EMBO/soda-nel huggingface dataset:
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 5

Figure 5. Endothelial NICD induction rescues HSC emergence from the VDA

A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) evi1 morphant embryos (top, Movie EV 2) or in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) crossed to Tg(5xUAS-E1b:6xMYC-notch1a) embryos prior to MO injection (bottom, Movie EV 3) from 28 to 42 hpf. Shown are three representative time-points, in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible. For each time-point merged images are shown. NICD induction controlled by endothelial fli.1 (lower panel) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA (arrows).

B. Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction. At least n=5 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d. (n.s., not significant, p<0.05 (*)).
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 6

Figure 6. evi1 regulates Notch independently of the Vegf pathway and can compensate for Vegf inhibition

A. WISH of dll4 (upper) and notch1b (lower) in control injected embryos (left), evi1 morphants (middle) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos at 32 hpf after heat-shock induction at 20 hpf (right).

B, C. WISH of runx1/c-myb (B) or notch1b (C) after treatment with DMSO (left), the VEGF receptor inhibitor SU5461 (middle) and respectively SU5461 after injection of UAS:mEvi1 (inducing endothelial evi1 expression) in Tg(fli.1:Gal4FF;UAS:RFP) embryos (right).

Two or more biological replicates are shown for each experiment. Lateral views are shown, anterior to the left, dorsal is up. Numbers indicate the amount of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Arrows indicate up- or down-regulation of the respective gene in each condition. For each analyzed gene, quantitation of results is shown, displaying the percentages of embryos with normal vs. decreased gene expression for each condition. A Fisher´s exact test was applied to calculate statistical significance (p<0.05 (*), p<0.01 (**), p<0.001 (***)).
Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence (2016)
Figure 7

Figure 7. evi1 regulates Notch signaling via pAKT.

A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.

B. Expression of notch1b after treatment with DMSO vehicle control (left) or Wortmannin (WM, middle). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.

C. WISH for runx1/c-myb in control injected embryos (upper left), evi1 morphants (upper right) or respectively evi1 MO with myr-AKT injected embryos with HS at 14 hpf (lower right) and evi1 MO with UAS-myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos.

D. Quantitation of results from (C). Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.

E. WISH for notch1b in evi1 Morpholino injected (left) and evi1 plus UAS:myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.

F. Schematic illustration of the role of Evi1 during HSPC emergence and the dual regulation of Notch by the shh-vegf and the evi1-pAKT axis.

Two or more biological replicates are summarized for each experiment. Lateral views are shown, anterior to the left, dorsal is up. Numbers indicate the amount of embryos with the respective phenotype/total number of embryos analyzed in each experiment. Arrows indicate up- or down-regulation of each analyzed gene. A Fisher´s exact test was applied to calculate statistical significance (n.s., not significant, p<0.05 (*), p<0.01 (**), p<0.001 (***)).
SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (2021)
Figure 1

Figure 1.SARS-CoV-2 activates delayed innate immune responses in lung epithelial cells (A-H) Measurements of replication and innate immune induction in Calu-3 lung epithelial cells infected with SARS-CoV-2 at MOIs 0.08, 0.4 and 2 TCID50VERO/cell. (A) Replication of SARS-CoV-2 genomic and subgenomic E RNAs (qRT-PCR). (B) Quantification of N staining from cells in (A) by flow cytometry. Mean percentage of N-positive of all live-gated cells is shown +/- SEM, n=2. (C) Representative example of immunofluorescence staining of N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue), cell mask (red). Scale bar represents 50µM. (D) Quantification of N staining in cells in (C) by immunofluorescence . (E) Infectious virus released from cells in (A) determined by TCID50 on Vero.E6 cells. (F-H) Fold induction of (F) interferons (IFNβ, IFNλ1 and IFNλ3) (G) IFN stimulated genes (CXCL10 and IFIT2) or (H) pro-inflammatory mediators (IL-6 and CCL5) each overlaid with SARS-CoV-2 E (qRT-PCR). All data from cells in (A) at MOI 0.4 TCID50VERO/cell. (A -H) Means from replicate wells shown +/- SEM n=2, full growth curve is representative of three independent experiments. (I-K) SARS-CoV-2 infection (MOIs 0.04 (closed symbols) and 0.0004 (open symbols) TCID50VERO/cell) in Calu-3 cells with addition of 10ng/ml IFNβ before or after infection at time points shown, measured by (I) E RNA copies (J) N positive cells, (K) released virus (TCID50VERO/cell) all measured at 24 hpi. Dotted line indicates untreated. Mean +/- SEM, n=3, One-way ANOVA Light and dark blue * indicates significance for high and low MOIs respectively.
SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (2021)
Figure 2

Figure 2. Peak SARS-CoV-2 replication precedes innate immune activation. (A-I) (A,C) Representative images of NF-κB p65 (A) (red) and IRF3 (C) (red) nuclear localisation in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. SARS-CoV-2 N protein (green. (E and G) Representative images of IL-6 mRNA (E) detected by FISH (red) and N protein (green) , or IFIT1 mRNA (G) (green) with N protein (red), both with nuclei (DAPI, blue) in mock or SARS-CoV-2 infected (MOI 0.4 TCID50VERO/cell) Calu-3 cells at 24 hpi. (B, D, F, H, I) Single cell analysis time course quantifying the Integrated Nuclear Intensity of NF-κB p65 (B), IRF3 (D) , or overall integrated intensity for IL-6 (F) or IFIT1 (H) mRNA over time in N protein positive cells and N protein negative cells (I). n=2. Kruskal-Wallis test with Dunn's multiple comparison. * (p<0.05), **** (p<0.0001). Scale bar represents 50µM. (J,K) Secretion of CXCL10 (J) and IL-6 (K) by infected Calu-3 cells (MOIs 0.08, 0.4 and 2 TCID50VERO/cell), (ELISA). (L) Lactate dehydrogenase (LDH) release into culture supernatants by mock and SARS-CoV-2 infected Calu-3 cells (MOIs 0.08, 0.4 and 2 TCIDVERO50/cell) quantified absorbance (492nm). (M) Quantification of live/dead staining of non-adherent cells recovered from supernatants of mock or SARS-CoV-2 infected Calu-3 cultures at 48 and 72hpi. Data information: (J-M) Means from replicate wells shown +/- SEM, n=2, representative of three independent experiments.
SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (2021)
Figure 3

Figure 3. SARS-CoV-2 is sensed by MDA5 and RIG-I. (A-D) Measurement of (A) viral genomic and subgenomic E RNA at 24 hpi, (B) fold induction of CXCL10 from (A), (C) IFIT2 and (D) IL-6 mRNA (qRT-PCR) from (A) after Remdesivir treatment (0.125-5 μM) of SARS-CoV-2 infected Calu-3 cells (MOI 0.04 TCID50/cell) with Remdesivir added 2h prior to infection. Mean +/- SEM, n=3. (E-H) Measurement of (E) viral genomic and subgenomic E RNA (F) fold induction of CXCL10, (G) IFIT2 and (H) and IL-6 at 24 hpi, of Calu-3 cells with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) with Remdesivir treatment (5μM) prior to, at the time of, or 8 h post-infection. Mean +/- SEM, n=3, One way ANOVA with Dunnett's multiple comparisons test to compare to untreated infected condition ('mock'), ** (p<0.01), *** (p<0.001), **** (p<0.0001). (I) Representative example of immunofluorescence staining of dsRNA (red) and N protein (green) after SARS-CoV-2 infection of Calu-3 at MOI 0.4 TCID50VERO/cell, at time points shown. Nuclei (DAPI, blue). Scale bar represents 50µM. (J) RNAi mediated depletion of MAVS, RIG-I or MDA-5, reduced their expression levels as compared to siControl (siCtrl) Mean +/- SEM, n=3. Two-Way ANOVA with Sidak's multiple comparisons test, **** (p<0.0001). (K-O) Fold induction of (K) IFNβ, (L) CXCL10, (M) IFIT2 (N) TNF and (O) IL-6 or in SARS-CoV-2 infected Calu-3 cells (MOI 0.04 TCID50/cell) 24 hpi. Mean +/- SEM, n=3, and compared to siCtrl for each gene by One Way ANOVA with Dunnett's multiple comparisons test, ** (p<0.01), *** (p<0.001), **** (p<0.0001), n.s. : non significant. (P) Live/dead stain counts for non-adherent cells, recovered at 48 hpi from supernatants of SARS-CoV-2 infected Calu-3 cells, depleted for MAVS or RNA sensors, compared to siCtrl. Non-adherent cell counts were determined by acquisition by flowcytometry for a defined period of time. Mean +/-SEM, n=3. Total numbers are compared to siCtrl by unpaired t-test, *** (p<0.001). (Q-R) (Q) Viral E RNA and (R) released infectious virus (TCID50VERO/cell) at 24 hpi of infected Calu-3 cells depleted for MAVs, RIG-I or MDA5. Mean +/- SEM, n=3. Each group compared to siCtrl by One Way ANOVA with Dunnett's multiple comparisons test, *, p>0.05, ** (p<0.01), n.s : non significant.
SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (2021)
Figure 4

Figure 4. NF-κB and JAK/STAT signalling drive innate immune responses. (A-C) Fold induction at 24 hpi of (A) CXCL10, (B) IFIT1 or (C) IL-6 mRNA (qRT-PCR) after Calu-3 infection with SARS-CoV-2 over a range of MOIs (0.004, 0.04, 0.4 TCID50VERO/cell) in the presence of 10 μM TPCA-1 or DMSO as shown. (D-F) Fold induction at 24 hpi of (D) CXCL10, (E) IFIT2 or (F) IL-6 mRNA (RT-qPCR) after Calu-3 infection with SARS-CoV-2 over a range of MOIs (0.0004, 0.004, 0.04, 0.4 TCID50VERO/cell) in the presence of 2 μM Ruxolitinib (Rux) or DMSO as shown. (G-H) Viral genomic and subgenomic E RNA at 24 hpi (RT-qPCR) with DMSO or TPCA (G) or Rux (H) treatment. Data information: (A-H) Mean +/- SEM, n=3, statistical comparisons are made by unpaired t test comparing inhibitor-treated to mock-treated SARS-CoV-2 infected conditions at each MOI and each timepoint. * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). (I) Live/dead stain count from non-adherent cells recovered from supernatants of SARS-CoV-2 infected Calu-3 cultures (MOI 0.04 TCID50VERO/cell) 48hpi (flow cytometry). Mean +/- SEM, (n=3). One Way ANOVA comparison of inhibitor-treated infected cells to mock-treated infected cells. *** (p<0.001).
SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (2021)
Figure 5

Figure 5. Epithelial responses to SARS-CoV-2 drive macrophage activation (A) Schematic of experimental design. (B-J) Calu-3 cells were transfected with siRNA targeting MAVS or non-targeting control (siCtrl) (B-D) or treated with DMSO vehicle or inhibitors 10 μM TPCA-1 (E-G) or 2 μM Ruxolitinib (Rux) (H-J) as shown, and were mock-infected or infected with SARS-CoV-2 at MOI 0.04 TCID50VERO/cell. Virus containing conditioned media (CoM) was harvested at 48 hpi. MDM were treated with Calu-3 virus containing CoM for 6 hpi, before washing and measuring MDM gene expression (B, E, H), and MDM activation markers by flowcytometry 48 h later (C,D,F,G,I,J) , plotting relative median fluorescent intensity (MFI) compared to mock-infected siCtrl (C, D) or mock-infected DMSO control (F, G, I, J). Legends in (B), (E) and (H) apply to (C,D), (F,G) and (I,J) respectively. The inhibitors in (E) and (H) were tested side-by-side with the same mock condition. Mean +/- SEM shown, data from 4-6 independent MDM donors is shown. Statistical comparison by two-tailed paired t-test comparing MDM exposed to control infected CoM to siMAVS/inhibitor treated infected CoM. * (p<0.05), ** (p<0.01), *** (p<0.001). (K-O) MDM were treated with either anti-IFNAR antibody (2.5ug/ml), an isotype control IgG antibody (IgG, 2.5ug/ml), Rux (2 μM), or mock treated during 6 h of exposure to CoM from infected, unmodified Calu-3 cells, before washing and measuring MDM gene expression (K, L, M), and MDM activation markers (N, O) by flowcytometry 48 h later. Both gene expression and relative MFI are compared to mock-treated MDM exposed to CoM from uninfected Calu-3s cells. Mean +/- SEM shown, data from 7-8 independent MDM donors is shown. Statistical testing by one-way paired ANOVA, comparing treated MDMs to untreated control by Dunnett's multiple comparison test, * (p<0.05), ** (p<0.01), *** (p<0.001).
SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (2021)
Figure 6

Figure 6. Pre-existing immune activation exacerbates SARS-CoV-2-dependent inflammation. (A) Schematic of experimental design. (B-H) MDM were primed with 100ng/ml LPS for 2h before exposure to SARS-CoV-2 (MOI 0.02 TCID50VERO/cell). (B) Expression of genomic and subgenomic viral E RNA at 48 h post exposure in indicated conditions. (C-G) Host gene expression of (C) CCL5, (D) ISG56, (E) IFIT2, (F) IL-6 or (G) IL-1b was measured 48hpi. (H) IL-1b secretion was detected in culture supernatants at 48 hpi by ELISA. (I) Schematic of experimental design. MDM were exposed to SARS-CoV-2 (MOI 0.02 TCID50VERO/cell) for 48 h and subsequently stimulated with 100ng/ml LPS for 24 h. (J-P) (J) Expression of genomic and subgenomic viral E RNA 72 h post-exposure in indicated conditions. (K-O) Host gene expression of (K) CCL5, (L) ISG56, (M) IFIT2, (N) IL-6 and (O) IL-1b. (P) IL-1b secretion was detected in culture supernatants at 48 hpi by ELISA. Data Information: (A-P) Gene expression is shown as fold induction over untreated controls. Data from 8-13 independent donors is shown. Groups were compared as indicated by Wilcoxon matched-pairs signed rank test, *, p<0.05, ** (p<0.01), *** (p<0.001). (Q-V) Calu-3 cells were infected with SARS-CoV-2 (MOI 0.04 TCID50VERO/cell) in the presence or absence of 10ng/ml IL-1b. (Q-T) Gene expression of (Q) IFNβ (R), CXCL10, (S) IL-6 and (T) IFIT1 was measured after 24h. Fold induction over untreated mock infection is shown, n=3. (U) Non-adherent cells were collected at 48h post infection and live/dead stained. Cells were acquired by flowcytometry and cell counts determined by time-gating. For statistical comparison, total cell numbers were compared. (V) Viral release into culture supernatants at 24 h was measured by TCID50 on VeroE6 cells. (Q-V) Mock and SARS-CoV-2 infected conditions were compared with or without IL1b-treatment, respectively, by unpaired T test (n=3). *, p<0.05; n.s., non-significant. Mean +/- SEM shown.
SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense (2016)
Figure 1

Figure 1. Sirt5 protects cells from oxidative stress.

(A) Sirt5 deficiency leads to higher ROS levels in MEFs. ROS levels were determined in the indicated MEFs as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).

(B) Sirt5 deficiency suppresses GSH production in MEFs. The ratio of [GSH/GSSG] was determined in cell extracts as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).

(C) Sirt5 deficiency inhibits NADPH production in MEFs. The ratio of [NADPH/NADP+] was determined in cell extracts as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).

(D) Sirt5 deficiency decreases cell growth. MEF cells were seeded in 6-well plates at a density of 50,000 cells/well, and cell growth rate was carefully monitored every 1-2 days by cell counting for 8 days. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).

(E) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat. The levels of cleaved PARP and Caspase-3 were determined by western blot analysis.

(F) Sirt5 protects MEFs from ROS-induced cell death. Wild-type and Sirt5 KO MEFs were treated with Paraquat (500 μM) for 24 hrs, and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).

(G-I) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection. Female Sirt5 KO and WT littermates (n=3~5 per group) were intraperitoneally injected with saline or paraquat (10 mg/kg) once a week for 3 consecutive weeks. The ratios of [NADPH/NADP+] and [GSH/GSSG] and the level of lipid peroxide were determined in tissue extracts. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).

(J-K) Sirt5 KO mice are more sensitive to Paraquat-induced nigrostriatal dopaminergic degeneration. Female Sirt5 KO and WT littermates were treated with paraquat as in (G-I). The brain sections were stained to detect tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNc region, as described in ‘Materials and Methods’. Representative immunohistochemistry images (original magnification, 100×; scale bar, 100 μm) and the corresponding quantifications are shown in (J) and (K), respectively. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).
SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense (2016)
Figure 2

Figure 2. SIRT5 preserves cellular antioxidant capacity partially through activating NADPH-producing G6PD and IDH2

(A)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).

(B) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).

(C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).

(D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis.

(E) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).

(F) Cartoon representation of major NADPH-producing enzymes in the cell. As shown, G6PD, 6PGD, ME1, IDH1 and MTHFD1 are located in cytosol, while IDH2 and MTHFD2 are located in mitochondria.

(G) The activities of G6PD/6PGD and IDH1/2 are reduced in stable HEK293T cells with SIRT5 knockdown. Activities of the indicated endogenous proteins were determined in cell extracts as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01, ***p<0.001, n.s.: not significant (two-tailed unpaired t-test).

(H) G6PD and IDH2 are activated by SIRT5 in vitro. Upon pre-incubation with SIRT5 in vitro, the activities of the indicated ectopically expressed proteins were determined as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments **p<0.01, n.s.: not significant (two-tailed unpaired t-test).
SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense (2016)
Figure 3

Figure 3. SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to activate these two enzymes and maintain NADPH homeostasis

(A) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).

(B) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in ‘Materials and Methods’. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).

(C) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation, respectively, and decreases both enzyme activity. Flag-G6PD or Flag-IDH2 was ectopically expressed in the indicated stable HEK293T cells. G6PD/IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).

(D) SIRT5 decreases G6PD glutarylation and IDH2 succinylation, and increases both enzyme activity. Flag-G6PD or Flag-IDH2 was co-overexpressed with HA-SIRT5 in HEK293T cells. G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01 (two-tailed unpaired t-test).

(E) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket (PDB ID: 1LWD) [33], made by Pymol (www.pymol.org). The protein is colored in light green and isocitrate in rainbow, with Lys 360 and Lys 413 residues in IDH2 being colored in red.

(F-G) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5. The indicated Flag-tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co-overexpression. Wild-type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide, followed by measurement of IDH2 enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, n.s.: not significant (two-tailed unpaired t-test).