EMBO/soda-nel huggingface dataset:
SIRT2-dependent IDH1 deacetylation inhibits colorectal cancer and liver metastases (2020)
Figure 1

Figure 1. SIRT2 deacetylates IDH1 on K224

(A) IDH1 acetylation levels upon treatment with NAM or TSA. Flag-tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period.

(B) Five putative lysine residues were mutated. Acetylation levels of Flag-bead-purified IDH1 were determined by Western blot analysis using a pan-anti-acetyllysine antibody. Relative IDH1 acetylation ratios were calculated after normalizing against Flag.

(C) Mutation of IDH1 K224R and K224Q resulted in altered acetylation levels using Western Blot.

(D) IDH1 catalytic activity of IDH1 K224 mutants in vitro. Wild-type IDH1 and K224R, K224Q mutants were expressed in HEK293 cells. Proteins were purified by immunoprecipitation (IP), IDH1 levels were normalized for protein, and activity assays were performed.

(E-F) Steady-state kinetic analysis of IDH1 and variants in HEK293T cells. Comparison of IDH1 WT and K224R and K224Q showed that the K224 site was important in catalysis.

(G) IDH1 directly interacted with SIRT2 but not SIRT1. SIRT2 deacetylated IDH1 effectively. Flag tagged IDH1 and HA tagged SIRT1 or SIRT2 were co-transfected into cells. The acetylation level of Flag-bead-purified IDH1 and the protein association between IDH1 and SIRT1 or SIRT2 were determined by Western blot.

(H) Co-immunoprecipitation showed endogenous proteins of IDH1 and SIRT2 interacted with each other.

(I) SIRT2 interacted with IDH1 in vitro. GST pull-down assay with GST-fused SIRT2 and Flag-tagged IDH1 proteins.

(J) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot analysis.

(K) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity. SIRT2 H187Y was a catalytically inactive mutant.

Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For multiple comparisons in (D), a one-way ANOVA with Newman-Keuls post hoc test was used to test for statistical significance. For (E-F), statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.
SIRT2-dependent IDH1 deacetylation inhibits colorectal cancer and liver metastases (2020)
Figure 2

Figure 2. SIRT2 is a tumor suppressor in CRC

(A) Colony formation ability after transfection of SIRT2 plasmids, AGK2 and SIRT2 siRNA.

(B) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2. Stained cells in the lower chambers were quantified.

(C) Cell migration ability was assessed by wound healing assay in HCT116 cells.

(D) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids, SIRT2 siRNA or AGK2.

(E) Immunohistochemical analysis of SIRT2 protein in representative colorectal tissue specimens is shown (magnification ×100). Weak cytoplasmic staining for SIRT2 was observed in CRC tissues and metastatic tissues, whereas strong cytoplasmic staining was observed in noncancerous tissues.

Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A, B, D, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. For figure C, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.
SIRT2-dependent IDH1 deacetylation inhibits colorectal cancer and liver metastases (2020)
Figure 3

Figure 3. IDH1 acetylation senses metabolite concentration and regulate cellular redox hemostasis

(A-B) IDH1 K224 acetylation level was enhanced with increasing glucose concentration. Flag-IDH1 was overexpressed in cells treated with increased concentrations of glucose for 6 h. IDH1 proteins were purified by Flag beads, and then IDH1 K224 acetylation level was determined by Western blot and IDH1 catalytic activity was assessed.

(C-D) IDH1 K224 acetylation level was upregulated and IDH1 activity was weakened with increasing glutamine concentration. Flag-IDH1 was overexpressed in cells treated with increased glutamine for 6 h. IDH1 proteins were purified by Flag beads, and the K224 acetylation level of IDH1 was determined by Western blot analysis and then IDH1 catalytic activity was evaluated.

(E-F) Downregulation of SIRT2 diminished the effect of glucose or glutamine on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glucose (E) and glutamine (F).

(G) IDH1 K224 deacetylation regulated cellular NADPH/NADP+ redox in cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of NADPH/NADP+ in cells was measured as followed by manufacture's instruction.

(H) IDH1 K224 deacetylation promoted GSH production in HCT116 cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of GSH/GSSG was assessed as followed by manufacture's instruction.

(I) IDH1 K224 deacetylation suppressed cellular ROS levels in HCT116 cells. ROS was determined in cells under non-stressed condition or exposed to menadione. *P<0.05 and **P<0.01

Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B, D, G, H, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. For figure I, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.
SIRT2-dependent IDH1 deacetylation inhibits colorectal cancer and liver metastases (2020)
Figure 4

Figure 4. IDH1 acetylation control HIF1α-SRC axis in CRC progression

(A) Gene expression profile array analysis in HCT116 cells. Representative genes with higher fold difference (up or down) compared IDH1 K224R cells with IDH1 WT cells.

(B) ChIP assays for HIF1α and its binding motif. Antibodies anti-IgG and anti- HIF1α were used in the ChIP assays. qRT-PCR was performed to quantify the binding activity.

(C-D) The transcriptional activity of the SRC promoter was greatly increased after co-expression of HIF1α and SRC reporter in wild type. SRC promoter constructs containing a potential HIF1α binding motif (-1636~-1629 bp and -1466~-1459 bp).

(E) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy-HIF1α. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, HIF1α and hydroxy-HIF1α was measured by Western blot assay.

(F) HCT116 cells of IDH1 K224Q were cotreated with Octyl-α-KG and NAC, the expression of HIF1α and SRC was further evaluated.

(G-H) HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose and lactate levels.

Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B, D, statistical significance was assessed with a two-tailed unpaired Student's t-test. For G, H, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.
SIRT2-dependent IDH1 deacetylation inhibits colorectal cancer and liver metastases (2020)
Figure 5

Figure 5. IDH1 K224 deacetylation suppresses CRC functions in vitro

(A) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after transfection of IDH1 K224R or knockout of IDH1. Quantification was performed by counting the stained cells that invaded to the lower chamber under a light microscopy.

(B) Three-dimension imaging analysis was to assessed velocity of cell movement.

(C) Cell migration ability was evaluated by wound healing assay in HCT116 cells. Quantification was performed by measuring the smallest clearance distance of the wound.

(D) Colony formation ability after deacetylation of IDH1 K224 or knockout of IDH1.

(E) Cell cycle distribution was assessed flow cytometrically after treatment with IDH1 K224R or knockout of IDH1.

Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A, B, D, E, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. For figure C, statistical significance was assessed with a two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.
Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress (2021)
Figure 1

Figure 1 - Tfcp2l1 shields 2i-ESCs from spontaneous cell death but is dispensable for renewal and pluripotency.

A. Z-score shows transcription factors enriched in 2i-ESCs and FBS-ESCs.

B. Immunoblotting shows protein levels of Tfcp2l1, Nanog, and H3K27me3 in 2i-ESCs and FBS-ESCs.

C, D. Real-time PCR (C) and immunoblotting (D) showing Tfcp2l1 deletion by CRISPR-Cas9. n=5 clones; error bars indicate standard error of the mean (SEM); p values, t-test; five clones were pooled for immunoblotting.

E. Alkaline phosphatase staining. The scale bar equals 100 µm. Left, representative stained colonies; right, a bar chart showing the percentage of differentiated and undifferentiated colonies of WT and Tfcp2l1_KO 2i-ESCs.

F. Hematoxylin and eosin staining of teratoma formed by WT and Tfcp2l1_KO 2i-ESCs in NSG mice. Scale bars=100 µm.

G, H. Propidium iodide staining to measure spontaneous cell death of WT and Tfcp2l1_KO 2i-ESCs (G) and ESC-FBS (H). A representative histogram is shown on the left and the bar chart on the right is the percentage of spontaneous dead cells. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using t-test.

I, J. Cumulative cell numbers of WT and Tfcp2l1_KO 2i-ESCs (I) and FBS-ESCs (J). Results are from three independent clones of WT and Tfcp2l1_KO ESCs.
Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress (2021)
Figure 3

Figure 3 - Tfcp2l1 deletion impairs fatty acid oxidation in 2i-ESCs.

A. Real-time fatty acid oxidation (FAO) profile of WT and Tfcp2l1_KO 2i-ESCs using the FAO stress test kit in Seahorse XF. Oxygen consumption rate (OCR) were measured. Error bars indicate SEM; n = 5 biological repeats. Palm-BSA, palmitate conjugated BSA, a substrate of FAO; Oligomycin, an inhibitor of ATP synthase (complex V); FCCP, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, a proton gradient uncoupler; Rotenone and Antimycin, a complex I inhibitor and a complex III inhibitor, respectively.

B. Basal respiration, maximal respiration, and spare respiration capacity were calculated from A. n=5 biological repeats; error bars indicate SEM; p values were calculated using student t-test.

C, D. Mito Stress Assay (C) and Glycolysis Stress Assay (D) in Seahorse XF. Error bars indicate SEM; n=3 biological repeats; no time point shows a statistically significant difference between WT and KO 2i-ESCs.

E. Real-time OCR profile using FAO substrate (Palm-BSA) or BSA control followed by FAO inhibitor, etomoxir. n=5 biological repeats; error bars indicate SEM.

F. Quantification of OCR changes after etomoxir addition. n=5 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.

G. Mitochondrial membrane potential (left) and amount (right) were measured by TMRM and Mitotracker Green, respectively. Representative histograms were shown. The numbers shown are mean fluorescence intensity; p-values were calculated using two-sample t-test.
Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress (2021)
Figure 4

Figure 4 - Tfcp2l1 regulates FAO and spontaneous cell death of 2i-ESCs through Cpt1a.

A. Genomic views of Cpt1a mRNA and genome binding of Tfcp2l1 in WT and Tfcp2l1_KO 2i-ESCs.

B. Real-time PCR showing Cpt1a mRNA levels in naïve WT and Tfcp2l1_KO ESCs. n=6 biological repeats; error bars indicate SEM; p-value was calculated using two-sample t-test.

C. Immunoblotting of Cpt1a and β-actin.

D. Luciferase reporter assay showing that Tfcp2l1 directly regulates Cpt1a transcription. Reporter and Tfcp2l1 expression vector were transfected into Tfcp2l1_KO 2i-ESCs. n=3 biological repeats; error bars indicate SEM; p-value was calculated using two-sample t-test.

E. Immunoblotting of Cpt1a in WT, Tfcp2l1_KO, and Tfcp2l1_KO 2i-ESCs with Cpt1a overexpression (OE).

F. Real-time FAO profile using the FAO Stress Test in Seahorse XF. n = 4 biological repeats; error bars indicate SEM; **, p-value <0.01; NS, not significant; t-test.

G. Percentage of dead cells measured by live-cell propidium iodide staining detecting spontaneous cell death in 2i-ESCs. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.

H. Cumulative cell number of WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using two-sample t-test.

I. MitoTracker Green staining. p-value was calculated using two-sample t-test. The numbers for each genotype are mean fluorescence intensity.
Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress (2021)
Figure 5

Figure 5 - Tfcp2l1 deletion and FAO defect are associated with the upregulation of glycolysis, the TCA cycle, and glutamine catabolism.

A. Clustering of a total of 572 metabolites in WT and Tfcp2l1_KO 2i-ESCs. Data were from 9 biological repeats for each genotype.

B. A schematic diagram of glycolysis and the TCA cycle. Most metabolites within these two pathways were detected by metabolomics and had significant changes between naïve WT and Tfcp2l1_KO ESCs. Metabolites in red, significantly upregulated in KO versus WT in 2i-ESCs; metabolites in green, significantly down-regulated; metabolites in black, not significantly changed.

C. Relative levels of metabolites in glycolysis and the TCA cycle.n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.

D. Live-cell propidium iodide staining to measure cell death in WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs after 5 mM 2-DG treatment for 24 hours. n=3 biological repeats; error bars indicate SEM; p-value were calculated using two-sample t-test.

E. A schematic diagram of glutamine metabolism. Most metabolites within this pathway were detected by metabolomics and had significant changes between WT and Tfcp2l1_KO 2i-ESCs. Metabolites in red, significantly upregulated in KO versus WT in 2i-ESCs; metabolites in green, significantly down-regulated; metabolites in black, not significantly changed.

F. Relative levels of metabolites in glutamine metabolism. n=9 biological repeats; upper whiskers, maximum-the third quartile; lower whiskers, the first quartile-minimum; boxes, the third quartile-the first quartile; the central line indicates the median; p-values were calculated using two-sample t-test.

G. Percentage of cell death of WT, Tfcp2l1_KO, and Tfcp2l1_KO with Cpt1a overexpression (OE) 2i-ESCs under glutamine deprivation. Error bars indicate SEM; n=3 biological repeats; p-values were calculated using two-tail t-test.
Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress (2021)
Figure 6

Figure 6 - The Tfcp2l1-Cpt1-FAO axis promotes the survival of 2i-ESCs in response to mTOR inhibition.

A. Pharmacological (INK-128) and physiological (glutamine deprivation) inhibition of the mTOR signaling.

B. Immunoblotting of phosphorylated and total S6K (a substrate of mTOR) showing the inhibitory effect of INK-128 (200 nM) and glutamine deprivation on mTOR activity.

C. Percentage of cell death of WT, Tfcp2l1_KO, and Tfcp2l1_KO 2i-ESCs with Cpt1a overexpression (OE) treated with 200 nM INK-128 for 24 hours. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.

D. 0.5 million of 2i-ESCs with indicated genotypes were seeded and treated with 200 nM INK-128 on day0. Cell numbers were counted on day2, 4, and 6. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.

E. Immunoblotting of WT and Tfcp2l1_KO 2i-ESCs untreated or treated with 200 nM INK-128.

F. Real-time PCR showing Cpt1a mRNA levels in WT and Tfcp2l1_KO 2i-ESCs in the presence and absence of 200 nM INK-128. n=3 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.

G, H. Percentage of cell death in 2i-ESCs treated with 50 µM Eto in the presence or absence of 200 nM INK-128 or 100 µM of hexanoate (G) or octanoate (H) as indicated for 24 hours. n=4 biological repeats; error bars indicate SEM; p-values were calculated using two-sample t-test.
Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis (2020)
Figure 1

FIGURE 1. Identification of the putative Cyt-bd inhibitor ND-011992.

(A) Molecular structure of ND-011992.

(B) Effect of ND-011992 on the intracellular ATP level in M. bovis BCG. The bacteria were treated with DMSO (blue triangle), 100 nM Q203 alone (green triangle), 20 μM ND-011992 alone (brown square), and a dose-range of ND-011992 in the presence of Q203 (red squares) for 15 hours before quantification of intracellular ATP levels.

(C) Oxygen consumption assay in M. bovis BCG using methylene blue as an oxygen sensor.

(D) Oxygen consumption assay in M. bovis BCG using the Seahorse XFe96 Extracellular flux analyser. 12 μM ND-011992 was injected alone (green triangles), 100 nM Q203 (blue circles), or in combination with Q203 (red squares). For each condition, OCR readings were normalised to the last basal OCR reading before drug injection. OCR: Oxygen Consumption Rate. The dotted lines indicate the timepoints of drug injection.

(E) Dose-dependent inhibition of M. bovis BCG OCR by ND-011992 (in the presence of 100 nM Q203) measured on a Seahorse XFe96 analyser platform. For each condition, OCR readings were normalised to the last basal OCR reading before drug injection.

(F) Effect of ND-011992 on the OCR of M. smegmatis IMVs using the Oroboros O2k fluorespirometer. IMVs OCR from the parental strain (green triangles), ∆cydAB knockout (blue circles), and ∆cydAB complemented with M. tuberculosis CydABDC+ (red squares) energized with NADH were determined. Q203 was used at 1 μM. 100% OCR was defined as the OCR of the untreated samples for each strain.

Data information: Data are expressed as the mean ± S.D. for each condition of a representative experiment. Experiments in (B) and (E) were performed with 2 technical replicates and independently repeated at least twice. In (D) and (F), each experiment was performed with 3 technical replicates and independently repeated at least once.
Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis (2020)
Figure 2

FIGURE 2. ND-011992 inhibits the mycobacterial Cyt-bd.

(A) Differential gene expression analysis of H37Rv treated with Q203, ND-011992, or combination (H37Rv-Combo), and ∆cydAB treated with Q203 compared to untreated controls. The log2-fold differences in gene expression of various conditions relative to the untreated control is indicated using a sliding scale where higher expression is reflected in red and lower expression is reflected in blue, with a midpoint signifying no difference in white.

(B) Effect of cydABDC over-expression on the Oxygen Consumption Rate (OCR) and intracellular ATP levels. in M. bovis BCG treated with ND-011992 in the presence of 100 nM Q203. IC50: Inhibitory Concentration 50%.

(C) Absence of the Cyt-bcc:aa3 sensitized H37Rv to ND-011992. M. tuberculosis H37Rv wildtype (Blue circles), ∆ctaE-qcrCAB (red squares) and complemented strain (green triangles) were treated with ND-011992. Growth inhibitory potency (MIC50) was recorded after 2 to 3 weeks of incubation.

(D) The reduced minus oxidized difference spectrum of M. smegmatis ΔqcrCAB inverted membrane vesicles (IMVs). The green line represents the spectrum without treatment, while the blue line represents the spectrum of the IMVs in the presence of 10 μM ND-011992.

Data information: Data are expressed as the mean ± S.D. of triplicates for each condition of a representative experiment. Each experiment was performed with 3 technical replicates and independently repeated at least once.
Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis (2020)
Figure 3

FIGURE 3. ND-011992 has a low spontaneous-resistance mutation frequency and is active against drug resistant M. tuberculosis clinical isolates.

(A) Combined potency of ND-011992 + Q203 against the drug-resistant clinical isolate M. tuberculosis 123-20-0047.

(B) Potency of the combination ND-011992 + Q203 against various clinical isolates in an agar plate MIC assay. A dose-range of ND-011992 was tested against the clinical isolates in the presence of Q203. Q203 was used at 100 nM against the strains H37Rv, N1274, N1283, 123-20-0015, 123-20-0091, 123-20-0041, 123-20-0047, and at 20 nM against the stains N0072, N0157, N0145, N0052, and N0136. "S" indicates pan-susceptibility to anti-TB drugs; "MDR": multi-drug resistant; "XDR": extensively drug-resistant.

(C) Growth inhibition assay in M. bovis BCG. 106 bacteria were plated onto 7H10 agar supplemented with DMSO, 25 nM Q203, 3 µM ND-011992, or 25 nM Q203 + 3 µM ND-011992.

(D) Fluctuation analysis in M. tuberculosis H37Rv. M. tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 (red). Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND-011992 or 2 µg/mL rifampicin (blue). The corresponding values of the median frequency of resistance is indicated in the graph.
Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza (2019)
Figure 1

Figure 1. The stability of the 1918 PB1-F2 protein is very low and it is degraded by the Ub-dependent proteasome pathway.

A A549 and U937 cells were infected with each IAV at MOI 1 or 5 for 24 h, and lysates were analyzed by Western blotting using polyclonal anti-PB1-F2 antibody.

B A549 cells were transfected with a plasmid encoding Flag-tagged PB1-F2 derived from the PR8 or 1918 strain and treated with the proteasome inhibitor MG132 (25 μM) or vehicle for 6 h before harvesting. PB1-F2 transcripts were analyzed by semi-quantitative RT-PCR (top panel), and The PB1-F2 protein was analyzed by Western blotting with monoclonal anti-Flag antibody.

C Representative immunofluorescence images showing expression of PB1-F2 (red). A549 cells were transfected with plasmids encoding indicated PB1-F2. At 18 h after transfection, cells were treated with MG132 for 6 h and subjected to immunofluorescence analysis. Magnification, ×100; scale bar, 100 μm.

D Each HA-tagged PB1-F2 was transfected into 293T cells with or without Flag-ubiquitin. At 18 h after transfection, cells were treated with MG132 for 6 h. The lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting with anti-Flag antibody. PR8, A/Puerto Rico/8/34 (H1N1); 1918, A/Brevig Mission/1/1918 (H1N1).

Results shown are representative of three independent experiments.