EMBO/soda-nel huggingface dataset:
Control of AGR2 dimerization links endoplasmic reticulum proteostasis to inflammation (2019)
Figure 6

Figure 6: Alteration of TMED2 expression and relationships with eAGR2-mediated monocytes attraction. A) Representative histological H&E staining of sections of the proximal colon and ileum of WT and TMED2 hypomorph mice. B) Immunohistological analysis of AGR2 and MUC2 expression in the proximal colon of WT and TMED2 hypomorph mice. C) Semi-quantitative analysis of AGR2 and MUC2 expression in the proximal colon of WT (blank bars) and TMED2 hypomorph mice (black bars) (n=6). D) Freshly isolated PBMCs were placed in Boyden chambers towards media conditioned by cells overexpressing AGR2 WT, E60A, ∆45 or AGR2 AA mutants and incubated for 24 h. Migrating cells were then characterized and quantified by flow cytometry using FCS/SSC parameters or CD14 monocyte marker. (**): p=0.0016. E and F) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells overexpressing TMED2 and either AGR2 WT (E) or AGR2 AA mutant (F) and incubated for 24 h. Migrating cells were then characterized and quantified as indicated in Figure 6D. For E (*): p=0.0461 (AGR2WT/TMED2) and 0.018 (AGR2WT/TMED2 + blocking Ab) respectively; (**): p=0.0053 and (***): p=0.0048; for F (***): p=0.0007. G) Freshly isolated PBMC were placed in Boyden chambers towards media conditioned by cells silenced for TMED2 and incubated for 24 h. Migrating cells were then characterized and quantified as indicated in Figure 6D. (*): p=0.0149. H) Freshly isolated PBMC were placed in Boyden chambers towards decreased doses of recombinant AGR2 and incubated for 24 h. CCL2 cytokine was used as positive control for monocyte migration. ns: non-statistically significant, (*): p=0.0171, (**): p=0.0084, (***): p=0.0003. I) Impact of AGR2 blocking antibodies on AGR2-mediated monocytes migration was tested using Boyden chambers as described above. The concentrations of recombinant human AGR2 was of 200 ng/ml and decreasing amounts of antibodies were used from 20 µg to 1 µg. The non-relevant antibody (Isotype) was used at the maximal dose of 20 µg. Data are representative of three independent experiments. ns: non-statistically significant, (*): p=0.0165 (Ab=20μg/ml), 0.0223 (Ab=10μg/ml) and 0.0493 (Ab=5μg/ml), respectively.
Control of AGR2 dimerization links endoplasmic reticulum proteostasis to inflammation (2019)
Figure 7

Figure 7: Expression levels of AGR2, TMED2 and CD163 in biopsies from healthy controls and patients with IBD. A) Selective enrichment of AGR2 modulators in colonic Crohn's disease (CD). Percentage of tested genes with altered expression in colonic biopsies from patients with Ulcerative Colitis (UC, n=8) and colonic CD (CC, n=15) and in ileal biopsies from patients with ileo-colonic CD (IC, n=9). B) Hierarchical clustering of the AGR2 modulator genes significantly deregulated in non-inflamed colonic mucosa from patients with Crohn's Disease (CD) versus healthy controls (C) (dChip software t-Test) C) TMED2 mRNA expression in healthy controls (CT), colonic Crohn's disease (CC) and ulcerative colitis (UC). D) Representative immunohistological analysis of TMED2 in non-inflamed colonic biopsies from healthy controls and patients with active CD. E) Representative immunohistological analysis of CD163 (scavenger receptor present in macrophages), AGR2, and TMED2 in non-inflamed colonic biopsies from healthy controls and patients with active or quiescent CD. F) Semi-quantitative analysis of CD163, AGR2, and TMED2 immunostaining in colonic mucosa from healthy controls (C) and patients with quiescent (Q) or active (A) CD. Three random sections from each patient of the four groups were scored. Final IHC scores (AxB range to 0-9) combine the percentage of positively labelled cells. A: % of IHC positive labeled cells - 0 (0%), 1 (<30%), 2 (30 to 60%), 3 (>60%)) and the intensity of the reaction product. B: 0 (no reaction), 1 (weak), 2 (mild), 3 (strong) in most of the examined fields. All values are mean ± S.E.M. G) Schematic representation of the role of AGR2 dimer alteration in intestinal epithelial cells under basal conditions or in Crohn's disease. In normal cells, the ratio of dimeric vs. monomeric AGR2 defines proteostasis boundaries in the ER, and those are altered in diseased conditions through the alteration of the expression of AGR2 dimer regulators, thereby leading to pro-inflammatory signals comprising the release of AGR2 in the extracellular milieu and to the induction of uncontrolled autophagy and the subsequent alteration of protein secretion.
The unfolded protein response links tumor aneuploidy to local immune dysregulation (2021)
Figure 3

Figure 3. The unfolded protein response is influenced by SCNA levels.

(A) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available. The boxplot indicates the median (central band) and the interquartile range (boxes) of the expression value and whiskers indicates values that outside of the middle 50% (interquartile). The barplot on the top shows the number of normal tissue samples for each corresponding tumor type. Asterisks indicate significant differences by student's t-test after Benjamini-Hochberg multiple testing correction (FDR < 0.05).

(B) A heat map showing the Spearman correlation coefficient between the log2 TPM expression of three UPR branch pathway parental genes (rows) and SCNA scores across 32 tumor types (columns). Red indicates positive correlation coefficients (r > 0) and blue indicates negative correlation coefficients (r < 0). Size indicates significance level of correlation. Gene names colored in blue belong to the PERK pathway, orange to the ATF6 pathway and green to the IRE1 pathway.

(C) A heatmap showing the partial correlation between the expression of UPR branch pathway genes with SCNA levels controlling for the effect of the gene copy number change using data from GISTIC2.0. Rows depict genes from the UPR branch pathways from REACTOME and columns depict the 32 tumor types. Cells are colored in red or blue if the gene showed significant correlation with SCNA level in that tumor type (partial correlation test setting copy numbers for genes as control, after Benjamini-Hochberg multiple hypothesis correction, FDR < 0.05). Color intensity corresponds to the partial correlation coefficient between gene expression and SCNA level. The left side bar indicates pathway membership of the genes. The bottom panel shows the variances of SCNA scores for each tumor type and the number of normal tissue samples available for partial correlation analysis.
The unfolded protein response links tumor aneuploidy to local immune dysregulation (2021)
Figure 6

Figure 6. Experimental aneuploidy triggers activation of the UPR pathways in cancer cells.

(A) Schematic representation of PCR-based analysis of Xbp1 splicing. During conditions of ER stress, a 26 base pair fragment is spliced from Xbp1 mRNA. Forward and reverse PCR primers (shown in blue) were designed to span the splice site. PCR amplification distinguishes between unspliced (Xbp1-u, upper band) and spliced (Xbp1-s, lower band) Xbp1 mRNA. To quantify ER stress, a ratio of spliced: unspliced Xbp1 was calculated.

(B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin (Tg) is shown as positive control.

(C) Schematic representation depicting cell-cell fusion between a B16 melanoma cell and mouse embryonic fibroblasts (MEF).

(D) Xbp1 splicing in B16 melanoma cells and eight B16:MEF fused clonal cell lines.

(E) Detection of mRNA expression (by RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine (Rv) at 4µM (DLD1 cells) or 0.5µM (SKOV3 cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD.

(F) Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control.

(G) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.
The unfolded protein response links tumor aneuploidy to local immune dysregulation (2021)
Figure 7

Figure 7. Cell nonautonomous effects of aneuploid cancer cells on bone marrow-derived macrophages and T cells.

(A) Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated DLD1 and SKOV3 cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma cells or their nonfused parental cells.

(B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM cultured in the CM of Rv-treated SKOV3 cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD.

(D) mRNA expression of Il6 (upper panel) and Arg1 (lower panel) in BMDM cultured in complete medium spiked with Rv (0.5 µM). Untreated BMDM were used as control. Data representative triplicate samples run in duplicate at the same time point repeated at least twice and expressed as means ± SD.

(E) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.

(F) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.

(G) IFNγ (left) and Granzyme B (right) production by human T cells activated by anti-CD3/anti-CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non-fused cells. Percentages in parenthesis refer to change from parental non-fused cells. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.

Expanded View Figure Legends
Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors (2021)
Figure 2

Figure 2: The TseTBg effectors of NGJ1 exhibit potent anti-bacterial activity. (A) Growth curve of recombinant BL21 (DE3) cells upon IPTG mediated induction of proteins, represented as colored line graphs.

(B) Bacterial two-hybrid assay reflecting interaction between proteins of T6SS apparatus and effector operons. Interaction between T25-zip and T18-zip was used as positive control while pKNT25 and pUT18C (empty vectors) was used as negative control. Appearance of blue color suggests positive interaction while absence of color suggests lack of interaction.

(C) Yeast two-hybrid assay reflecting interaction of TseTBg1 and TseTBg2 effector proteins with the corresponding PAAR (PAARTseTBg) proteins encoded in their operon. Interaction between P53 and SV40 large T-antigen (T) proteins was used as positive control. The pGBKT7 and pGADT7 (empty vectors) were used as negative control.

Data information: In (A), graphs represent mean ± SD of three biological replicates. Asterisk ** indicates significant difference at P<0.01 and 'ns' indicates non-significant difference (estimated using one-way ANOVA) at the given time point. In (B, C), similar results were obtained in at least three independent experiments.
Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors (2021)
Figure 3

Figure 3: The TseTBg effector proteins of NGJ1 harbour potent DNase and RNase activity.

(A, B) Effect of purified TseTBg (TseTBg1/TseTBg2) and one of the variants (TseTBg2D116A,K129A) proteins on degradation of bacterial genomic DNA.

(C, D) RNase activity of TseTBg effector proteins, in presence or absence of cognate TsiTBg immunity proteins.

(E) Effect of TseTBg1, TseTBg2, TseTBg2D116A,K129A proteins on degradation of pHM1, a broad host-range plasmid isolated from E. coli and NGJ1.

(F) Effect of TseTBg1 and TseTBg2 proteins on degradation of pHM1, reintroduced into E. coli from NGJ1.

(G) Effect of ectopic expression of DNA adenine methylase (Dam) protein on TseTBg proteins mediated degradation of plasmid DNA.

Data information: In (A-G), similar results were obtained in at least three independent experiments. M; DNA marker (1kb); -; negative control.
Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells (2021)
Figure 1

Figure 1. The centrosomes in quiescent NSCs are immature

A) An illustration of the larval brain at early larval stages and the alignment of the quiescent NSC along the apico-basal axis.

B) Larval brains at 6 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Sas-4, Dpn, and GFP.

C) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with γ-tubulin (γ-tub), Asl, and GFP and imaged under super-resolution microscopy.

D) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Centrosomin (CNN), Asl and GFP and imaged under spinning disc super-resolution microscopy.

E) Larval brains at 0 h ALH from Jupiter-GFP (G147) were labeled with GFP, Msps, and Dpn.

F) Larval brains at 24 h ALH from Jupiter-GFP were raised on normal food (fed) and food depleted of amino acids (nutritional restriction/NR) and labelled with GFP and Dpn.

G) Quantification graph showing Jupiter-GFP protein levels in qNSCs by normalizing to that in MB NSCs in (F). n=10 brain lobes (BL) for normal food condition; n=14 BL for NR food condition. ****p<0.0001.

H) Larval brains at various time points from grh-Gal4; UAS-CD8-GFP were labeled with Msps, Dpn, and GFP.

I) Quantification graph of Msps intensity in qNSCs by normalizing against that in MB NSC at 0 h ALH (n=45 NSCs) and 6 h ALH (n=81 NSCs) in (H). ****p<0.0001.

Data information: Quiescent NSCs in the central brain are shown (B-H). Arrows, centriole(s)/centrosomes (B). In (G, I), data are presented as mean ± SD. In (G, I), statistical significances were determined by two-tailed Student's t-test. Scale bars (B, E, F, H): 10 µm. Scale bars (C, D): upper panel 1µm; lower panel 0.1µm.
Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells (2021)
Figure 2

Figure 2. Microtubules in the cellular extension of qNSCs are predominantly plus-end-out orientated

A) Larval brains at 16 h ALH, in which kin-lacZ was expressed under the control of insc-Gal4, tub-Gal80ts, were labelled with β-Gal, Msps, and Dpn. The primary protrusion of qNSCs was marked by Msps. Quiescent NSCs at the ventral nerve cord (VNC) are shown. Arrows indicate the localization of Kin-lacZ in qNSCs.

B) Larval brains at 6 h ALH from insc-Gal4; UAS-nod-lacZ were labeled with β-Gal, Msps, and Dpn. Quiescent NSCs at both CB and VNC are shown. Arrows indicate the localization of Nod-lacZ in qNSCs.

C) Representative kymographs decorated with and without coloured lines depicting movements of EB1-GFP comets in the primary protrusion of qNSCs at 0 h ALH.

D) Representative kymographs decorated with and without coloured lines tracking movements of EB1-GFP comets in the primary protrusion of qNSCs at 6 h ALH.

E) Quantification graph of the percentage of anterograde and retrograde movements of EB1-GFP comets in the primary protrusion of qNSCs at 0 h ALH and 6 h ALH. n=28 at 0 h ALH; n=64 at 6 h ALH. 3 biological replicates per time point. p=0.9096 (ns).

F) Quantification graph of the velocity of EB1-GFP comet movement in the protrusion of qNSCs at 0 h ALH, 6 h ALH and 10 h ALH, as well as in interphase NSCs (aNSCs) at 72 h ALH. n=250 for 0 h qNSCs (anterograde); n=30 for 0 h qNSCs (retrograde); n=211 for 6 h qNSCs (anterograde); n=24 for 6 h qNSCs (retrograde); n=171 for 10 h qNSCs (anterograde); n=103 for interphase NSCs (anterograde). ****p<0.0001; * p=0.0285.

Data information: In C-D, the horizontal arrow indicates the anterograde movement direction from the cell body to the tip of the primary protrusion in qNSCs. In (E, F), data are presented as mean ± SD. In (E), statistical significance was determined by two-way ANOVA with multiple comparisons. In (F), statistical significance was determined by one-way ANOVA with multiple comparisons. Scale bars: 10 µm.
Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells (2021)
Figure 3

Figure 3. Msps is essential for NSC reactivation

A) Larval brains at 24 h ALH from control (insc-Gal4; UAS-dicer2/UAS-β-Gal RNAi) and msps RNAi (VDRC#21982) controlled under insc-Gal4 were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira.

B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for both control and msps RNAi. ****p< 0.0001.

C) Larval brains at 24 h ALH from wild-type and various msps loss-of-function alleles and msps810 with a genomic rescue construct (g-msps) were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira.

D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (C). n=13 BL for control; n=13 BL for msps810; n=17 BL for msps924; n=14 BL for mspsP18; n=6 BL for mspsP; n=12 BL for g-msps; msps924. ****p< 0.0001; p=0.7502 (ns).

E) Quantification graph of the diameter of the cell body in NSCs at 24 h ALH from various genotypes. n=238 NSCs for control; n=219 NSC for msps924; n=170 NSCs for mspsP. ****p< 0.0001.

F) Quantification graph of the percentage of qNSCs with a primary protrusion in wild-type, msps924, and mspsP18 larval brains. The protrusion was labelled by grh>CD8-GFP. n=5 BL for control; n=6 BL for msps924; n=4 BL for mspsP. ****p< 0.0001.

G) Larval brains at 24 h ALH from wild-type and tacc loss-of-function tacc74 were examined for EdU incorporation. NSCs were marked by Dpn and Mira.

H) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (G). n=17 BL for control; n=15 BL for tacc74. ****p< 0.0001.

I) Quantification graph of qNSCs retaining primary protrusion per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001.

J) Quantification graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001.

Data information: Arrows indicated EdU-negative NSCs in A, C, and G. In (B, D-F, H-J), data are presented as mean ± SD. In (B, H-J), statistical significance was determined by two-tailed Student's t-test. In (D-F), statistical significance was determined by one-way ANOVA with multiple comparisons. Scale bars: 10 µm.
Msps Governs Acentrosomal Microtubule Assembly and Reactivation of Quiescent Neural Stem Cells (2021)
Figure 5

Figure 5. Delocalization of E-cadherin at NSC-neuropil contact sites in qNSCs upon msps depletion

A) Larval VNCs at 24 h ALH from the control (grh-Gal4 UAS-CD8-GFP) and msps810 with grh-Gal4 UAS-CD8-GFP were labeled with E-cadherin, Dpn, and GFP. NSC-neuropil contact were marked by dashed lines, and NSC-neuropil contact points were circled.

B) Quantification of E-cadherin basal localization at NSC-neuropil contact sites in qNSCs from genotypes in (A). "No E-cad" means absent or strongly reduced E-cad observed at the basal region of qNSCs. n=32 NSCs for control; n=61 NSCs for msps810. **p=0.0028.

C) Quantification graph of the basal E-cad intensity at NSC-neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in (A). n=34 NSCs for control; n=40 NSCs for msps810. ****p<0.0001.

D) Larval VNCs at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP) and msps924 with grh-Gal4 UAS-CD8-GFP were labeled with E-cadherin, Dpn, and GFP. NSC-neuropil contact were marked by dashed lines, and NSC-neuropil contact sites were circled.

E) Quantification of E-cadherin basal localization at NSC-neuropil contact sites in qNSCs from genotypes in (D). "No E-cad" means absent or strongly reduced E-cad observed at the basal region of qNSCs. n=77 NSCs for control; n=30 NSCs for msps924. **p=0.0080.

F) Quantification graph of the basal E-cad intensity at NSC-neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in (D). n=60 NSCs for control; n=37 NSCs for msps924. ****p<0.0001.

G) An illustration of targeted GFP Reconstitution Across Synaptic Partners (t-GRASP).

H) Larval brains at 16 h ALH from control t-GRASP under the control of single driver grh-Gal4 or nSyb-QF2 (Q-system) and t-GRASP under the control of driver pairs grh-Gal4 and nSyb-QF2 (Q-system) were analyzed for E-Cad, GFP (reconstituted GFP), and Dpn. Arrows pointing at the cell body of qNSCs on the two different focal planes. NSC-neuropil contact sites were circled by dashed lines.

I) A representative Intensity plot of E-cadherin (in blue) and GRASP-GFP (in green) in the primary protrusion of a qNSC from (H). Pixel intensity of GFP and E-cad in qNSCs were measured between the PIS region and the tip of the primary protrusion.

Data information: in (B-C, E-F), data are presented as mean ± SD. In (B, E), statistical significances were determined by two-way ANOVA with multiple comparisons. In (C, F), statistical significances were determined by two-tailed Student's t-test. Scale bars: 10 µm.