of n = 3 biological replicates. Statistical significance was tested by one-way ANOVA followed by a Tukey's multiple comparison test (ns: not significant, ***: P < 0.001, ****: P < 0.0001, vs WT).

(I) GST-pull down assay using GST-EBNA2 446-474 as a bait to purify cellular proteins from DG75 cells followed by Western blotting and quantification of signals obtained by GST and p300 specific antibodies. Relative binding affinities (rel. p300 bindg.) are normalized to the wt signal. Since 3 lanes of the original blot were excised a broken line marks the borders between two parts in the figure.
PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice (2021)
Figure 4

Figure 4

PLK1 inhibits EBNA2 activity by phosphorylating residues S457/T465

(A) Flag-tagged active (PLK1) and kinase inactive PLK1 (K82M) were co-expressed with HA-EBNA2 wt, docking site mutant (S379A) and phosphorylation mutant (S457A/T465V) and tested for activation of an EBNA2/CBF1 responsive promoter reporter in DG75 cells. Activation of the reporter firefly luciferase gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to EBNA2 wt activity. Data were presented as the mean ± S.E.M. of n = 3 biological replicates. Statistical significance was tested by two-way ANOVA followed by a Tukey's multiple comparison test (ns: not significant, *: P < 0.05, **: P < 0.01).

(B) 30 µg of cellular extracts that were produced for luciferase assays were tested for protein expression using Flag-, HA- and GAPDH-specific antibodies.
PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice (2021)
Figure 5

Figure 5

EBV strains expressing EBNA2 mutants deficient for PLK1 docking or phosphorylation accelerate B cell proliferation and express elevated levels of the viral LMP1 protein.

(A) Primary B cells were stained with cell trace violet before they were infected, and cell cultures were started. Loss of the fluorescent cell tracking dye was recorded by flow cytometry to monitor proliferation on day 0, 4 and 6 post-infection. Mean values for 3 biological replicates, standard deviation and significance of changes in signal loss of mutants compared to wild-type controls Data were presented as the mean ± S.E.M. of n = 3 biological replicates. Statistical significance was tested by two-way ANOVA followed by a Tukey's multiple comparison test (*: P < 0.05, ***: P < 0.001, ****: P < 0.0001, vs WT).

(B) Long-term growth transformed B cell cultures infected with recombinant EBV mutants were seeded at a starting concentration of 2 x 105 cells per ml. Data were presented as the mean ± S.E.M. of n = 4 biological replicates. Statistical significance was tested by two-way ANOVA followed by a Tukey's multiple comparison test (*: P < 0.05, **: P < 0.01, ****: P < 0.0001, vs WT).

(C) Expression of EBNA2, LMP1, PLK1, MYC, and GAPDH was analyzed by Western blotting and immunostaining of whole cell extracts of cell lines established from 3 individual donors.
PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice (2021)
Figure 6

Figure 6

EBV strains carrying EBNA2 mutants with diminished PLK1 binding or resistant to PLK1 phosphorylation cause lymphomas in humanized mice more frequently than EBV wt.

(A) Experimental set-up. (Images of animals, syringes and blood collection tubes are derived from Servier Medical Art.)

(B) Survival curve of humanized mice infected with 105 GRU EBV wt (n = 13), EBV S379A (n = 11) or EBV S457A/T465V EBV (n = 9). Log-rank test.

(C) Percentage of mice having macroscopically visible tumors at the day of sacrifice. Numbers within bars indicate the total number of mice with or without tumors in the respective groups. Fisher's Exact test.

(D) Viral loads in spleen of infected humanized mice at the day of sacrifice. Error bars indicate mean ± SEM. Number of biological replicates per group: EBNA2 WT EBV: n = 13; EBNA2 S379A EBV: n = 11; EBNA2 S457A T465V EBV: n = 9.

(E) Development of viral loads in blood of infected mice over a period of five weeks. Error bars indicate mean ± SEM. Number of biological replicates per group: EBNA2 WT EBV week 2 - 5: n = 8; EBNA2 S379A EBV week 2 - 5: n = 7; EBNA2 S457A T465V EBV week 2: n = 9, week 3: n = 8, week 4 and week 5: n = 7

(F) Flow-cytometric analyses of CD8+/CD4+ T cell ratios in the blood of infected mice over a period of five weeks. Error bars indicate mean ± SD. Number of biological replicates per group: EBNA2 WT EBV week 0, 2, 4, 5: n = 13, week 3: n = 8 ; EBNA2 S379A week 0, 2, 4, 5: n = 11, week 3: n = 7 ; EBNA2 S457A T465V week 0, 2: n = 12, week 3: n = 7, week 4: n = 10, week 5: n = 9.

Data information: The shape of data points indicates to which cohort (experiment 1, 2 or 3) the respective animal belongs while the color indicates the EBV EBNA2 mutant with which the animal was infected. Statistical significance was tested using the Mann-Whitney U Test with Holm-Sidak correction for multiple comparisons if not indicated differently.

EXPANDED VIEW FIGURE LEGENDS
Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress (2020)
Figure 3

Figure 3. GSNOR induction by hydrogen peroxide is dependent on the redox activation of ATM and kept sustained over time.

A. HEK293 cells treated for 4 h with 100 μM H2O2. Phospho-histone H2A.X (γH2A.X) was assessed by immunofluorescence analysis. Nuclei (blue) were stained with Hoechst 33342. scale bar: 10μM. 4X digital magnification is shown at the bottom left of each image. B. Additionally, γH2A.X was also assessed by Western blot at the indicated time points. GAPDH was used as loading control (top panel). Densitometry of each lane intensity is normalized to GAPDH, selected as loading control, and expressed as % of γH2A.X in H2O2-treated versus untreated cells. Values represent the means ± SD of two independent experiments (bottom panel). C. HEK293 cells were treated for 8 or 24 h with 100 μM H2O2 or, alternatively, for 2 h with 2 mM hydroxyurea (HU), selected as positive control of ATR/CHK1 axis activation. Basal and phospho-active forms of ATR and CHK1 (at Ser345) were assessed by Western blot. GAPDH was used as loading control. D. HEK293 cells were treated for 8 h with 20 μM neocarzinostatin (NCS). (left) Western blot analysis of basal and phospho-active forms of ATM, CHK2, ATR, CHK1, p53, and GSNOR in HEK293 cells treated for 8 h with 20 μM neocarzinostatin (NCS). Vinculin and GAPDH were used as loading controls. (right) Phospho:basal level ratios of ATM, CHK2, ATR, CHK1, along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. E. HEK293 cells were treated for 2, 4 and 8 h with 20 μM neocarzinostatin (NCS). After treatment, cells were incubated with 2',7'-H2DCF-DA to cytofluorometrically assess the intracellular production of H2O2. Values are shown as units of DCF fluorescence relative to untreated cells. Values are shown as units of fluorescence relative to NCS-untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. n.s., not significant. F. HEK293 cells were transfected with plasmids coding for the wild-type (WT), C2991L redox mutant (CL) of ATM, or with an empty vector (Empty) (used as negative control) for 40 h and treated for additional 8 h with 100 μM H2O2. Basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot analysis. Vinculin and GAPDH were used as loading controls. Two different exposures (low and high) were selected to highlight differences in P-ATM levels in different experimental settings. Phospho:basal level ratios of ATM, along with densitometry of GSNOR immunoreactive bands are expressed as arbitrary units relative untreated cells. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.
Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress (2020)
Figure 4

Figure 4. GSNOR induction by hydrogen peroxide involves p53 as downstream effector of ATM/CHK2 phosphorylation axis.

A. HEK293 cells were treated for 24 h with 100 μM H2O2 in the presence or absence of 20 μM pifithrin-α (Pft). Basal and phospho-active forms of ATM, CHK2, p53, and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM, CHK2 and p53 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. B. HEK293 cells were transfected with a pool of siRNA against p53 (sip53) or with control siRNA (siScr) for 24 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, as well as p53 and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant. C. HCT116 cells expressing the wild type form of p53 (p53wt), or an empty vector (Empty, selected as negative control), were treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, p53 and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2, along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; n.s., not significant.
Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress (2020)
Figure 5

Figure 5. ATM/GSNOR axis drives mitophagy upon H2O2 treatment.

A, B. HEK293 cells treated for 2, 4 and 8 h with 100 μM H2O2. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001. C. HEK293 were transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterwards, they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [i.e., NDUFB8 (complex I), SDHB (complex II), MTCO2 (complex IV)]. Basal and phospho ATM, as well as GSNOR were used to check the efficiency of siRNA-mediated knock down. Vinculin was used as loading control. D. Densitometry of mitochondrial protein immunoreactive bands of panel C (normalized to Vinculin) is indicated as H2O2-treated versus untreated cells (CTR) and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant. E. In the same experimental settings, mitophagy was also assessed by RT-qPCR relative quantitation of D-loop (selected as measure of mtDNA) normalized to genomic actin (gActin). Results shown are the means ± SD of n = 8 experiments ***p<0.001; n.s., not significant. F. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 4 h with 100 μM H2O2. Mitophagy was assessed by RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 6 experiments. *p<0.05; ***p<0.001. G, H. In the same experimental settings, mitophagy was also assessed at 8 h by fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 7 different cells analyzed. *p<0.05; **p<0.01; ***p<0.001.
Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress (2020)
Figure 6

Figure 6. ATM/GSNOR axis drives mitophagy upon CCCP treatment.

A, B. ATMWT U2OS cells were treated with 10 μM CCCP for 8 h. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. ***p< 0.001. C. In the same experimental settings, basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. D-H. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. Mitophagy was evaluated by: (D) RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 4 experiments. *p<0.05; **p<0.01; ***p<0.001. (E, F) fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 9 different cells. **p<0.01; ***p<0.001; n.s., not significant. (G, H) Western blot of different subunits of mitochondrial proteins, i.e., NDUFB8 (complex I), SDHA and SDHB (complex II), MTCO2 (complex IV) and voltage-dependent anion channel (VDAC). Tubulin, LDH and Vinculin were used as loading controls. Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant.
Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress (2020)
Figure 7

Figure 7. Redox activation of ATM/GSNOR axis is required to counteract nitroxidative stress-induced cell death.

A. Scheme of the protocol designed for the combined treatment of HEK293 cells with H2O2 and DPTA. B. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) in the presence or absence of the ATM inhibitor KU55933 (KU). Analysis of dead cells were performed with Trypan blue exclusion assay. Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency. Data, shown as fold change of dead cells relative to untreated cells (arbitrarily set to 1), represent the mean count ± SEM of n = 4 experiments done in duplicate *p<0.05; with respect to Empty cells. C. Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU. D. Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C. Tubulin was used as loading control. E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p<0.01; n.s., not significant.

F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h. G, H. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. Scale bar = 50 µm. Data, shown as % of dead (red) cells, represent the mean count ± SD of n = 3 different fields of three independent experiments. *p<0.05; n.s., not significant with respect to WT MAFs.
Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome. (2015)
Figure 1

Cellular locations of native and virally mediated Kcnq1 expression in the cochlea

A Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in (B–F).

B–F Immunolabeling results of Kcnq1 (green) in cochlear cryosections are shown for uninjected WT (B), untreated Kcnq1−/− (C), injected WT (D), and Kcnq1−/− mice given injections into the ST (E) or SM (F). Cell nuclei were outlined by counterstaining with either DAPI (B–D, F) or Qnuclear deep red (E). Scale bars represent approximately 100 μm. Meaning of white arrows are given in the text.
Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome. (2015)
Figure 2

Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cells

Kcnq1 and cell nuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.

Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.

Cryosection through the SV of a Kcnq1−/− mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1 immunolabeling in fibrocytes outside the SV.

Cryosection through the lateral wall of Kcnq1−/− mice showing intracellular distribution of Kcnq1 immunolabeling (green) in fibrocytes.

Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/− mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).

Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cell nuclei. Scale bars represent appropriately 50 μm.
Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome. (2015)
Figure 3

Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SV

The membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.

A Immunolabeling results (Kcnq1 labeled in green) in WT mice.

B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/− mice, middle (B) and apical (C) turns, respectively.

D The percentage of marginal cells having positive Kcnq1 immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/− mice (black bars, right). Data are given as mean ± SD (n = 6).

E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/− mice (F) are compared.

Data information: Scale bars represent approximately 50 μm.
Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome. (2015)
Figure 4

Comparison of cochlear morphology

A–C Cochlear sections obtained from WT (A), nontreated Kcnq1−/− (B), and treated Kcnq1−/− mice (C) were compared. Major landmarks of the cochlear sections are labeled and pointed by arrows. Scale bars represent approximately 50 μm.
Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome. (2015)
Figure 5

Comparison of the ABR data from WT, treated, and untreated Kcnq1−/− mice

Waveforms of ABRs are compared in WT, untreated Kcnq1−/−, and treated Kcnq1−/− mice, as labeled above data traces. Series of averaged ABR data traces were evoked from tone-burst sounds with intensities ranging from 20 to 90 dB SPL.

Summary of averaged ABR thresholds at various frequencies for different groups of mice; untreated Kcnq1−/− (open triangles), treated Kcnq1−/− (filled triangles), injected WT (filled circles), and uninjected WT (filled squares). Plot legends are given in the figure. The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1−/− mice. Error bars represent standard error of the mean.

Click-evoked ABR thresholds of WT (filled squares), treated Kcnq1−/− mice (filled triangles), untreated Kcnq1−/− mice (open squares) measured 4–30 weeks after mice were born. Error bars represent standard error of the mean. Upward arrows indicate that click-ABR thresholds were at the maximal sound level that could be reliably measured by the system.