EMBO/soda-nel huggingface dataset:
Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction (2019)
Figure 1

Figure 1: Inhibition of IIS extends the lifespan of OXPHOS deficient nematodes. (A) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction. (B-C) Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants and (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves. (D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red). (E) Schematic representation of IIS pathway including results and predictions from IPA analysis of phosphoproteome of gas-1(fc21) compared to wt. Shades of red and green are proportional to fold change, as calculated by IPA. (F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction. (H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction. (I) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction. (K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.
Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction (2019)
Figure 2

Figure 2: IIS inhibition affects the transcriptome and proteome of mitochondrial deficient nematodes. (A) Left panel: Venn Diagram of the number of genes significantly dysregulated in age-1; gas-1 versus wt (green) and gas-1 versus wt (red). Right panel: Scatter-plots of the expression of genes significantly downregulated (left) or upregulated (right) in gas-1 and age-1; gas-1 mutants compared to wt. In black: expression levels of genes that are significantly up- or down-regulated in gas-1 mutants compared to wt. In red: expression levels of the same genes in age-1; gas-1 mutants compared to wt. (B) IPA analysis indicating the overrepresented canonical pathways in age-1; gas-1 versus gas-1 mutants. (C) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves (D) Volcano plot of TMT/iTRAQ-based quantitative proteomic analysis. In red, proteins significantly dysregulated in age-1; gas-1 compared to gas-1 (FDR< 20%; |log2 (fold change)| >0.47). Dashed lines indicate cutoffs. Proteins involved in carbohydrate (green) and lipid (blue) metabolism are labeled. (E) STRING analysis on dysregulated proteins in age-1; gas-1 vs gas-1 nematodes (20% FDR; |log2(fold change)| >0.47). (F) IPA analysis of TMT/iTRAQ scan results and prediction of downstream effects. Legend indicates color-coding of molecules and connections. Shades of red and green are proportional to fold change, whereas shades of blue and orange are proportional to the strength of the prediction, as calculated by IPA. (G) STRING network for proteins involved in carbohydrate (green) and lipid (blue) metabolism. Legend indicates the nature of each indicated connection. (H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.
Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction (2019)
Figure 3

Figure 3: Increased AMPK/AAK2 and PKA/KIN-1/2 activity occur in the long-lived age-1; gas-1 mutants. (A-B) Schematic representation with overlaid Molecule Activity Predictor (MAP) analysis of (A) AAK-2/AMPK- and (B) KIN-1/PKA-regulated metabolic processes in age-1; gas-1 versus gas-1 mutants. Color intensity corresponds to effect intensity or prediction strength. (C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates. (D) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves. (E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band). (F) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate. (G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves. (H-I) ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes; (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test. (J) Left panel: curves represent FRAP in age-1; gas-1 animals grown on RNAi from larval L1 (or from egg for fzo-1 RNAi) to L4 stage. Each point: mean recovery of bleached regions (2-5 regions per animal, 10-20 animals per condition) ± SEM. Right panel: Maximum (total) recovery per condition are shown, ± SEM. *p value< 0.05, **p value< 0.01, ****p value< 0.0001, Kruskal-Wallis test, Dunn's post-hoc correction. For all lifespan curves: the legend shows average median lifespan ± SEM across all biological replicates.
Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction (2019)
Figure 4

Figure 4: Xanthine accumulates in age-1; gas-1 mutants. (A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5). (B) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7. (C) Schematic representation of purine degradation pathway with overlaid Molecule Activity Predictor (MAP) analysis showing increased purine degradation in age-1; gas-1 mutants and the consequent xanthine accumulation. Shades of red and green are proportional to fold change, whereas shades of blue and orange are proportional to the strength of the prediction, as calculated by IPA. (D) Left panel: qRT-PCR of xdh-1 (normalized to wt). Error bars: Mean ± SEM, n=6. ****p value< 0.0001, *p value< 0.05, ns= non-significant, Kruskal-Wallis with Dunn's post hoc test. Right: xanthine oxidase activity levels normalized to animal number. Error bars: mean ± SEM, n=2, ns= non-significant, Mann Whitney U test. (E) Schematic representations of xanthine and the methylxanthines used in this study. (F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control). (G) Immunoblot analysis of phospho-PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated (actin loading control). For all immunoblot panels: numbers correspond to band densitometries across 2-3 biological replicates.
Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction (2019)
Figure 5

Figure 5. Methylxanthines have lifespan-extending effects in gas-1 mutant nematodes. (A) Effect of methylxanthines on wt lifespan. Average median lifespans ± SEM from all replicates indicated beneath representative curve. (B) Locomotory activity of 10-day-old gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines. Values represent arbitrary units from 3 biological replicates with 4 technical replicates each, 35 worms per technical replicate. Error bars: mean ± SEM, **p value< 0.01, ***p value< 0.001, one-way ANOVA, Dunnett's post-hoc correction. (C) Effect of methylxanthines on the survival of gas-1 mutants. Average median lifespans ± SEM from all replicates indicated beneath representative curves. (D) Pharyngeal pumps/minute in young adult gas-1 mutant nematodes treated with vehicle or methylxanthines. Error bars: mean ± SEM, n=18. ****p value< 0.0001, one-way ANOVA, Dunnett's post-hoc correction. (E-F) Immunoblot analysis of (E) KIN-1 phospho-substrates and (F) phospho-AAK-2 in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control). (G) Left panel: FRAP in vehicle or PTX-treated gas-1 animals. Each point: mean recovery of 14 bleached regions (2 regions per animal, 7 animals per condition) ± SEM. Right panel: maximum (total) recovery per condition ± SEM. **p value< 0.01 unpaired t test. For all immunoblot panels: numbers correspond to band densitometries across biological replicates and for all lifespan curves: the legend shows Mean median lifespan ± SEM across all biological replicates. (H) Schematic drawing showing the critical biological processes underlying the lifespan extension of age-1; gas-1 mutant nematodes. OXPHOS deficient nematodes display decreased lifespan, diminished ATP production, fragmented mitochondrial network and a phosphoproteome signature associated with enhanced IIS signaling. As a result of IIS inhibition, AMPK and PKA drive a catabolic shift underlying lifespan extension of nematodes carrying OXPHOS defects. Supplementation of xanthine derivatives stimulates survival of mitochondrial mutant animals.
A Malectin-Like Receptor Kinase Regulates Cell Death and Pattern-Triggered Immunity in Soybean (2020)
Figure 1

Fig 1. Identification and characterization of the GmLMM1 gene

(A) Phenotype appearance of the Gmlmm1-1 mutant. Typical leaves (left panel; scale bar, 1 cm) and whole plants (right panel; scale bar, 10 cm.) were photographed 60 and 118 days after seeding, respectively.

(B) Enhanced resistance of Gmlmm1-1 and Gmlmm1-2 to Psg infection. Leaves from 2-week-old soybean plants were infected with Psg and the bacteria number was determined at 0 and 4 dpi (Mean ± SD, n = 8, n represents sample number, ***, p < 0.001, Student's t-test).

(C) Physical locations of markers defining the GmLMM1 region. The chromosomal positions (Pd, physical distance in Mb) of each marker are indicated on chromosome 13 (Chr13) according to the soybean reference genome. Recombination ratio (Rr, %) at the corresponding physical location was calculated using a formula (2 × plant number of genotype HD 12 + plant number of heterozygote genotype) / (2 × total number of plants) for rough mapping. Numbers of recombination (Nr) refers to the number of heterozygous plants at a physical location in the 92 plants for gene mapping. The candidate genes were harbored in a 131 kb region spanned by MOL3754 and MOL3780, in which six homologous genes were tandemly repeated and belonged to the malectin-like gene family in the shadows. The direction of the arrow represents the direction of the gene on the chromosome. Genes with high similarity are indicated by arrows of the same color. The number under the genes corresponds to the gene locus on chromosome 13. The Glyma.13G054400 (44) gene harbors a mutation (C to T) according to re-sequencing.

(D) Comparison of Arabidopsis FERONIA, ANXUR1 and GmLMM1. SP and TM indicate the predicted signal peptide and transmembrane domain, respectively. Two corresponding mutated sites in Gmlmm1-1 and Gmlmm1-2 are indicated by arrows.

(E) Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein.

(F) Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site.

Data information: The experiments were performed three times (A, B, E) or two times (F), as biological replicates, with similar results.
A Malectin-Like Receptor Kinase Regulates Cell Death and Pattern-Triggered Immunity in Soybean (2020)
Figure 3

Fig 3. Gmlmm1 mutant plants exhibit enhanced resistance to bacterial and oomycete pathogens

(A) ROS accumulation in Gmlmm1-1 and Gmlmm1-2 mutant plants. Soybean leaves (aged 3 weeks) were subjected to ROS accumulation examination by DAB staining. Scale bar, 1 cm.

(B) ROS accumulation upon P. sojae treatment. The leaves (aged 3 weeks) were inoculated with P. sojae isolate P7076, and DAB staining was performed at 16 hpi. The left panel was photographed under bright field (scale bar, 1 cm). The middle and right panels were photographed under a microscope (scale bar, 50 μm).

(C) Enrichment of defense-related genes in the Gmlmm1-1 mutant. The Gmlmm1-1 and WT plants (aged 2 weeks) were subjected to RNA-seq analysis. The genes that were highly expressed in Gmlmm1-1 compared with the WT (2-fold changes) are presented. The enrichment (%) of the GO terms indicated is presented, and the p-values are calculated by Fisher's tests. Detailed data are shown in the source data.

(D, E) The increased resistance of the mutants to Psp (D) and P. sojae (E) infection. The indicated plants (aged 2 weeks) were infected with the indicated pathogens (Mean ± SD, n ≥ 8, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The lesions are shown by trypan blue staining. Scale bar, 1 cm.

Data information: The experiments were performed three times (A, B, D, E), as biological replicates, with similar results. The exact number (n) and p values are shown in the source data.
A Malectin-Like Receptor Kinase Regulates Cell Death and Pattern-Triggered Immunity in Soybean (2020)
Figure 4

Fig 4. GMLMM1 negatively regulates PTI responses

(A) Flg22-induced ROS burst in Gmlmm1-1, Gmlmm1-2 and Williams 82. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the peak relative luminescence unit (RLU) value was recorded (Mean ± SEM, n ≥ 12, n represents sample number).

(B) Flg22-induced ROS burst in the CRISPR lines C9-24-13 and DN50. The indicated leaves (aged 2 weeks) were subjected to flg22-induced ROS examination, and the relative RLU value was recorded (Mean ± SEM, n ≥ 9, n represents sample number).

(C) Suppression of flg22-induced ROS production in Arabidopsis by GmLMM1. The GmLMM1 gene was introduced into Arabidopsis WT (Col-0) plants by Agrobacterium-mediated transformation. Two independent T2 lines were subjected to flg22-induced ROS examination (Mean ± SD, n ≥8, n represents sample number, ***, p < 0.001, Student's t-test). The protein expression is shown.

(D E) Suppression of flg22-induced (D) and chitin-induced (E) ROS production in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed by Agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination. GmLMM1K564E contains a mutation at the predicted ATP binding site without kinase activity. GFP was used as a control (Mean ± SD, n = 6, n represents sample number, **, p < 0.01, ***, p < 0.001, Student's t-test). The protein expression is shown in the lower panel.

(F) Inhibition of XEG1-induced cell death in N. benthamiana by GmLMM1. The indicated constructs were transiently expressed in N. benthamiana 1 day before infiltration of Agrobacterium containing the XEG1 or GFP vector. The cell death phenotypes were visualized 5 days later. Protein expression of GmLMM1-HA and GmLMM1K564E-HA is shown in the lower panel.

Data information: All the experiments were performed three times (biological replicates) with similar results. The exact n, SEM values and p values are shown in the source data.
A Malectin-Like Receptor Kinase Regulates Cell Death and Pattern-Triggered Immunity in Soybean (2020)
Figure 5

Fig 5. GMLMM1 is involved in the PRR complexes

(A) Identification of GmLMM1-interacting proteins by IP-MS analysis. GmLMM1-FLAG was transiently expressed in Arabidopsis protoplasts, purified by anti-FLAG M2 agarose, and subjected to mass spectrometry analysis. The complete list is shown in the source data.

(B) Interactions of GmLMM1 with PRR receptors. The indicated constructs were transiently expressed in N. benthamiana leaves and subjected to luciferase complementation assay. GmFLS2 (Gm08G083300) is the closest homolog of FLS2 in soybean. GmILPA1 was used as a negative control. The protein interaction intensity is shown by the relative luminescence unit (RLU) (Mean±SD, n ≥ 6, n represents sample number, p<0.05, Student's t-test, different letters indicate signiﬁcant difference). Immunoblots show the levels of protein expression.

(C) Co-IP of GmLMM1 with NbFLS2 and GmFLS2. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days and the protein interactions were examined by Co-IP assay.

(D) GmLMM1 interacts with NbRXEG1 in N. benthamiana plants.

(E) Interaction of GmLMM1 with NbBAK1, GmBAK1a, and GmBAK1b. The experiment and statistical testing were performed as in (B). Immunoblots show the levels of protein expression.

(F) GmLMM1 interacts with NbBAK1, GmBAK1a, and GmBAK1b. The indicated constructs were transiently expressed in N. benthamiana and the interactions were examined by Co-IP assays.

Data information: The experiments were performed three times (B-E) or two times (F), as biological replicates, with similar results.
A Malectin-Like Receptor Kinase Regulates Cell Death and Pattern-Triggered Immunity in Soybean (2020)
Figure 6

Fig 6. GMLMM1 interferes with flg22-induced FLS2-BAK1 association

(A) Effect of flg22 on the NbFLS2-GmLMM1 interaction. The indicated constructs were transiently expressed in N. benthamiana by Agrobacterium-mediated transient expression for 2 days, and the leaves were treated with flg22 (1 μM) or water for 10 min before being subjected to Co-IP assay.

(B) Regulation of NbBAK1-GmLMM1 interaction by flg22.

(C) Suppression of flg22-induced NbFLS2-NbBAK1 interaction by GmLMM1. NbFLS2 and NbBAK1 were co-expressed in N. benthamiana along with GmLMM1 or GmLMM1L407H, treated with H2O or flg22 and subjected to co-IP assays.

(D E) Suppression effect of GmLMM1 on the flg22-induced interaction of GmFLS2 with GmBAK1a (D) and GmBAK1b (E).

(F) A model chart illustrating GmLMM1 mediation of PTI signaling balance in soybean. Flg22 induces the FLS2-BAK1 interaction to trigger downstream immune signaling. GmLMM1 constitutively interacts with BAK1 both before and after immune activation. GmLMM1 can suppress the FLS2-BAK1 interaction to control an appropriate FLS2-BAK1 interaction level. Thus, GmLMM1 works as a molecular switch to regulate moderate immune activation.

Data information: The experiments were performed three times (A-C) or two times (D, E), as biological replicates, with similar results.

Expanded View Figure Legends
CDK12 drives breast tumor initiation and trastuzumab resistance via WNT and IRS1-ErbB-PI3K signaling (2019)
Figure 1

Figure 1. CDK12 amplification is a candidate of druggable target that is associated with poor prognosis in breast cancer.

A The schematic diagram showing the process to determine candidate target genes from chr17q12 amplicons in the METABRIC dataset.

B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS (top right) and OS (bottom right) of breast cancer patients in the METABRIC dataset. Genes located at the HER2 amplicon were nominated according to hazard-ratio levels (all P-values < 0.01). The HER2 co-amplification percentage of the indicated genes is represented as bar graphs (left).

C The frequency of CDK12 amplification in HER2-amplified- and HER2-nonamplified breast cancer. The bar graphs indicate the frequency of HER2 and CDK12 co-amplified cases among the patients with HER2 gene amplification (left). Tables show the percentage of CDK12 amplification in the indicated cohorts (right). Amp, amplification; non-amp, non-amplification.

D Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as the Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). P-values were calculated using one-way ANOVA, followed by post-hoc LSD test.

E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan-Meier method with the log-rank test.

F The results of NPI- and AOL-adjusted analysis showing the association between CDK12 expression and the risk of metastatic relapse (MR) in breast cancer datasets generated using bc-GenExMiner 4.0 (http://bcgenex.centregauducheau.fr).