EMBO/soda-nel huggingface dataset:
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 4

Figure 4. p38γ and δ deficiency in myeloid cells improves glucose metabolism in an obesity model.

Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a high-fat diet (HFD) for 10 weeks. (A) Body weight measured at the indicated times during HFD treatment. (B) Fat mass and lean mass determined by MRI at the end of the diet period. (C and D) Liver mass and white fat (WF) mass. (E) Respiratory exchange quotient, energy expenditure, and locomotor activity, detected in metabolic cages. Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 5

Figure 5. p38γ/δLyzs-KO mice are protected against steatohepatitis induced by HFD.

Lyzs-Cre and p38γ/δLyzs-KO mice were fed a ND or a HFD for 10 weeks. (A) Representative H&E and oil red stained liver sections (Scale Bar: 50μm) and (B) liver triglycerides (n=5-10). (C) Plasma ALT at the end of the diet period. (D) Glucose tolerance measured at the end of the diet period. Blood glucose concentration was measured in mice given an intraperitoneal glucose injection (1g/kg) after overnight fasting (n=5-10). (E) Basal blood glucose in overnight-fasted ND and HFD-fed Lyzs-Cre and p38γ/δLyzs-KO mice (n=5-10). Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 refers to p38γ/δLyzs-KO versus Lyzs-Cre; ##P<0.01; ###P<0.001 refers to ND versus HFD (2-way ANOVA coupled to Bonferroni’s post tests or Newman-Keuls post test for liver triglycerides).
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 6

Figure 6. p38γ/δLyzs-KO hematopoietic cells protect mice against HFD-induced steatosis.

Lethally irradiated WT mice were reconstituted with BM from Lyzs-Cre (Cx BM Lyzs-Cre) or p38γ/δLyzs-KO mice (Cx BM p38γ/δLyzs-KO). Two months after the transplant, mice were fed the HFD for 10 weeks. (A) Freshly prepared CD45.2 whole BM mononuclear cells (2 × 107) were transplanted into lethally irradiated B6.SJL (CD45.1) mice, and engraftment by CD45.2 cells (%) was analyzed by antibody staining and FACS of peripheral blood and liver CD45+ cells. Charts show CD45.1 and CD45.2 expression in blood cells (left) and liver cells (right) isolated from transplanted mice (n = 3). Representative FACS dot plots of CD45.1 and CD45.2 expression are shown beneath the charts. (B) Representative H&E and oil red-stained liver sections. Scale bar: 50 μm. (C) Liver triglyceride content. (D) Plasma transaminase ALT activity. (E) Fasted glucose, detected at the end of the diet period in overnight-fasted mice. Data are means ± SEM. (n=5-10) *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 7

Figure 7. p38γ/δLyzs-KO neutrophils have deficient migration to the liver.

(A) Flow cytometry analysis of liver myeloid subsets (CD11b+ Gr-1high, CD11b+ Gr-1intermediate, CD11b+ Gr-1-) isolated from Lyzs-Cre and p38γ/δLyzs-KO mice fed a MCD for 3 weeks or HFD for 10 weeks. Representative dot plots are shown, and bar charts show the diet-induced increase in each population as a percentage of the total intra-hepatic CD11b+ leukocyte population. Myeloid infiltrating cells isolated from livers were sorted by FACS and stain with H&E. Representative cells were shown next to the appropriate myeloid subsets. (B and C) Neutrophils and monocytes as a percentage of total circulating leukocytes, measured in total blood in animals fed the MCD diet for 3 weeks (B) or the HFD for 10 weeks (C). (D and E) WT mice fed the MCD diet were i.v. injected with a 1:1 mix of DiO-labeled Lyzs-Cre neutrophil and DiD-labeled p38γ/δLyzs-KO neutrophils (6x106 cells in total; n=10). One hour after injection, liver-infiltrating neutrophils were assessed by flow cytometry (D) and fluorescence micrography on liver sections (E). (F) Intravital microscopy quantification of the rolling and adhesion frequencies and rolling velocities of neutrophils recruited to venules irrigating inflamed (TNFα-injected) cremaster muscle. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 8

Figure 8. Neutrophil depletion protects against steatosis.

Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre and p38γ/δLyzs-KO mice. These animals were fed a ND or MCD for 3 weeks. (A) Neutrophils and monocytes as a percentage of circulating leukocytes, measured in total blood. (B) Representative H&E and oil red-stained liver sections after 3 weeks of treatment. Scale Bar: 50μm. (C) Liver triglyceride and (D) plasma transaminase activity (ALT) at the end of the diet period. (E) Total RNA was extracted from livers, and chemokine and cytokine mRNA levels were determined by qRT-PCR. mRNA expression was normalized to the amount of Gapdh mRNA. Data are means ± SEM. (n=5-10). *P<0.05; **P<0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).
p38γ and p38δ reprogram liver metabolism by modulating neutrophil infiltration (2015)
Figure 9

Figure 9. Neutrophils control liver metabolic changes in steatosis development.

(A, B and C) Plasma levels of 3-hydroxybutyrate in WT and p38γ/δ-/- (A), Lyzs-Cre and p38γ/δLyzs-KO mice (B) and Lyzs-Cre Ly6G Ab treated after MCD diet (C). (D) Overlap of gene expression changes between Lyzs-Cre mice treated with anti-Ly6G and p38γ/δLyzs-KO at the end of the MCD diet period (n=8). (E) Hierarchical clustering of the expression profiles of genes differentially expressed both, between p38γ/δLyzs-KO and MCD-diet–fed Lyzs-Cre and between Lyzs-Cre treated with anti-Ly6G and MCD-diet–fed Lyzs-Cre mice. Genes up-regulated in both comparisons (red cluster) were mainly regulated by PPARα, SREBF1 and SREBF2, while the enriched upstream regulators of genes down-regulated in both conditions versus the control group (blue cluster) were IL1β, TNFα and PDGFB β. (F) IPA (http://ingenuity.com) functional categories enriched in the set of genes differentially between p38γ/δLyzs-KO and MCD-diet–fed Lyzs-Cre and between Lyzs-Cre treated with anti-Ly6G and MCD-diet–fed Lyzs-Cre mice (n=4). (G) Genes differentially expressed between p38γ/δLyzs-KO and MCD-diet–fed Lyzs-Cre and between Lyzs-Cre treated with anti-Ly6G and MCD-diet–fed Lyzs-Cre mice and involved in liver steatosis according with IPA. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P<0.001 (2-way ANOVA coupled to Bonferroni’s post tests).
Structural basis of Focal Adhesion Kinase activation on lipid membranes. (2020)
Figure 1

Figure 1. Cryo-EM structure of the FERM-kinase region of FAK on a PI(4,5)P2 membrane.

A Domain structure of FAK. Important phosphorylation sites are indicated and the FAK region used in the cryo-EM study is boxed with a dashed line.

B Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5)P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5x zoom of the main image.

C Local resolution map of the refined Apo and AMP-PNP particle, as calculated with Blocres (Heymann & Belnap, 2007).

D 14 FAK molecules, each containing a FERM and kinase domain are fitted into the EM maps obtained from the AMP-PNP dataset. Each molecule is in a different color. Middle: Membrane distal view; right: Membrane proximal view; left: zoomed view of central symmetric dimer from the membrane distal view with locations of FERM (F) and kinase (K) domains indicated. The equivalent figure for the Apo structure is shown in Fig. EV1d.

E Linear oligomeric assembly of FAK formed by three symmetric FAK dimers in the AMP-PNP particle shown from the membrane distal view. FERM (F) and Kinase (K) domains are labelled. FERM and kinase of the same molecule are in the same color with the kinases shaded in a lighter tone.

F Model of the FAK oligomeric assembly placed on a lipid bilayer. The membrane is placed by considering the KAKTLRK basic sequence in the FERM F2 lobe as main membrane binding site (Cai et al., 2008; Goni et al., 2014).
Structural basis of Focal Adhesion Kinase activation on lipid membranes. (2020)
Figure 4

Figure 4. In vitro phosphorylation and catalytic activity of interaction mutants.

A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles (PC; phosphatidylcholine) or PI(4,5)P2 containing vesicles. Plotted are mean values from two independent autophosphorylation reactions, each determined in duplicates (n=4). Mutant nomenclature is as shown in Table 2.

B Catalytic turnover activity as measured in a coupled kinase assay is shown. Plotted are mean values from at least three independent experiments.

C To determine whether autophosphorylation occurs in cis or trans, autophosphorylation was measured for WT or a kinase dead form of FAK (KD, contains K454R mutation) in presence or absence of a substrate deficient FAK mutant (Y397F) and/or PI(4,5)P2 vesicles. PI(4,5)P2 negative experiments contain control PC vesicles instead. Note that the scales of the two plots are different, i.e. autophosphorylation is increased in presence of PI(4,5)P2. Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). Left: Schematic of possible cis- and trans-autophosphorylation (red arrows) in mixtures of FAK-WT/FAK-Y397F and FAK-KD/FAK-Y397F. See also Fig. EV4A-B for experiments at different protein concentrations and time points.

D Src phosphorylation of Y577 in FAK in presence of PC or PI(4,5)P2 vesicles as determined by an ELISA method. The plot on the left is obtained in presence Src containing only the kinase domain (Src254-536) and the plot on the right with Src containing additionally the SH3 and SH2 domains (Src84-536). Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4).

Data information: A-D Error bars: s.d.
Structural basis of Focal Adhesion Kinase activation on lipid membranes. (2020)
Figure 5

Figure 5. PI(4,5)P2 binding.

Interactions between WT or interface mutant FAK with a PI(4,5)P2 containing membrane was probed using a pulldown assay with membrane coated silica beads. Plotted are bound protein relative to the binding for WT at the highest PI(4,5)P2 concentration. Mutant naming is as in Table 2. For WT the non-specific binding to a PC membrane is shown for comparison. Plotted are mean values from at least three independent experiments. Error bars: s.d.
Structural basis of Focal Adhesion Kinase activation on lipid membranes. (2020)
Figure 6

Figure 6. FAK signaling in SCC cells.

A FAK phosphorylation of WT or interface mutant FAK in SCC cells. Whole cell lysates were subjected to western blot analysis with anti-pFAK Y397, anti-pFAK Y576/577, anti-pFAK Y861, anti-pFAK Y925 and anti-FAK. Anti-GAPDH served as a loading control. The graph shows densitometric analysis of relative pFAK/FAK ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.

B Downstream signaling in SCC cells expressing WT or interface mutant FAK. Whole cell lysates are analyzed by western blot analysis with the indicated antibodies. The graph shows densitometric analysis of relative phospho/total protein ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.

C Src phosphorylation and interactions between FAK and Src or pSrc Y416 in SCC cells . FAK, Src or pSrc Y416 were immunoprecipitated from whole cell SCC lysates using anti-pSrc Y416, anti-FAK, anti-Src or non-specific IgG-agarose, followed by western blot analysis with anti-FAK, anti-pSrc Y416 and anti-Src. Anti-GAPDH served as a loading control. For full blots see source data.
Structural basis of Focal Adhesion Kinase activation on lipid membranes. (2020)
Figure 7

Figure 7. FAK and Src localization in SCC cells expressing interface mutant FAK.

A,B FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-FAK (A), or anti-pSrc Y416 (B), anti-Paxillin and DAPI (A-B). Representative immunofluorescence images are shown. Scale bars, 20 μm. The graph (B) shows the quantification of internalized active Src from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.d. * = p < 0.01.

C Subcellular fractionation of SCC cells expressing FAK-WT or interface mutant FAK. Whole cell lysates (WCL) and subcellular fractions were subjected to western blot analysis with anti-FAK, anti-Tubulin (marker for cytoplasmic fraction; C), anti-RCAS (marker for perinuclear fraction; PN) and anti-H4 (marker for nuclear fraction; N). The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.
Structural basis of Focal Adhesion Kinase activation on lipid membranes. (2020)
Figure 8

Figure 8. FAK mutant SCC cells in 3D proliferation and invasion.

A FAK-WT, interface mutant FAK or FAK -/- SCC cells were resuspended in methylcellulose solution in growth medium on a layer of agarose and observed for 3D sphere formation. Images were taken from 10 random fields after nine days (representative images are shown) and the relative colony size (top graph) as well as the number of colonies (bottom graph) were assessed from three independent experiments. Scale bars, 200 μm. Student's t-test was carried out to calculate the statistical significance. Error bars s.d. * = p < 0.01, # = p < 0.05.

B FAK-WT, interface mutant FAK or FAK -/- SCC cells were seeded on growth factor-reduced Matrigel in serum-free media. After 72 h, invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel. Representative images of the invasion assay are shown. Scale bars, 200 μm. The graph shows relative invasion from five independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.e.m. * = p < 0.01, # = p < 0.05.
MDM2 regulates RB levels during genotoxic stress (2020)
Figure 1

Figure 1. MDM2 enhance RB tumour suppressor expression.

A) H1299 cell line was transfected with increasing concentrations of MDM2, and the RB+5´UTR exogenous (left panel) and endogenous (right panel) levels were evaluated.

B) Evaluation of the RB expression as in (A) but inducing DNA damage with doxorubicin 1 µM during 16 h.

C) Evaluation of the RB levels in an MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line.

D) Evaluation of the RB levels in a MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line.

E) Evaluation of the endogenous RB expression using H1299 cell line, under normal conditions, transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2(S395D).

F) Evaluation of the endogenous RB as in (E) but using C33 cell line.

Data information: The bar diagrams show the quantification of the western blots data. Data are means ± s.d. of at least three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01, ***p< 0.001).
MDM2 regulates RB levels during genotoxic stress (2020)
Figure 2

Figure 2. The positive effect of MDM2 on RB affects the translation of the protein.

A) Evaluation of RB mRNA levels, normalised by GAPDH, in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Endogenous levels (upper panel); exogenous levels (middle panel); endogenous levels of RB but using the MDM2(S395D) under normal conditions (lower panel).

B) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h. The cells were treated with cichlohexamide (CHX) was added to stop translation, under normal conditions (upper panel) and treated with doxorubicin (lower panel). The cells were transfected with MDM2.

C) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence (upper panel) or in absence (lower panel) of CHX.

D) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX.

Data information: The bar diagrams show the quantification of the western blots data. Data are means ± s.d. of at least three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).
MDM2 regulates RB levels during genotoxic stress (2020)
Figure 3

Figure 3. MDM2 recognises the 5´ UTR of RB mRNA.

Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data.

A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner.

B) Cells were transfected with the RB large pocket construct.

C) Cells were transfected with RB full length without the 5´URT (named RB-HA).

D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR).

E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO.

F) RT-qPCR of RB and GAPDH of the MDM2-immunoprecipitation from the polysomal fractionation (see EV2D). Data are representative of three similar independent experiments.

G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm.

H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.

Data information: Data are means ± s.d. of at least three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).
MDM2 regulates RB levels during genotoxic stress (2020)
Figure 4

Figure 4. MDM2 induces the G1 cell cycle phase under DNA damage conditions. C33 and H1299 cells were stable transfected with MDM2 (see EV3).

A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase.

B) The cell cycle analysis of synchronised C33 cell line during the time.

C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector).

D) The same as in (C) but under normal cellular conditions.

E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.

F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).
MDM2 regulates RB levels during genotoxic stress (2020)
Figure 5

Figure 5. Physiological consequences of the MDM2-RB mRNA interaction.

A) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2/M cell cycle phases under stress conditions.

B) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2(S395D).

C) In vitro RB ubiquitination by MDM2(S395D) in presence or absence of RB mRNA.

D) A representative clonogenic assay. Increasing concentrations of MDM2 were transfected in H1299-RBKO stable transfected with RB-HA or RB+5´UTR. The cells were treated with doxorubicin to induce genotoxic stress conditions. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05).

E) Evaluation of cyclin E mRNA levels in an MDM2 dose-dependent manner. The

H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, ***p< 0.001).

F) Working model of the MDM2 mechanism of RB regulation. Under genotoxic stress conditions MDM2 induce the G1 cell cycle phase via RB tumour suppressor. In the G1 cell cycle phase MDM2 increases the synthesis of RB, allowing the RB-E2F1 binding, promoting in this way the G1 cell cycle arrest. During G2/M, MDM2 ubiquitinate and degrades RB promoting the cell cycle progression.

Expanded View Figure Legends
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 1

Figure 1. Wnt signaling regulates the stability of FoxM1 and GSK3 kinase regulates the degradation of FoxM1 protein.

A. LN229 and U87 cells treated with Wnt 3a (0, 10, 20, 50, 100 ng/ml) for 4 hr. The indicated proteins were analyzed by Western blotting.

B. LN229 cells treated with or without Wnt3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS-PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments.

C. Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 (100 ng/ml) for indicated times. Noted that the basal levels of FoxM1 and β-catenin in (A) and (C) are similar but look different due to variations on the exposure time of the Western blots.

D. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 µg/ml) for indicated times.

E. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 µg/ml) for indicated times, and FoxM1 and GSK3αβ expression levels were determined by Western blotting.

F. Flag-FoxM1 and GSK3βKA or GSK3βKD plasmids were co-transfected into 293T cells for 48 hr. The cells were then treated with CHX (100 µg/ml) for indicated times. Exogenous Flag-FoxM1 expression level was determined by Western blotting.

All Western blots in (A), (C)-(F) are representative of three independent experiments.