EMBO/soda-nel huggingface dataset:
A regulatory region on RIPK2 is required for XIAP binding and NOD signaling activity (2020)
Figure 3

Figure 3: Characterization of RIPK2 diGly- and phosphosite mutations.

A Activation of the NF-κB pathway by RIPK2 Lysine- and phosphosite mutants. RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants were stimulated with L18-MDP, harvested at indicated time points and activation of the NF-κB pathway was analyzed by immunoblotting.
B IL-8 production of wild-type THP-1 and RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 4-8 experiments. Shown is average ± SEM. * = P ≤ 0.05; two-way ANOVA.
C) RIPK2 ubiquitination determined by UBA pulldown in RIPK2-deficient cells reconstituted with wild-type or mutant RIPK2 after stimulation with L18-MDP.
D) Detection of K63- and M1-linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type-specific antibodies in RIPK2-deficient cells reconstituted with RIPK2 K209R after stimulation with L18-MDP.
A regulatory region on RIPK2 is required for XIAP binding and NOD signaling activity (2020)
Figure 4

Figure 4: Functional studies of RIPK2 mutations introduced in close proximity to K209R.

A: Structural features of the RIPK2 kinase domain (left) and location of K209 within a hydrophobic pocket between helices αEF and αE (right). Shown is chain B of the RIPK2 kinase in complex with ponatinib from PDB:4C8B. The electrostatic interaction potential is shown as a blue to red gradient.
B Conservation of amino acids creating a hydrophobic pocket between helices αEF and αE. Degree of conservation among 227 vertebrate species indicated by background colour saturation.
C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting. ­
D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.
E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.
A regulatory region on RIPK2 is required for XIAP binding and NOD signaling activity (2020)
Figure 5

Figure 5: RIPK2 K209 and I212 mediate XIAP binding.

A: Thermal stability assay using selected RIPK2 mutants. THP-1 cells expressing RIPK2 were subjected to heat treatment and the non-denatured fraction was analysed by immunoblotting.
B: Binding of RIPK2 to XIAP-BIR2. Lysates from RIPK2-deficient THP-1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP-BIR2 and analysed by immunoblotting.

Expanded View Figures & Legends
Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients (2018)
Figure 2

Figure 2: Detection of SCNAs in CSF is influenced by tumor grade, cfDNA concentration and contact between the tumor and CSF. A) Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light grey. B) Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL).
Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics (2020)
Figure 1

Figure 1: Mito-C, a new chemical compound targeting NEETs proteins, induces mitochondrial network fragmentation

A, Reaction scheme for synthesis of Mito-C and structure of the compound.

B, HeLa cells were treated with DMSO or 2µM Mito-C for 15 min and immunostained with anti-TOM20 (green) antibody and DAPI; cropped areas show the mitochondria morphology changes.

C, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in B) from HeLa cells treated with DMSO or 2µM Mito-C for 15 min or 2h, expressed as ratio of fragmented mitochondria (percentage of total). Errors bars show the standard error of the mean (SEM) (n=100, technical replicates).

D, Profile of [2Fe-2S] cluster release from recombinant NAF-1 (expressed and purified according to the procedure described in Conlan et al()) was determined in untreated control (DMSO) or in presence of Mito-C by monitoring absorbance at 458 nm as a function of time. Errors bars show the standard deviation (SD) of 3 independent experiments.

E, Time lapse video-microscopy snapshot on HeLa cells transfected with NAF-1-mRFP (red) and treated with fluoMito-C (green). The distance/intensity fluorescence quantification graph illustrates the codistribution of fluoMito-C and NAF-1-mRFP.

Data information: all scale bars = 10µm, except magnifications areas (4µm in B and 2µm in E). For evaluating significance of differences observed in C, t-test was used (*** indicates p<0.0001); for differences observed in D one-way Anova followed by Dunn's post-test was used (*** indicates p<0.0001).
Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics (2020)
Figure 2

Figure 2: Knocking down NEETs causes mitochondrial fragmentation

A, Western blot analysis of MitoNEET expression in HeLa cells transfected with CISD1 siRNA; Bar Chart (right panel) shows replicates quantification of MitoNEET expression. Errors bars show the standard deviation (SD) of 3 independent experiments.

B, Western blot analysis of NAF-1 expression in HeLa cells transfected with CISD2 siRNA; graph (right panel) shows the replicates quantification of NAF-1 expression. Errors bars show the standard deviation (SD) of 3 independent experiments.

C, RT-qPCR analysis of CISD3 mRNA levels from HeLa cells transfected with siRNA targeting CISD3. Errors bars show the standard deviation (SD) of 9 independent experiments.

D, HeLa cells transfected with siRNA to reduce expression of MitoNEET (CISD1), NAF-1 (CISD2) or MiNT (CISD3) and immunostained with anti TOM20 antibody. Arrowheads indicate fragmented mitochondria.

E, Quantification of mitochondria morphology (based on TOM20 immunostaining as shown in D) from HeLa cells transfected with siRNA targeting mitoNEET (siCISD1), NAF1 (siCISD2) or MiNT (siCISD3). Quantification is expressed as ratio of tubular and fragmented mitochondria. Errors bars show the standard error of the mean (SEM). (n =125-130 cells).

F, Electron microscopy pictures from 150 INS-1 cells transfected as described in G.

G, Western blot analysis of NAF-1 expression in cells stably transfected with control shRNA, shRNA targeting reduction of NAF-1 protein expression (shCISD2), or shRNA targeting reduction of NAF-1 protein expression complemented with plasmid derived expression of NAF-1 (shCISD2+CISD2); western blot quantification of NAF-1 is shown on the accompanying bar chart. Errors bars show the standard deviation (SD) of 3 independent experiments.

H, Quantification of mitochondrial length in EM images from cells transfected as described in G. Errors bars show the standard error of the mean (SEM) (n=260-270, technical replicates).

Data information: to evaluate significant differences observed in A, B, C, and G a t-test was used (for A ** indicates p<0.001; for B * p is 0.045; C ** p is 0.005; for G p is 0.0001); for differences observed in D and E one-way Anova followed by Bonferroni's post-test was used (*** indicates p<0.0001); in H one-way Anova followed by Dunn's post-test was used (*** indicates p<0.0001). Immunofluorescence scale bars = 10µm, except 4µm in magnification area in D. Scale bars from EM analyses = 0.5 µm.
Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics (2020)
Figure 3

Figure 3: Mito-C causes DRP1 dependent mitochondrial fission and ER-mitochondria contact sites increase

A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20).

B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates).

C, Western blot analysis of DRP1, GAPDH (cytosolic marker) and TOM20 (mitochondrial marker) protein in cytosolic and mitochondrial fractions treated or not, with 2µM Mito-C as indicated.

D, Quantification of western blots showed in C and expressed as a distribution of DRP1 in the cytosolic and mitochondrial fractions. Errors bars show the standard deviation (SD) of 5 independent experiments.

E, HeLa cells were transfected with DRP1K38A mutant and mCherry and treated or not, with Mito-C (T for transfected, NT for not transfected).

F, Quantification of the mitochondrial phenotypes observed in E. Errors bars show the standard error of the mean (SEM). (30-35 images each with an average of 15-20 cells from triplicate independent experiments were analyzed).

G, Electron micrographs (EM) from HeLa cells treated with 2µM Mito-C or DMSO for 120 minutes. Empty arrowheads indicate ER-mitochondria contact sites.

H, Quantification of ER-mitochondria membrane contact sites events (reported in 50µm² section; 50 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM).

I, Quantification of ER-mitochondria membrane contact sites density (in µm; 30 images from an average of 30 cells from triplicate independent experiments were analyzed). Errors bars show the standard error of the mean (SEM).

Data information: to evaluate significant differences observed in D, Mann-Whitney test was used (** p is 0.003); to evaluate statistical differences shown in B,F,H and I a one-way Anova test was used (** indicates p<0.001 *** indicates p<0.0001). All scale bars = 10µm, except magnifications areas (4µm in A and 6 µm in E). Scale bars in G = 0.5µm.
Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics (2020)
Figure 4

Figure 4: Mito-C counteracts dengue virus replication

A, Huh7 cells were treated with Mito-C at the indicated concentrations and simultaneously infected with dengue virus (MOI = 5). Infectious titers are presented as % of DMSO in Mito-C concentration of 0.625, 2.5 and 10µM conditions, upon 48h of viral infection. Errors bars show the standard error of the mean (SEM) (n=3).

B, Huh7 cells were treated with or without (DMSO) Mito-C, infected with dengue virus and fixed after 48h of infection. Cells were then immunostained for endogenous TOM20 (green channel), viral non-structural protein NS5 (red) and DAPI (Blue). Cropped areas illustrate the mitochondrial morphology in described conditions.

C, Quantification of the mitochondrial phenotypes observed in b. Errors bars show the standard error of the mean (SEM). (15-20 images each with an average of 20-25 cells from triplicate independent experiments were analyzed).

Data information: to evaluate significance of differences observed in A, we used one simple t test (theoretical mean of 100%, NS, for non-significant, p=0,0375 ( >0.2), *p=0,0019 (<0.01), ***p= 0,0002 (<0.001)). To evaluate significance of differences observed in C, two-way Anova followed by Bonferroni's post-test was used. (NS, for non-significant, *** indicates p<0.0001). Scale bars = 10µm, except 4µm in magnification area in B.

Expanded view figures legends
Vegan diet in young children remodels metabolism
and challenges the statuses of essential nutrients (2020)
Figure 2

Figure 2.Biomarkers of micronutrient statuses and cholesterol metabolism in omnivores, vegetarians and vegans
A. Serum, plasma or urine levels of vitamin D, vitamin A, folate, zinc, iron and iodine status biomarkers in diet groups. Solid lines indicate cut-offs for deficiency, and dashed lines indicate cut-offs for insufficiency. The RBP limits for vitamin A insufficiency and deficiency are validated for the method at 1.17 µmol/l and 0.83 µmol/l, respectively. Vitamin A status was calculated based on RBP, transthyretin, and CRP.(Talsma et al, 2015).
B. Cholesterol metabolism biomarkers in the diet groups. The subfigure summarizes cholesterol metabolism in humans and highlights cholesterol-related metabolites measured in this study with black font. (Nelson & Cox, 2012; Risley, 2002).

Data information: The summary bar is presented as mean ± SEM. Differences between diet groups were evaluated with age- and sex-adjusted permutation tests with Benjamini-Hochberg correction for multiple testing. Only significances p < 0.05 are displayed. n = 24 omnivores, 10 vegetarians, 6 vegans except for panel A iodine, where n = 13 omnivores, 9 vegetarians and 6 vegans. *p < 0.05, **p < 0.01, ***p < 0.001. Exact p-values are provided in Table EV6. Abbreviations: DMAPP = dimethylallyl pyrophosphate, HDL = high-density lipoprotein, IPP = isopentenyl pyrophosphate, LDL = low-density lipoprotein, OMN = omnivore, RBP = Retinol-binding protein, VGN = vegan, VGTR = vegetarian, VLDL = very low-density lipoprotein.
Phytosulfokine peptide optimizes plant growth and defense via glutamine synthetase GS2 phosphorylation in tomato (2022)
Figure 2

Figure 2. Tomato CPK28 protein interacts with PSKR1.

Y2H assay. The interactions between the GAL4 DNA activation domain (AD) fusion of CPK28VK, and GAL4 DNA binding domain (BD) fusion of PSKR1JK was detected. The transformed yeast cells were grown on a synthetic dropout medium lacking Trp (T), Leu (L), Ade (A), and His (H). CPK28VK, consisting of the variable N-terminal domain and the adjacent protein kinase domain. PSKR1JK, consisting of the inner juxtamembrane domain and the protein kinase domain.

In vitro pull-down assays. GST or GST-PSKR1JK fusion proteins coupled with glutathione beads were used to pull down His-CPK28. The amount of fusion proteins loaded on the gel was indicated by Coomassie brilliant blue (CBB) staining. Asterisks indicate the specific bands of fusion proteins.

BiFC assay. CPK28-nYFP, PSKR1-cYFP and FLS2-mCherry (plasma membrane localization marker) were co-expressed in N. benthamiana leaves. The YFP and mCherry signals were visualized using confocal microscopy. Scale bar, 50 μm.

Co-IP assays. PSKR1-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated using GFP-Trap. Both immunoprecipitated and input samples were subjected to western blotting using an anti-HA or anti-GFP antibody.
Phytosulfokine peptide optimizes plant growth and defense via glutamine synthetase GS2 phosphorylation in tomato (2022)
Figure 5

Figure 5. CPK28 interacts with GS2 to enhance tomato plant growth but repress defense against Pst DC3000.

A. Co-IP assay showing that PSK promotes CPK28-GS2 interaction. GS2-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA and anti-GFP antibody.

B. Co-IP assay showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. GS2-HA and CPK28-GFP were co-expressed with or without PSKR1-FLAG in N. benthamiana leaves. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA, anti-GFP or anti-FLAG antibody.

C. In vitro pull-down assays showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. Fixed amounts of His-CPK28 and GST-GS2 fusion proteins were incubated with an increasing amount of MBP-PSKR1 fusion protein and MBP protein. Asterisks indicate the specific bands of fusion proteins.

D. GS2 gene silencing efficiency in virus-induced gene silencing (VIGS) tomato leaves. Leaves were sampled 3 weeks after A. tumefaciens infiltration. Eight efficient silencing plants were selected and used for further experiments.

E. GS enzyme activity in tomato leaves four weeks after the VIGS experiment.

F. Identification of GS2 over-expression lines by western blotting using an anti-HA antibody. Ponceau S staining was used as the loading control.

G. GS enzyme activity in GS2 over-expression lines. Tomato leaves were collected from four-week-old OE-GS2 plants.

H-K. GS2 gene positively regulates growth. The representative plant image (H, J) and dry weight data (I, K) of four-week-old TRV:0, TRV:GS2, WT and OE-GS2 plants are shown. Scale bar, 5 cm.

L-O. Pst DC3000 infection assay. Trypan blue staining for cell death at 2 dpi (L, N). Bacterial population in tomato leaves at 2 dpi (M, O). Scale bar, 100 μm.

Data information: In (D, E, G, I, K, M and O), data are represented as means ± s.d., n = 8 (D, I and K), n = 5 in (E, G), and n = 6 (M, O). Different letters depict statistically significant differences (P < 0.05, Tukey's test). n refers to biological repeats. Experiments in (A-C) were repeated twice, and experiments in (D-O) were repeated three times with similar results.
Phytosulfokine peptide optimizes plant growth and defense via glutamine synthetase GS2 phosphorylation in tomato (2022)
Figure 6

Figure 6. CPK28 phosphorylates GS2 at S334 and S360, each is associated with tomato plant defense regulation and growth response, respectively.

A. PSK induces the phosphorylation of GS2 by CPK28 in vivo. GS2-GFP was co-expressed with CPK28-HA (empty vector as a negative control) in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The phosphorylated GS2 protein was precipitated by GFP-Trap, and then immunoblotted with anti-pSer/pThr and anti-pSer antibodies.

B. Schematic diagram showing the phosphorylation sites of GS2 identified by MS. Gray and yellow bar indicate the GS2 transit peptide and catalytic domain, respectively.

C. Site-directed mutation of S334 and S360 residues in GS2 inhibited its phosphorylation by CPK28.

D. Identification of OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background by western blotting using an anti-GLN1/GLN2 antibody. Ponceau S staining was used as the loading control.

E. GS enzyme activity in the OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background. Leaves were collected from four-week-old plants.

F, G. Growth phenotype of OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background with or without PSK treatment. Representative plant image (H) and root length data (I) of complementation lines grown on medium for 4 days are shown. Scale bar, 2 cm.

H-J. Defense phenotype of OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background with or without PSK treatment. Representative leaf image for disease symptoms at 5 days post inoculation (dpi)(J). Trypan blue staining for cell death at 2 dpi (K). Bacterial population in tomato leaves at 2 dpi (L). Scale bar, 1 cm (J), 100 μm (K).

Data information: In (E, G and J), data are represented as means ± s.d. in, n = 5 in (E), n = 20 in (G), and n = 6 in (J). n refers to biological repeats. Different letters indicate statistically significant differences (P < 0.05, Tukey's test). Experiments in (E-J) were repeated three times with similar results.
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy (2022)
Figure 1

Figure 1. Autophagy activation is dependent on gene transcription.

A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.

C-E Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.

F WT cells were grown in YPD to log phase and then cultured in SD-N media for 4 h. Collected yeast cells were subject to genome mRNA sequencing and quantification.

G Genome-wide analysis of differential gene expression in yeast cells cultured in SD-N compared to cells in rich medium (p value <0.05, blue: downregulated, red: upregulated). Significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test (n = 3 biological replicates).

H Yeast cells were grown in YPD to log phase and then shifted to SD-N for 4 h. The total RNA was extracted to detect mRNA levels of autophagy genes by qRT-PCR (quantitative real-time polymerase chain reaction). Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (n = 5 biological replicates).