EMBO/soda-nel huggingface dataset:
Macrophagic AMPKa1 orchestrates regenerative inflammation induced by glucocorticoids (2022)
Figure 4

Figure 4. An AMPK-GR-FOXO3 trinity regulates glucocorticoid induction of restorative macrophages.

(A) Transcriptional regulators of differentially regulated identified by oPOSSUM were stratified into known interactors with AMPK. (B) Heat map from RNA-seq data performed in WT and FOXO3-/- macrophages treated or not with Dex clustered to show genes responsive in WT. (C) Box plot of log2 fold change of genes up-regulated in response to Dexamethasone (Dex) of genes in FOXO3-/- macrophages, stratified by AMPK dependency (clusters C4 and C2 of Fig.1E). (D-E) FOXO3 (D) and GR (E) enrichment at GR binding sites for 4 AMPK dependent GC regulated genes. (F) WT and AMPKα1-/- Bone Marrow Derived Macrophages (BMDMs) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated. Westen-blot for FOXO3, AMPKα1, actin and the phosphorylated form of FOXO3 were done. Quantification of the band intensity is provided normalized to those of actin and total FOXO3, a representative example is shown. (G) FOXO3-/- BMDMs were treated or not with Dex and cultured with CSFE-fluorescently labelled apoptotic thymocytes for 2.5 h. The number of macrophages with ingested apoptotic cells was evaluated by flow cytometry. Results are expressed as percentages of positive cells. (H) WT and FOXO3-/- BMDMs were treated with LPS for 24 h prior addition of Dex for 6 h; immunofluorescence for pro- (iNOS, CCL3) and anti- (CD163, CD206) inflammatory markers was performed. (I) WT and AMPKα1-/- BMDMs were treated with either Lipopolysaccharide (LPS) or muscle Damaged Associated Molecular Patterns (DAMPs) for 24 h and further treated with the AKT inhibitor MK2206 for 1 h prior addition of Dex for 6 h; before immunofluorescence for pro- (iNOS) and anti- (CD206) inflammatory markers was performed. Results are expressed as percentages of positive cells. Data information: results are z-score of 4 (B), or box and whiskers in which the central band represents the median (D,E,F,G,I) or means +/- SEM (I). N= 4-6 (D,E), 4-5 (F,G), 3 (H), 4 (I) biological replicates. * p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001 using Mann-Whitney-Wilcoxon test (C) or ANOVA and Kruskal-Wallis tests (D-I).

Expanded View Figure Legends
The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner (2022)
Figure 1

Figure 1. MASTL modulates the AKT-mTORC1 signaling pathway.

A. Schematic representation of the protocol followed for depletion of MASTL in human cell lines and different nutrient starvations. MASTL was knocked down (RNA interference; shMASTL) or knocked out (inducible CRISPR/Cas9; isgMASTL) and cells were nutrient starved for 1-6h before analysis (time 0'). In case of re-stimulation with glucose or insulin, cells were analyzed 10-60 min after re-stimulation.

B. MASTL was ablated in MDA-MB-231 cells using the isgMASTL system. 72 h after Dox, cells were starved of glucose in the media in presence of 10% dFBS for 1 h (-) and then re-stimulated with 25 mM of glucose for 10 min before recovery (+). Whole-cell lysates were blotted with the indicated antibodies. * in indicates unspecific band. β-Actin was used as a loading control.

C. MASTL was depleted in MDA-MB-231 cells using shRNAs against MASTL (+) or scrambled shRNAs (−) as control. Cells were starved from glucose for 2 h and re-stimulated with 5 mM glucose for 15 min and 1 h. Total extracts were recovered and tested for the indicated antibodies. β-Actin was used as a loading control.

D. Quantification of phospho-AKT T308 levels in glucose deprivation or glucose stimulation (15 min) conditions in MDA-MB-231 control cells and MASTL-depleted cells either using shRNA or the inducible sgRNA. Bars displays mean data + SEM from three independent experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).

E. Immunoblot analysis with the indicated antibodies in BT-549 cells upon glucose deprivation and 15 min glucose stimulation. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. β-Actin was used as a loading control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant).

F. Immunoblot analysis with the indicated antibodies in MCF-7 cells upon glucose deprivation and glucose stimulation for 15 min. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).
The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner (2022)
Figure 2

Figure 2. MASTL controls AKT phosphorylation in response to insulin signaling.

A. Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum-starved control and MASTL knockout MDA-MB-231 cells using the isgMASTL system. The plot shows the quantification of phospho-AKT T308 after 30 min of insulin stimulation. Plots displays mean data + SEM from 5 independent experiments. *P<0.05, Student's t-test.

B. TSC2 was knocked down in control or MASTL-null MDA-MB-231 cells using specific (+) or Scrambled (−) shRNAs. Cells were starved for glucose for 1 h. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.

C. MDA-MB-231 control (−) or MASTL null (+) cells were treated with 100 nM rapamycin or vehicle as control. Cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min; rapamycin was added 15 min before glucose stimulation. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.
The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner (2022)
Figure 3

Figure 3. Metabolic alterations in MASTL-depleted cells.

A. Immunofluorescence for GLUT4 (magenta) in MDA-MB-231 cells in conditions of serum starvation and insulin stimulation. DAPI (DNA) is in cyan and a cell mask is in yellow. Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane (PM). Representative magnifications are also shown. Scale bars, 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM. Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA. Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies.

B. Cells were treated with specific shRNAs against MASTL (+) or scrambled shRNAs (−), and then serum-starved before incubation with 2-deoxy-D-[1-3H]-Glucose. Bars show mean glucose uptake + SEM of 3 independent experiments. *P<0.05, Student's t-test.

C. Control (shScr.) and MASTL-depleted (shMASTL) HepG2 cells were incubated with 10 mM glucose and metabolites in the media were quantified by NMR at the indicated time points. Time course of glucose consumption (upper panel) and lactate production (lower panel) in cell media are indicated. Data are mean ± SD of 5 independent plates. *P<0.05; **P<0.01, ***P<0.001, unpaired Student's t-test.

D. Glycolytic index at 24 h comparing control and MASTL-depleted cells treated as in C calculated as -0.5*([Lac]/([Glc]0-[Glc]). Data are mean ± SD of 5 independent plates. ns, not significant, unpaired Student's t-test.
The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner (2022)
Figure 5

Figure 5. MASTL mediates mTORC1/S6K1-mediated AKT inhibition through the ENSA/ARPP19-PP2A/B55 pathway.

A. Co-depletion of ENSA and ARPP19 proteins in the MDA-MB-231 cell line using specific siRNAs or control siRNAs against luciferase (Luc). 72 h after transfection cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min. Total cell extracts were collected and analyzed for the indicated antibodies.

B. isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG- and HA-tagged versions of ENSA and ARPP19 phospho-mimetic mutants (S67D and S62D) or GFP as a control. Cells were treated with Dox to induce MASTL CRISPR/Cas9-dependent knock-out, and 72 h later cells were treated as specified in A. The plot displays mean + SEM from 3 independent experiments. Significance determined by Student's t-test comparing ENSA/ARPP19 overexpression versus control cells (ns, not significant; *P<0.05).

C. Control (Dox −) and MASTL knockout (Dox +) cells were treated with 5 µM fostriecin, 50 nM okadaic acid (OA) or vehicle (Veh) 10 min before insulin stimulation for 30 min. Total lysates were analyzed for the indicated antibodies and P-T308-AKT signal was quantified. β-Actin was used as a loading control. The plot represents mean + SEM from 3 independent experiments, except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown. *P<0.05; Student's t-test.
The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner (2022)
Figure 6

Figure 6. The MASTL-PP2A/B55 pathway controls the feedback loop at the level of adaptor proteins.

A. MDA-MB-231 cells were co-transduced with specific siRNAs against the PPP2R2A (B55α) and PPP2R2D (B55δ) transcripts (B55αδ) or against luciferase (Luc) as a control. Asynchronous cells were collected and blotted for the indicated antibodies. The histograms represent the quantifications of the specified phospho-residues. Plots show mean + SEM from 3 independent experiments. *P<0.05, Student's t-test.

B. Immunodetection of the indicated antigens in control (−) and MASTL knockout (+) cells starved from glucose and re-stimulated with glucose for 15 min.

C. siRNA-mediated genetic depletion of B55alpha and delta subunits (B55αδ) in control (Dox −) and MASTL-null (Dox +) cells. The phosphorylation status of the different proteins was scored in conditions of glucose re-addition. n=3 independent experiments. The plot shows mean + SEM from 3 independent experiments. *P<0.05, 1-way ANOVA.

D. Immunoblot with the indicated antibodies in skeletal muscle (gastrocnemius) extracts. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1, and phospho-S6K1 T389 (normalized to total S6K1 level; n=6 Mastl(+/+) and 7 Mastl(∆/∆)). β-Actin or vinculin were used as a loading control. Data are mean + SEM; *P<0.05, unpaired Student's t-test.
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 1

Secretagogin locus in the paraventricular nucleus of the hypothalamus

A–A3 Secretagogin+ neurons populate the paraventricular, dorsolateral, ventromedial, periventricular and arcuate nuclei of the mouse hypothalamus (Paxinos & Franklin, ). Red circles denote the localization of neuronal perikarya and their relative densities. AHP, anterior hypothalamus; Arc, arcuate nucleus; DM, dorsomedial nucleus; ME, median eminence; Pe, periventricular nucleus; PVN, paraventricular nucleus, PVNm, magnocellular part; SON, supraoptic nucleus; VM, ventromedial nucleus.
B–B2 Largely non‐overlapping distribution of AVP+, oxytocin+ and secretagogin+ (sgcn+) neurons in the magnocellular PVN. The mouse supraoptic nucleus (SON) harbored vasopressin+ and oxytocin+ but not secretagogin+ neurons. Open arrowheads pinpoint single‐labeled neurons. Solid arrowhead denotes AVP/secretagogin dual‐labeling. lv, lateral ventricle; PVNm, magnocellular part of the paraventricular nucleus; scgn, secretagogin.
B3, B4 Secretagogin+ neurons had smaller somatic diameters than AVP+ or oxytocin+ neurons yet without a difference in their diameter quotient, a measure of ovoid profiles (*P 0.05, Student's t‐test).
C–D3 Terminal‐like profiles in the posterior pituitary (arrowheads in C) suggesting that secretagogin can co‐exist, even if infrequently with oxytocin. Solanum tuberosum lectin (Sol. tub) was used to identify blood vessels.

Data information: Scale bars: 100 μm (B), 10 μm (B1, B2), 2 μm (C–D3)
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 2

Ultrastructural analysis suggests the prevalence of membrane‐bound secretagogin

Secretagogin (scgn) distribution at the ultrastructural level as revealed by pre‐embedding silver‐enhanced immunogold labeling. Secretagogin (arrowheads) was localized to membranous organelles in the perikarya (A), particularly the plasmalemma (A1) and endoplasmic reticulum (ER) in neuronal soma (s; A2). Open rectangles in (A) denote the location of insets. Semi‐transparent shading is used to visually dissociate subcellular compartments in (A–C).
Pre‐embedding secretagogin labeling (arrowheads) was also seen in dendrite (d) segments.
In axo‐dendritic terminals (ax), secretagogin was closely associated with synaptic vesicles along the plasmalemma (arrowheads).
Quantitative analysis of subcellular secretagogin distribution upon electron microscopy detection of silver‐enhanced gold particles. Particles were considered as membrane bound when they were at 50 nm of a membrane (plasma membrane or endomembrane; i.e., secretory vesicle, Golgi or endoplasmic reticulum). In the soma of PVN neurons, significantly more particles were found in the cytosol as along the plasma membrane (**P 0.01). In contrast, membrane association predominated in axonal nerve endings in the median eminence (*P 0.05). Note that a significant proportion of particles was found adjacent to endomembranes in all subcellular compartments studied.

Data information: Scale bars:, 1 μm (A), 500 nm (B, C), 200 nm (A1, A2).
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 3

Electrophysiological classification of secretagogin+ parvocellular neurons

A–A3 Biocytin‐filled (arrow) neuron immunonegative for secretagogin yet containing AVP/oxytocin (a mixture of magnocellular markers was used in triple‐labeling experiments). Scale bars: 50 μm (A), 8 μm (A3). lv, lateral ventricle; PVN, paraventricular nucleus.
B–B2 Secretagogin+ biocytin‐filled neuron (arrow) lacking AVP/oxytocin immunosignal. Scale bars: 8 μm.
C, C1 Dendritic reconstruction of secretagogin− and secretagogin+ neurons at their actual location in the paraventricular nucleus of the hypothalamus.
D Electrophysiological characteristics of magnocellular and parvocellular neurons. The waveform of repetitive action potential firing is shown on the left, while differential channel characteristics are depicted on the right. Examples of secretagogin+ neurons are shown in red.
E Pie diagrams showing the grouping of neurons in the PVN (upper row) and in adjacent pre‐autonomic areas (lower row) cumulatively based on electrophysiological criteria listed in Supplementary Table S1. Secretagogin+ neurons typically belonged to Ia and IIa subtypes.
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 4

Molecular identity of secretagogin+ paraventricular neurons

Single‐cell mRNA transcriptome profiling of dissociated cells from the mouse paraventricular nucleus.

A Differential clustering based on secretagogin (Scgn), neuropeptide, hormone and hormone receptor mRNA expression. Secretagogin‐expressing(+) neurons typically contained corticotropin‐releasing hormone (Crh) and Nr3c1 mRNA transcripts.
A1 Clusters of gene transcripts from 130 cells reveal the phenotypic segregation of PVN neurons. Increasing mRNA copy numbers were depicted by a color gradient from deep blue (not detected) to red (high numbers). Secretagogin+ neurons are indicated by red arrows.
B Venn diagrams depicting the (non‐)overlapping relationships of select mRNA transcripts used to molecularly define PVN neurons.
B1 Cumulative ratio of phenotypic diversity among secretagogin+ neurons. Note that Crh and/or GABA (derived from Gad1, Gad2 and Slc32a1 mRNA expression) co‐existed with secretagogin in parvocellular neurons.
C, C1 Secretagogin co‐localization with GFP in the PVN (solid arrowheads) of adult CRH‐GFP (BAC) reporter mice (Alon et al, ). Open arrowhead pinpoints a single‐labeled neuron.
D–I3 We found secretagogin (scgn) co‐expressed with CRH in both neuronal soma (solid arrowheads; D1–D3) and axon terminal‐like specializations (arrowheads; E–E2) in the PVN. Likewise, secretagogin+ neurons harbored, yet infrequently, tyrosine hydroxylase (TH; solid arrowheads; F–F3), GABA (G–G2), somatostatin (Sst; H–H3) and galanin (I–I2). Open rectangles denote the general location of insets. Open arrowheads indicate the lack of co‐localization.

Data information: Scale bars: 40 μm (D, F, H), 25 μm (C1), 10 μm (D3, F3, G2, H3), 4 μm (E2, I2).
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 5

Secretagogin is a Ca2+ sensor protein

A–A2 Secretagogin co‐existed in the majority of CRH+ nerve endings in the median eminence (ME). Open rectangle denotes the general location of (B, B1).
B, B1 Large axon terminals in the median eminence were immunopositive for secretagogin with silver‐intensified immunogold particles (open arrowheads) associated with axonal membrane and dense‐core vesicles (see Fig D for quantitative data). Solid arrowheads denote silver deposit particles proximal to the plasmalemma.
C Immunoprecipitation using an anti‐secretagogin antibody in Ca2+‐free and Ca2+‐loaded conditions was subtractively used to decipher the Ca2+‐dependent protein interactions. Silver‐stained gel is shown.
C1 Ontology classification of the 99 protein hits based on primary function assignments. Unbiased MALDI‐TOF proteomics was used to identify interacting proteins. The most abundant hits (Supplementary Table S3 is referred to for details on individual proteins) are proteins implicated in vesicle fusion, trafficking, transport and formation and the regulation of vesicle exocytosis. “Other” refers to a group of proteins without known function.
D Single‐cell transcriptomics was used to validate the above proteome data by clustering mRNA transcripts encoding proteins that underpin vesicular release processes (red color labels highest mRNA abundance, whereas dark blue color indicates the lack of mRNA expression).
E–E3 Rab3, a family of vesicular fusion and transport proteins (Schluter et al, ), was found co‐localized with secretagogin (arrowheads) in the median eminence. Neuronal soma in the PVN lacked appreciable co‐localization. Note that neither our MALDI‐TOF analysis nor our histochemical probing allowed the precise identification of individual Rab3C‐E family members.

Data information: Scale bars: 10 μm (A1, E3), 7 μm (E), 500 nm (B), 150 nm (B1).
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 6

Secretagogin regulates CRH release in vitro and in vivo

A, A1 siRNA‐mediated secretagogin (scgn) knockdown in cultured hypothalamic neurons, as indicated by reduced secretagogin immunoreactivity (A) and decreased CRH content in the culture medium (A1).
B–B2 Transient overexpression of secretagogin in immortalized CRH‐expressing mHypoE‐N44 hypothalamic cells significantly reduced CRH immunofluorescence intensity in secretagogin+ cell bodies, indirectly supporting enhanced CRH release. All experiments were performed in triplicate. Scale bar: 50 nm.
C, C1 siRNA‐mediated in vivo silencing of secretagogin mRNA expression in the PVN provoked somatic CRH accumulation (arrowheads). Scale bar: 150 μm.
C2 Quantitative analysis demonstrating significantly increased somatic CRH contents. Note that somatic secretagogin levels remained unchanged, which we interpret as data on a neuronal contingent not affected by siRNA silencing. The lack of secretagogin/CRH co‐localization suggests that secretagogin expression fell below detection threshold in many CRH+ neurons.
D, D1 Individual data points show maximal CRH fluorescence intensity (gray scale arbitrary unit (a.u.) expression) in PVN neurons that have low or no secretagogin expression after siRNA infusion.

Data information: Data in (C2) were normalized to those in non‐targeting siRNA controls. *P 0.05 versus control.
A secretagogin locus of the mammalian hypothalamus controls stress hormone release (2014)
Figure 7

Secretagogin+ neurons gate the CRH–ACTH axis in response to acute stress

A, A1 Paraventricular secretagogin‐immunoreactive (scgn+) neurons weakly expressed CRH (open arrowheads) but not c‐fos under control conditions. Scale bars: 300 μm (A), 25 μm (A1).
B, B1 Stress induced by subcutaneous injection of formalin‐triggered co‐expression of c‐fos and CRH in secretagogin+ paraventricular neurons (arrowheads). Scale bars: 300 μm (B), 25 μm (B1).
C, D In vivo siRNA‐mediated silencing of secretagogin expression in the PVN occluded the stress‐induced surge of serum ACTH levels (C) and significantly reduced the increase in plasma corticosterone (D). *P 0.05 versus control.
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 1

USP45 co‐immunoprecipitates with components of the SLX4 complex

Schematic representation of the domain architecture of human USP45 protein.
Endogenous USP45 was immunoprecipitated (IP) from wild‐type (WT) and USP45 knockout (KO) KBM7 cells lines. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue. The gel was divided into the indicated sections, and proteins identified by mass spectrometry are detailed in Supplementary Table S1. The key proteins that we focus on in this study are indicated.
Endogenous USP45 was immunoprecipitated from the WT and KO KBM7 cell lines and subjected to immunoblotting with the indicated antibodies.
Endogenous USP45 was immunoprecipitated from WT and USP45 KO U2OS cells lines and analysed as in (B). Mascot scores of the interactors are detailed in Supplementary Table S2.
Endogenous USP45 was immunoprecipitated from the WT or KO U2OS cell lines and subjected to immunoblotting with the indicated antibodies. Similar results were obtained in at least two separate experiments.
Schematic representation of the SLX4 complex.

Source data are available online for this figure
USP45 deubiquitylase controls ERCC1–XPF endonuclease‐mediated DNA damage responses (2014)
Figure 2

USP45 interacts directly with ERCC1

A Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10/300 GL preparative grade column (GE Healthcare) in buffer containing 0.15 M NaCl, and every second fraction denatured and analysed by immunoblotting with the indicated antibodies. The white dotted lines indicate that the samples were run on different polyacrylamide gels.
B Endogenous USP45 or ERCC1 was immunoprecipitated from HEK293 cell lysates and subjected to immunoblotting with the indicated antibodies.
C Yeast two‐hybrid (Y2H) assays were performed with a GAL4 DNA‐binding domain fusion and/or activation domain for each protein indicated in the table to detect interaction between these proteins. Cells grown on media lacking LEU, TRP and HIS (to select for bait and prey plasmids) or tested for lacZ reporter gene activity.
D, E Endogenous SLX4 and ERCC1 were immunoprecipitated from extracts of WT or ERCC1 KO MEFs (D), or WT or SLX4 KO MEFs (E) and subjected to immunoblotting with the indicated antibodies.
F Extracts of WT or SLX4 KO MEFs were analysed by gel filtration as described in (A). Similar results were obtained in at least two separate experiments.