EMBO/soda-nel huggingface dataset:
SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense (2016)
Figure 4

Figure 4. SIRT5 desuccinylase activity, but not its expression, is regulated by oxidative stimuli

(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).

(C) Schematic representation of the experimental workflow for Figs. 4D and 4E.

(D) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in ‘Materials and Methods’. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).

(E) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in ‘Materials and Methods’. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).

(F) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in ‘Materials and Methods’, normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).

(G) A proposed model for a role of SIRT5 in mediating oxidative stress to increase IDH2 and G6PD activities, NADPH production, and cellular protection. SIRT5 is activated by oxidative stress by a yet unknown mechanism. The active SIRT5 stimulates IDH2 and G6PD by desuccinylation and deglutarylation, respectively, thereby increasing NADPH production and protecting cells from ROS damage.
N(6)-methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay (2021)
Figure 4

Figure 4. YTHDF1 knockdown promotes the expression of BZLF1 and BRLF1

A. The relative mRNA levels of BZLF1 and BRLF1 at 24 hpi following EBV infection of HK1 cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV-GFP and analyzed by qRT-PCR at 24 hpi. All the gene expression levels were normalized to the housekeeping gene ACTB. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. *P<0.05 and **P<0.01 compared to siNC according to unpaired Student's t-test.

B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. *P<0.05, **P<0.01 and ***P<0.001 compared to siNC according to unpaired Student's t-test.

C. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. The total RNA was harvested from the cells at 24 h after siRNA transfection. All gene expression levels were normalized to the housekeeping gene ACTB. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. *P<0.05, **P<0.01 and ***P<0.001 compared to siNC according to unpaired Student's t-test.

D. The protein levels of YTHDF1 and BZLF1 in the EBV-infected (24 hpi) HK1 cells, CNE2EBV cells with induced EBV reactivation (24 h post NaB treatment) and C666 cells transfected with YTHDF1-specific siRNA (siY1-1 and siY1-2) or the siNC control. The whole-cell protein was extracted at same time as the RNA.

E. The relative mRNA expression levels of YTHDF1 in CNE2EBV cells following EBV reactivation (24 h post NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. **P<0.01 according to unpaired Student's t-test.

F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.
N(6)-methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay (2021)
Figure 6

Figure 6. YTHDF1 interacts with the RNA degradation complex

A. The interaction partners of YTHDF1 were identified by pull-down assays and mass spectrometry. The Flag-YTHDF1 pull-down assay was performed in CNE2EBV cells transfected with a plasmid encoding Flag-tagged YTHDF1. The proteins in the differentially abundant bands were identified by mass spectrometry.

B, C. The interactions between YTHDF1 and ZAP (B) or DDX17 (C) were measured by co-IP in 293T cells. The results were detected using specific antibodies.

D. The interaction between YTHDF1 and DCP2 was detected by co-IP in 293T cells.

E. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP using a YTHDF1-specific antibody in CNE2EBV cells.

F. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2-, YTHDF3-specific siRNAs or the siNC control. YTHDF1, YTHDF2, YTHDF3, ZAP, DDX17, and DCP2 were detected in the immunoprecipitated complexes using specific antibodies, respectively.
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 3

Figure 3| Endothelial kugeln are non-nucleated with a filamenteous actin-enriched neck.

A Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead).

B Double transgenic showing endothelial membrane (red) and endothelial F-actin (green). F-actin was found to localize at the neck of the kugeln (arrowhead; see also: EV Movie3).

C Double transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead).

D Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead) and erythrocytes were not observed inside kugeln (arrowhead).

E Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).

F Latrunculin B treatment statistically significantly reduced kugel diameter (*p = 0.0164; control n=169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n=361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).

G Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 µM 1h; ****p <0.0001; control n=22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n=24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).

H Blebbistatin treatment had no effect on kugel diameter (p = 0.3731; control n=83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n=26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 4

Figure 4 | The relationship between endothelial kugeln and blood flow

A MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted).

B MIP of the same embryo after exsanguination, which did not alter kugel size.

C Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT).

D Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).

E Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).

F Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 5

Figure 5| Kugeln do not interact with brain lymphatic endothelial cells (BLECs) in Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916, nor do they take up injected IgG-conjugated Alexa 674.

A Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916.

B BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads).

C No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats).

D Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads).

E Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).

F Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).

G Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).

H Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 7

Figure 7| Kugel number is increased by VEGF inhibition, and kugeln contain NO.

A VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).

B Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).

C DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.

D Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).

E Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative "filled" (Lysotracker positive) and "unfilled" (Lysotracker negative) kugeln.

F The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).

G Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).
Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish (2019)
Figure 8

Figure 8| Kugel number is decreased by Notch inhibition but increased by Wnt inhibition or activation.

A Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).

B Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).

C A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats).

D Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats).

E Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).

F Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)

G Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).

H The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test).

Expanded View Figure Legends
Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing ara-C efficacy (2019)
Figure 1

Figure 1 | RNR inhibitor and ara-C synergy is dependent upon functional SAMHD1 in cancer cell models.

Schematic detailing proposed interplay between RNR and SAMHD1.

Immunoblot of lysates prepared from the indicated SAMHD1-proficient (+/+), deficient (-/-) and rescue (WT, H233A) cell line pairs with the indicated antibodies. Representative of 2 independent experiments.

Proliferation inhibition analysis of ara-C and RNRi combination treatment in SAMHD1+/+ or -/- THP-1 cells. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.

Ara-C EC50 values plotted as a function of RNRi concentration in SAMHD1+/+, -/- and rescue (WT, H233A) THP-1 cell line pairs. EC50 values in the absence of RNRi are indicated by the black and red dotted line. Error bars indicate s.e.m. of two (HU and dF-dC) or three (3-AP) independent experiments, each performed in duplicate.

Drug synergy plots for ara-C and the indicated RNRi in SAMHD1+/+, -/- and rescue (WT, H233A) cell line pairs. Each data point indicates an average delta score from a single dose-response matrix experiment performed in duplicate. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. The horizontal line and the error bars indicate the mean and s.d., respectively, statistical significance was determined using a two-tailed unpaired t-test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Spearman correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C versus HU or dF-dC in a panel (n = 9) of haematological cancer cell lines. Error bars indicate s.e.m. Each data point corresponds to SAMHD1 protein levels determined by immunoblot analysis (n = 4 for each cell line, representative blot shown in Appendix Fig S2) and an average delta score from repeated dose-response matrix experiments each performed in triplicate: THP-1, n =4; HuT-78, n = 2; HL-60/iva, n = 1; KBM-7, n = 2 (HU) and 3 (dF-dC); K562, n = 3 (HU) and 4 (dF-dC); CCRF-CEM, n =3 (HU) and 4 (dF-dC); MV-4-11, n = 2 (HU) and 3 (dF-dC); Jurkat, n = 2 (HU) and 3 (dF-dC); MOLT-4, n = 2 (HU) an 3 (dF-dC).

Immunoblot analysis of lysates prepared from SAMHD1+/+ or -/- THP-1 cells treated for 24 h with ara-C and HU, as indicated. Representative of 3 independent experiments.
Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing ara-C efficacy (2019)
Figure 2

Figure 2 | RNR inhibition overcomes the SAMHD1-mediated barrier to ara-C in AML mouse models

Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- THP-1 cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group. For further data and analysis see Appendix Fig S6.

Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ or -/- HL-60/iva cell clones (day 0) and treated with ara-C and/or HU as indicated (day 6). n = 6 per treatment group. For further data and analysis see Appendix Fig S7.

Kaplan-Meier analysis of NOD/SCID mice injected i.v. with luciferase-expressing SAMHD1+/+ THP-1 cell clone (day 0) and treated with ara-C and/or dF-dC as indicated (day 6). n = 7 per treatment group. For further data and analysis see Appendix Fig S8.

Kaplan-Meier analysis of CD45.2 C57BL/6J mice injected i.v. with murine MLL-AF9-transformed AML blasts (day 0) and treated with ara-C and/or HU days 20-24. n = 5 per treatment group, except for vehicle (n = 4). For further data and analysis see Appendix Fig S8 and Appendix Fig S9.

Data information: Tick marks indicate censored animals. Statistical significance determined using Mantel-Cox log-rank test: ns, not significant, P ≥ 0.05; *, P < 0.05; **, P < 0.01.
Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing ara-C efficacy (2019)
Figure 3

Figure 3 | Figure 3 | RNR inhibition enhances ara-C efficacy in primary patient AML blasts in a SAMHD1-dependant manner

Drug synergy plots for ara-C and HU or dF-dC in primary patient-derived AML blasts. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate, n = 16 for HU and n = 9 for dF-dC. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy. Median, upper and lower quartiles, and range of delta scores are indicated by box-and-whisker plots. For proliferation inhibition curves for each sample see Appendix Fig S10A and B, and for patient characteristics see Appendix Table S2.

Pearson correlation of relative SAMHD1 protein abundance and synergy delta scores for ara-C and HU or dF-dC in primary patient-derived AML blasts (n = 23). For immunoblot analysis of SAMHD1 protein abundance see Appendix Fig S10C.

Immunoblot of primary patient-derived AML blasts treated with control (dX) or Vpx-containing (X) virus-like particles (VLPs): patient A2953 (C), ALG17_001 (E). Accompanying proliferation inhibition analysis of ara-C and indicated RNRi combination in these samples: patient A2953 (D), ALG17_001 (D). Error bars indicate s.d. of single experiment performed in triplicate.

Paired drug synergy plot for ara-C and RNRi (HU, n = 7; dF-dC, n = 5) in primary patient-derived AML blasts pre-treated with control (dX) or Vpx-containing (X) VLPs. Zero, >0, or <0 corresponds to additive, synergy, or antagonism, respectively, whilst >5 indicates strong synergy and <5 indicates strong antagonism. Each data point indicates an average delta score from a single patient sample subjected to a dose-response matrix experiment performed in triplicate. Statistical testing was performed using two-way ANOVA.