EMBO/soda-nel huggingface dataset:
Carfilzomib modulates tumor microenvironment to potentiate immune checkpoint therapy for cancer (2021)
Figure 7

Figure 7. Proteasome inhibitors are capable of reprogramming human M2 macrophage into M1-like macrophage

A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).

B. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory related cytokines in M2 macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24hours to differentiate into M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib, Bortezomib (500 nM) or MLN9708 (500 nM). The secretion of IL-1β, IL-6 and TNFα were detected through ELISA experiments 24 hours after stimulation. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).

C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).

G. Carfilzomib, Bortezomib and MLN9708 enhance the phagocytic ability of macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated with Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM) for 12 hours. Starved for 2 hours and incubated with L1210-GFP cells as targets in serum-free medium for another 2 hours. Macrophages were thoroughly washed and stained with fluorochrome-conjugated anti-CD11B antibody and analyzed through Flow cytometry. Phagocytosis efficiency was determined as the percentage of GFP positive cells in CD11B+ population.

H. Statistics represent the percentage of phagocytes and data are means ± SD of three independent experiments. *p < 0.05 (student's t-test).
OTULIN deficiency in ORAS causes LUBAC degradation, dysregulated TNF signalling, and cell death (2019)
Figure 1

Figure 1. Mutations in OTULIN in a new case of OTULIN-Related Autoinflammatory Syndrome (ORAS).

A Segregation of the inflammatory symptoms (filled symbols) and the c.841G>A substitution in OTULIN in the affected kindred. , females; , males; double lines, consanguineous relationship. Probands I.2 and I.3 are monozygotic twins. Roman numerals indicate generations.

B Schematic representation of the symptoms and clinical presentation of patient III.2.

C OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.
OTULIN deficiency in ORAS causes LUBAC degradation, dysregulated TNF signalling, and cell death (2019)
Figure 2

Figure 2. Mutation of Gly281 to Arg unfolds OTULIN's substrate-binding site and reduces its catalytic activity.

A Overall structure of OTULING281R (left, PDB: 6I9C) and OTULINC129A bound to M1-linked diUb (middle, PDB: 3ZNZ (Keusekotten et al, 2013)) and a superimposition of the two structures (right). The Cα positions of Gly281/Arg281 are shown as spheres.

B Close-up view of the M1-linked Ub-binding site from OTULING281R (light blue) and OTULIN's catalytic domain (blue) with M1-linked diUb bound (proximal Ub, orange; distal Ub, red) showing a clash between proximal Ub by His282 upon G281R mutation (arrow).

C Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. K­D­, dissociation constant.

D M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.

E First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.­
OTULIN deficiency in ORAS causes LUBAC degradation, dysregulated TNF signalling, and cell death (2019)
Figure 3

Figure 3. LUBAC degradation and accumulation of M1-linked Ub in OTULING281R fibroblasts

A Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts. Data are representative of three independent experiments.

B Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.

C Schematic representation of the AQUA-MS/MS-based proteomics approach for quantification of cellular Ub linkage composition.

D AQUA-MS/MS data from TUBE-based purification of cellular polyUb conjugates from untreated primary fibroblasts from a healthy control or patient III.2 harbouring the OTULING281R mutation. K27, K29, linkages could not be detected and K33 could not be accurately quantified in all samples. Data are representative of two independent experiments (see Figure EV2F and EV2G).

E-F Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts. Data are representative of three independent experiments.

G Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.
OTULIN deficiency in ORAS causes LUBAC degradation, dysregulated TNF signalling, and cell death (2019)
Figure 4

Figure 4. TNF-induced NF-κB and MAP kinase activation is impaired in OTULING281R fibroblasts due to reduced LUBAC recruitment to the TNF-RSC.

A Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.

B Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.

C Densitometry analysis of p65/RelA phosphorylation as presented in (A) from three independent experiments.

D ELISA analysis of IL-8 secretion in response to TNF stimulation (10 ng/mL) in healthy control or OTULING281R primary fibroblasts. Bars represent mean ± SEM (n=4).

E Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.
OTULIN deficiency in ORAS causes LUBAC degradation, dysregulated TNF signalling, and cell death (2019)
Figure 5

Figure 5. OTULIN-deficiency in human THP-1 monocytes leads to increased NF-κB activation and spontaneous TNF secretion

A Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments.

B Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.

C ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.

D Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.

E Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.
OTULIN deficiency in ORAS causes LUBAC degradation, dysregulated TNF signalling, and cell death (2019)
Figure 6

Figure 6. OTULIN-deficiency sensitises cells to TNF-induced apoptosis

A-D Viability of healthy control or OTULING281R primary fibroblasts (A-B) and shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays. Results were normalised to untreated samples. Each experiment was performed in duplicate. Bars represent mean ± SEM (A, n=9; B, n=6; C, n=8; D, n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.

E Increased apoptotic cell  death in the skin of ORAS patient III.2. Serial sections of normal skin (top panels) and a skin biopsy from patient III.2 taken at an inflammatory flare (bottom panels) were immunostained for cleaved caspase-3 (centre panels) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (right panels). Arrowheads indicate cleaved caspase-3-positive mesenchymal cells. None  of these markers were present in the healthy control skin. H&E, haematoxylin and eosin. Boxes in H&E panels indicate the areas magnified in the cleaved caspase-3 and TUNEL panels. Scale bars, 100 μm.  Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III.2.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 1

Figure 1. The Angptl2-/- mouse colon shows decreased β-catenin signaling

A, B. Representative images of H&E- (A) and PAS- (B) stained colon sections from wild-type and Angptl2-/- mice. Scale bar = 100μm.

C, D. Representative images of colon crypts as assessed by Ki-67 (C) and pH3 (D) staining. Scale bar = 100 μm.

E. mRNA levels of indicated proliferation and ISC markers in isolated crypts from wild-type (n=4) and Angptl2-/- (n=4) mice based on qRT-PCR analysis.

F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 served as an internal control. Numbers below panels represent normalized expression of proteins.

G. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice. Scale bar = 50 μm.

Data information: Data is represented as means ± SEM. *p < 0.05.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 2

Figure 2. ANGPTL2 functions in intestinal regeneration following DSS treatment

A. Survival rates of wild-type (n=33) and Angptl2-/- (n=33) mice supplied with drinking water containing 2.5 % DSS for 6 days and then untreated water for 6 more days, from two different experiments. ***p < 0.001, by log-rank test.

B. Representative images of colons from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).

C. Colon length of wild-type (n=7) and Angptl2-/- (n=6) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).

D, E, F. Representative images of colon crypts as assessed by H&E (D), Ki-67 (E), and pH3 (F) staining in wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 100 μm.

G, H. The number of regenerating crypts as quantified using Ki-67 (G) and pH3 (H) staining. Each data point represents the number of Ki-67-positive viable crypts or the number of pH3-positive cells in a single field of view. Multiple areas in at least 6 mice per genotype were quantified. Numbers below panels represent the average.

I. IHC for expression of active β-catenin in colon or wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12). Scale bar = 50 μm.

J. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).

Data information: Data are represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 3

Figure 3. ANGPTL2 functions in intestinal regeneration after irradiation

A. Weight loss of wild-type (n=4) and Angptl2-/- (n=5) mice in days following 12 Gy irradiation.

B. Small intestine and colon length from wild-type (n=4) and Angptl2-/- (n=5) mice 5 days after 12 Gy irradiation.

C, D. Representative images of crypts of small intestine as assessed by H&E (C) and Ki-67 (D) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.

E. The number of regenerating crypts in small intestine as quantified by Ki-67 (D) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.

F, G. Representative images of colon crypts as assessed by H&E (F) or Ki-67 (G) staining in wild-type and Angptl2-/- mice 5 days after 12 Gy irradiation. Scale bar = 100 μm.

H. The number of regenerating colon crypts as quantified by using Ki-67 (G) staining at days 3 and 5. Each data point represents the number of Ki-67-positive viable crypts in a single field of view. Multiple areas in at least 3 mice per genotype were quantified. Numbers below panels represent average.

Data information: Data are represented as mean ± SEM. *p < 0.05; ***p < 0.001.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 4

Figure 4. ANGPTL2 localizes to the intestinal mesenchyme

A. Angptl2 mRNA levels in small intestine (proximal, mid and distal), cecum and colon (proximal and distal) of wild-type mice based on qRT-PCR analysis. n=5.

B. ANGPTL2 IHC in colon from wild-type and Angptl2-/- mice. The Angptl2-/- colon image serves as a negative control. Scale bar = 50 μm.

C. mRNA levels of indicated genes in wild-type (n=4) mice based on qRT-PCR analysis.

D. Representative IF images of distal colon from wild-type mice as assessed with anti-ANGPTL2 (red) and anti-α-SMA (green). Nuclei are counterstained with DAPI (blue). Scale bar = 100 μm.

Data information: Data are represented as mean ± SEM.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 5

Figure 5. Bmp2 and Bmp7 transcripts are upregulated in Angptl2-/- ISEMFs

A, B, C. mRNA levels of indicated genes in ISEMFs from wild-type (n=4) and Angptl2-/- (n=3) mice based qRT-PCR analysis.

D. Representative staining for P-Smad1/5 in crypts from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Dashed lines indicate half of the crypt and arrowheads show P-Smad1/5-positive cells in lower halves of crypts. Scale bar = 50 μm.

E. The number of P-Smad1/5-positive cells in the crypt base (n=60) from wild-type and Angptl2-/- mice following DSS treatment for 6 days and then untreated water for 6 more days (DSS) or untreated water for 12 days (Untreated). Multiple areas in 3 mice per genotype were quantified.

F. Western blotting analysis of isolated colon crypts from wild-type and Angptl2-/- mice. HSC70 serves as an internal control. Numbers below panels represent normalized protein expression.

Data information: Dates are represented as mean ± SEM. *p < 0.05; ***p < 0.001.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 6

Figure 6. Integrin α5β1/NF-κB signaling downregulates Bmp expression

A. mRNA levels of indicated genes in colon and cultured ISEMFs from wild-type mice (n=4) based on qRT-PCR analysis. Inta5, Integrin α5; Intb1, Integrin β1.

B. Representative IHC of integrin α5 and β1 in colon from wild-type mouse. Scale bar = 50 μm.

C, D. mRNA levels of Bmp2 and Bmp7 in ISEMFs from wild-type mice 24 h after treatment with integrinα5β1 antibody or various signaling pathway inhibitors. n=3. U0126, ERK inhibitor; LY294002, PI3K inhibitor; BAY11-7085, NF-κB inhibitor.

Data information: Data are represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis (2016)
Figure 7

Figure 7. Co-culture with Angptl2-/- ISEMFs decreases organoid formation in vitro

A. Colon organoid cultures treated with vehicle or rANGPTL2 protein. Scale bar = 200 μm.

B. Organoid growth efficiency and size following treatment with vehicle or rANGPTL2. rA2, recombinant ANGPTL2. n=4. Data from vehicle/Wnt (+) was set at 1.

C. Schematic illustration of co-culture of colon organoids with ISEMFs in direct contact.

D. Colon organoid cultures in presence of ISEMFs with or without Noggin (Nog). Scale bar = 50 μm

E. Organoid growth efficiency and size in presence of ISEMFs with or without Noggin (Nog). n=4. Data from Wild-type organoids/Wild-type ISEMFs/Nog (+) was set at 1.

F. Model of ANGPTL2 activity in the intestinal stem cell niche. ISEMF-derived ANGPTL2 inhibits BMP signaling in an autocrine manner through the integrin α5β1-NF-κB pathway to maintain ISC stemness by regulating β-catenin and Smad 1/5 signaling.
Temporal specificity and heterogeneity of Drosophila immune cells (2020)
Figure 1

Figure 1: Hemocytes display distinct properties at E16 and WL stage.

A) Transcriptome comparison of hemocytes from stage 16 embryos (E16) and wandering 3rd instar larvae (WL). The x-axis is the average genes expression levels (n=3) and the y-axis the Log2 Fold Change WL/E16. P-values are indicated with the color code.

B) Gene Ontology (GO) term enrichment analysis in E16 (green) and WL (red) hemocytes. The fold enrichments for a subset of significant GO terms are displayed, the number of genes and the p-value of the GO term enrichment are indicated in brackets.

C-E) Scatter plots as in (A) highlighting in black subsets of known genes expressed in hemocytes (C) or genes associated with the GO terms extracellular matrix (D) and phagocytosis (E).

F-G) Phagocytosis assay on E16 (F) and WL hemocytes (G) srp(hemo)-moesin-RFP. The beads (in green) are phagocytosed by the hemocytes (in red). The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure. Full stacks are displayed and the scale bars represent 20 µm.

Data information: Related to Appendix Figure S1, S2, Dataset EV1, EV4.
Temporal specificity and heterogeneity of Drosophila immune cells (2020)
Figure 3

Figure 3: Localisation of the NI hemocytes clusters.

A) Identification of the position (circulating/resident) of the clusters within the larva by qPCR. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment. Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment (n=5, mean +/- SD are represented on the graph). The p-values are estimated by bilateral paired student test and indicated as follow: ns = non-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[..

B) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm.

C) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC-Lsp1beta-MI05460 larvae. The OregonR value indicates the background level. The number of cells included in the analysis is displayed on the x-axis label and were quantified from two independent preparation, p-values were estimated after variance analysis with bilateral student test for equal variance.

D) Resident hemocytes located around the oenocytes or along the muscles (top and bottom panels, respectively) from WL srp(hemo)-moesin-RFP, that express moesin tagged with RFP in hemocytes. The immunolabelling was done with anti-RFP (in red), anti-Pendulin (Pen, in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, Pen and RFP, the middle panels RFP alone and the right panels Pen alone. Arrow heads in the top panels indicate cells co-expressing RFP and Pen. The scale bars represent 50 µm.

Data information: Related to Appendix Figure S5, Table EV1.
Temporal specificity and heterogeneity of Drosophila immune cells (2020)
Figure 6

Figure 6: Characterisation of the two lamellocyte clusters.

A) Scatter plot comparing the transcriptome of the two lamellocyte clusters, deduced from the WI dataset. The x-axis is the average expression levels of the genes and the y-axis the log2 fold change (LM-1/LM-2). The genes significantly enriched in the LM-1 cluster are highlighted in red (p<10-6), those significantly enriched in the LM-2 cluster in orange (p<10-6).

B) GO term enrichment analysis on the genes enriched in lamellocytes compared to all other clusters (top panel) and on the genes specific to LM-1 (middle panel) or LM-2 (lower panel). The bars represent the fold enrichment and the color gradient indicates the p-value of the GO term enrichment (as in Figure 2C).

C) Expression levels (gradient of color) and the percentage of cells (size of the dots) for the top 10 markers of lamellocytes (in black), of markers specific to LM-1 compared to LM-2 (in red) and specific to LM-2 compared to LM-1 (in orange). Note that most markers enriched in LM-1 compared to LM-2 are exclusively expressed in LM-1 while most markers enriched in LM-2 compared to LM-1 are also expressed in the plasmatocyte and crystal cell clusters.

D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm.