EMBO/soda-nel huggingface dataset:
Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilisation (2019)
Figure 4

Figure 4.

MDGI silencing induces cell death via lysosomal membrane permeabilization (LMP).

A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3).

B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.

C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.

E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.

G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.

I. Percentage of LGALS1-positive puncta staining in the control (Scr) and MDGI-silenced (sh1) cells. In total 0.4-1.2x104 LGALS1-stained cells were analysed from 50 mm2 coverslip areas (n = 6). Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.

J. Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining (n = 100) in control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.

K. Cytoplasmic cathepsin B activity in the control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01; **P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.

L. Number of live cells in MDGI-silenced BT12 and BT13 cells incubated without (sh1) or with (sh1+K777) the pan-cathepsin inhibitor (K777) after 1, 3 and 5 days of incubation. Data shows the percentage of live cells compared to the control (Scr) cells set as 100 %. Data are represented as mean ± SD. *P < 0.05; **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.
Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilisation (2019)
Figure 5

Figure 5.

MDGI silencing induces changes in lysosomal membrane lipid composition.

A. Proportions (mol %) of saturated (Sat), monounsaturated (Mono), diunsaturated (Di; total of 2 double bonds in the acyl chains), and polyunsaturated (Poly; total of 3-7 double bonds in the acyl chains) lipid species from the two main phospholipid classes (phosphatidylcholine PC and phosphatidylethanolamine PE) of the lysosomal fractions of the BT12 and BT13 control (Scr) and MDGI-silenced (shMDGI) cells. A representative graph of two independent experiments is shown.

B. Fold change of the main PC and PE species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) cells (Log2, by mol % data). Abbreviations: [Lipid class] [total number of acyl chain carbons]:[total number of double bonds in the chains]. A representative heatmap of two independent experiments is shown.

C. Profiles of ceramide (Cer) species (mol %) in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. Abbreviations: Cer [number of acyl chain carbons]:[number of double bonds in the acyl chain]; all principal species containing a sphingosine chain. A representative graph of two independent experiments is shown.

D. Ratio of the 24:1 to 24:0 (mol %.mol %) Cer species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. A representative graph of two independent experiments is shown.
Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilisation (2019)
Figure 6

Figure 6.

Antihistamine treatment induces glioma cell death via lysosomal membrane permeabilization (LMP). A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.

B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.

C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm.

D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.
Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilisation (2019)
Figure 7

Figure 7.

Clemastine treatment eradicates invasive glioma cells.

A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.

B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.

D. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.

E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.

F, G. Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.

G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm.

H. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.

Expanded view Figure Legends
The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins (2022)
Figure 1

Figure 1. Toll pathway mutants succumb to A. fumigatus infection even though it is not required to limit the proliferation and dissemination of the pathogen, an immune function mediated by melanization.

(A) survival curves of MyD88 flies injected with different doses of A. fumigatus conidia (c/f: conidia injected per fly; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).

(B) fungal loads of single MyD88 mutant and wild-type flies (50 conidia injected per fly).

(C-D) GFP-labeled A. fumigatus (50 conidia per fly) injected in wild-type (C) or MyD88 mutant (D) flies form hyphae in the thorax of the flies (arrows). Scale bars 50 μm.

(E) expression level of Drosomycin induced by different doses of injected A. fumigatus conidia measured by RT-qPCR; M. luteus (OD=10) represents the positive control (pooled data of n=3 experiments, biological replicates).

(F-H) Hayan flies are susceptible to A. fumigatus. Survival (F), time course of fungal loads of single Hayan mutant flies (500 conidia per fly) (G), and fungal load upon death (500 conidia per fly) (H) of Hayan mutant flies; Hayan 0h vs. 120h, (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each). (I-J) A. fumigatus hyphae extrusion (arrows) from Hayan (I) and MyD88 (J) mutant cadavers; scanning electron micrographs. Scale bars 750 μm.

Data information: in (B,C,G,H), the middle bar of box plots represents the median and the upper and lower limits of boxes indicate respectively the first and third quartiles; the whiskers define the minima and maxima; data were analyzed using the Mann-Whitney statistical test. Survival curves were analyzed using the LogRank test. **p=0.004; ****p < 0.0001, and NS: not significant.
The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins (2022)
Figure 2

Figure 2. Secondary metabolism is critical for the virulence of A. fumigatus in Drosophila MyD88 immunodeficient flies.

(A) survival of MyD88 flies injected with ∆pptA and wild-type (Af) A. fumigatus conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each).

(B) fungal loads of single flies after the injection of 500 ∆pptA conidia.

(C-D) hyphae of ∆pptA A. fumigatus observed in thorax of wild-type and MyD88 flies (arrow) after Uvitex-B negative staining, air sacs and tracheae are stained by Uvitex-B (arrowheads). Scale bars 50 μm.

(E) dose response of MyD88 flies after ∆gliP (gliotoxin) mutant or wild-type [∆akuB] A. fumigatus infection; error bars represent mean ± SD of the survival of biological triplicates of 20 flies each; wild-type flies are used as a control for the dose of 250 conidia.

(F-H) dose response of MyD88 and wild-type flies after gliotoxin (F), fumagillin (G), and helvolic acid (H) injection at the indicated concentrations (20 flies per condition).

Data Information: in (B), the middle bar of box plots represents the median and the upper and lower limits of boxes indicate respectively the first and third quartiles; the whiskers define the minima and maxima; data were analyzed using the Mann-Whitney. Survival curves were analyzed using the LogRank test. ****p < 0.0001, and NS: not significant.
The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins (2022)
Figure 4

Figure 4. Restrictocin functions as a ribotoxin in vitro and in vivo and affects MyD88 mutant and not wild-type flies.

(A) survival of MyD88 or wild-type flies to 250 injected ∆aspf1 (restrictocin mutant) or wild-type [∆akuB] A. fumigatus conidia (20 flies per condition); MyD88: ∆aspf1 vs. ∆akuB (***p=0.0007).

(B) survival of MyD88 flies after the injection of different concentrations of restrictocin (R) (20 flies per condition ).

(C-D) survival of antibiotics-treated (C) and axenic (D) MyD88 mutant flies after restrictocin injection (20 flies per condition).

(E-F) ribosomal RNA cleavage measurement after restrictocin or PBS injection in wild-type (E) and MyD88 (F) flies; the arrowheads show the position of the 28S RNA-derived α-sarcin fragments whereas arrows on the right show its electrophoretic band position.

(G) fluorescence (arbitrary units) emitted by transgenic pUbi-Gal4-Gal80ts>UAS-GFP whole flies induced at the same time as the challenge and measured at the indicated time points; PBST vs. Af: **p=0.002 (pooled data of n=3 experiments, biological replicates).

(H) SDS-PAGE analysis of 35S-labelled translated proteins produced in a rabbit reticulocytes lysate from a m7G-capped reporter RNA containing the 5'UTR of β-globin followed by the Renilla luciferase coding sequence (arrow), in the presence of increasing concentrations (0.125 to 25 nM) of restrictocin.

(I-J) fluorescence analysis from in vitro translated eGFP from an IGR (CrPV)-driven reporter in noninduced (blue) and Toll-induced (red) ERTL lysates: translation kinetics of in vitro synthesized eGFP in the presence of 1 nM restrictocin showing fluorescence values normalized to untreated translation reactions (I) and a histogram representing the end-point fluorescence quantification of I (J).

Data Information: in (G), the middle bar of box plots represents the median and the upper and lower limits of boxes indicate respectively the first and third quartiles; the whiskers define the minima and maxima; data were analyzed using the Kruskall-Wallis and Dunn's post-hoc test. In (I), error bars represent mean ± SEM (n=3, technical replicates); in (J), error bars represent mean ± SEM (n=3, technical replicates). Survival curves were analyzed using the LogRank test. ****p < 0.0001, and NS: not significant.
The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins (2022)
Figure 5

Figure 5. Distinct Bomanins mediate resilience to specific A. fumigatus mycotoxins.

(A-B) survival (A) and fungal load (B) of Bom∆55C (55C) deficient flies compared to wild-type and MyD88 after injection of 250 conidia (error bars represent mean ± SD of the survival of biological triplicates of 20 flies each); ****p < 0.0001. (B): the fungal burden does not increase in Bom∆55C-deficient flies; wt 0h vs. 48h, **p=0.001; 55C 0h vs. 48h, **p=0.007; pooled data of n=3 experiments, biological replicates.

(C-D) rescue of the sensitivity of Bom∆55C flies to verruculogen (C) or to restrictocin (D) by the transgenic expression of individual 55C locus genes (caption in D also applies to (C)). 55C flies vs. BomS1, *p=0.0495, vs. BomS6 *p=0.011 for verruculogen assay (C); 55C flies vs. BomBc1 or BomS3, ****p<0.0001, 55C flies vs. BomS6, **p=0.0028 for restrictocin assay (D) (20 flies per condition).

(E-F) expression levels of BomBc1, and BomS3 measured by RT-digital PCR 48 hours after challenge; BomBc1 PBST vs. Af, *p=0.015, PBST vs. restrictocin (R), *p=0.015; BomS3: PBST vs. Af, *p=0.02, PBST vs. R, *p=0.03 (pooled data of n=3 experiments, biological replicates).

Data Information: in (B, E-F), the middle bar of box plots represents the median and the upper and lower limits of boxes indicate respectively the first and third quartiles; the whiskers define the minima and maxima; data were analyzed using the Mann-Whitney statistical test. Survival curves were analyzed using the LogRank test.
The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins (2022)
Figure 6

Figure 6. Bomanin S6 mediates resilience to verruculogen in the nervous system of Drosophila.

(A-B) tremor rate (A) and survival (B) of flies (20 flies per condition) overexpressing Tl[10B] in neurons compared to wild-type after injection of verruculogen. (A) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. elav> UAS-Toll10B, **p=0.002.

(C-D) tremor rate (C) and survival (D) of flies (20 flies per condition) overexpressing Tl[10B] in glia compared to wild-type after injection of verruculogen. (C) Each dot corresponds to the tremor rate measured in a batch of 20 flies; tremor rate wt vs. repo> UAS-Toll10B, **p=0.002. (D) Survival wt V vs. repo> UAS-Toll10B V, **p=0.005.

(E-G) recovery time from tremor (E,F) and survival (G) of single flies overexpressing BomS6 ubiquitously (E,G) or in neurons (F-G) (biological replicates) compared to wild-type after injection of verruculogen; in (G) the inset represents the survival of vehicle control groups.

(H-I) recovery time from tremor (H) and survival (I) of single flies overexpressing BomS6 in glia (pooled data from n=3 experiments, biological replicates) compared to wild-type after injection of verruculogen; in (I) the inset represents the survival of vehicle control groups. Recovery time (H) repo>mCherry vs. repo>BomS6, p=0.058, survival (I) repo>mCherry V vs. repo>BomS6 V, *p=0.016.

(J-M) expression of BomS4 (J) and BomS6 (K) in head after verruculogen powder challenge, and BomS4 (L) and BomS6 (M) in head after A. fumigatus (Af), restrictocin or M. luteus injection (pooled data from n=3 experiments, biological replicates).

Data Information: in (A,C,E,F,H,J-M), the middle bar of box plots represents the median and the upper and lower limits of boxes indicate respectively the first and third quartiles; the whiskers define the minima and maxima; data were analyzed using the Mann-Whitney statistical test. Survival curves were analyzed using the LogRank test. *<0.05, **<0.01, ****p < 0.0001, and NS: not significant. In this figure, concentration of injected verruculogen or restrictocin was 1 mg/ml.

Expanded View figure legends
Tudor Staphylococcal Nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis (2021)
Figure 1

Figure 1. Tandem affinity purification and characterization of the Arabidopsis TSN2-interacting proteins.

A, Schematic illustration of the expression cassette in TAPa vector. The vector allows translational fusion of TSN or GFP at their C-termini to the TAPa tag. The expression is driven by two copies of the cauliflower mosaic virus 35S promoter (2x35S) and a tobacco mosaic virus (TMV) U1 X translational enhancer. The TAPa tag consists of two copies of the protein A IgG-binding domain (IgG-BD), an eight amino acid sequence corresponding to the 3C protease cleavage site (3C), a 6-histidine stretch (His), and nine repeats of the Myc epitope (myc). A Nos terminator (Nos ter) sequence is located downstream of each expression cassette.

B, Immunolocalization of TSN2-TAPa and GFP-TAPa fusion proteins in root cells of 5-d-old seedlings. The seedlings were grown under no stress (NS) conditions (23ºC) or incubated for 40 min at 39ºC (HS) and then immunostained with α-Myc. Scale bars = 10 μm.

C, Schematic representation of three classes of TSN-interacting proteins, i.e. stress-dependent, stress-sensitive and stress-independent interactors, identified upon the comparison of TSN2_NS and TSN2_HS protein pools.

D, Venn diagram showing the comparison of TSN2_NS interactome with human and yeast SG proteomes Jain et al., 2016().

E, Venn diagram showing the comparison between TSN2_NS and TSN2_HS protein pools. TSN2-interacting proteins are divided into three classes: HS-sensitive, HS-independent and HS-dependent. Within each class, the proteins are further classified into two groups. Group 1 contains known human or yeast SG proteins (Jain et al., 2016), including SG remodelers (marked in grey color), whereas group 2 represents components of recently isolated Arabidopsis RBP47-SG proteome (Kosmacz et al., 2019). The full lists of TSN2-interacting proteins, including previously uncharacterized and potentially novel SG components not belonging to either group 1 or group 2, are provided in Dataset EV1.

F, Frequency of RBPs and proteins with prion-like domains or ATPase activity found in TSN2_NS and TSN2_HS protein pools in comparison with yeast and human SG proteomes Jain et al., 2016().
Tudor Staphylococcal Nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis (2021)
Figure 3

Figure 3. TSN2 and its interactors are localized in heat-induced SGs.

A, A list of TSN-interacting proteins selected for the co-localization analysis.

B, Co-localization of RFP-TSN2 (red) with GFP-ADH2 (negative control) and GFP-UBP1 (positive control) in N. benthamiana protoplasts incubated under 23°C (NS) or at 39ºC for 40 min (HS). Scale bars = 5 μm.

C, Pearson and Spearman coefficients (rp and rs, respectively) of co-localization (PSC) of RFP-TSN2 with individual GFP-tagged TSN-interacting proteins listed in A and with both negative and positive control proteins (denoted by red arrowheads) under HS. Data represent means ± SD of at least 5 replicate measurements from three independent experiments.

D, BiFC between cYFP-TSN2 and nYFP-TSN-interacting proteins in N. benthamiana leaf cells or protoplasts after HS (39ºC for 40 min). BiFC analysis of cYFP-TSN2 and nYFP-TSN-interacting proteins (TIPs) with empty vectors (EV) encoding nYFP and cYFP, respectively was used as a negative control. Only one representative example of BiFC between cYFP and nYFP-TIP is shown. Scale bars = 5 μm.
Tudor Staphylococcal Nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis (2021)
Figure 4

Figure 4. The highly disordered region of TSN2 is required for interaction with SG proteins.

A, B, % IDR (A) and propensity for LLPS (B) in TSN2_NS and TSN2_HS interactomes versus corresponding GFP-TAPa controls (C_NS and C-HS) using IUPred and PSPredictor algorithms, respectively. Upper and lower quartiles, medians and extreme points are shown. The number of protein sequences included to the analyses was 566, 277, 995 and 149 for C_NS, TSN2_NS, C_HS and TSN2_HS, respectively. P values denote statistically significant differences for comparisons to controls (two-tailed t-test).

C, Disorder profiles of TSN2 generated by PONDR-VLXT, PONDR-VL3, PONDR-VSL2, IUPred-short, IUPred-long and PONDR-FIT and a consensus disorder profile (based on mean values of six predictors). SN, staphylococcal nuclease region composed of four N-terminally situated SN domains. C-terminally situated Tudor region is composed of the domain of the same name and a partial SN domain.

D, BiFC between cYFP-TSN2 (full-length), cYFP-SN or cYFP-Tudor and nYFP-TSN-interacting proteins in N. benthamiana protoplasts after HS (39ºC for 40 min). Scale bars = 10 μm. Boxplots show quantification of the reconstituted YFP signal. AU, arbitrary units. Upper and lower box boundaries represent the first and third quantiles, respectively, horizontal lines mark the median, and whiskers mark the highest and lowest values. Three independent experiments, each containing seven individual measurements, were performed. ***P < 0.001 vs Tudor (one-way ANOVA).
Tudor Staphylococcal Nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis (2021)
Figure 5

Figure 5. Interactomes of Arabidopsis TSN1 and TSN2 largely overlap.

A, Venn diagram showing the overlap between TSN1 and TSN2 interactomes isolated by TAPa from Arabidopsis plants grown under NS conditions. Common interactors of TSN1 and TSN2 are classified into three groups: (1) homologues of human and/or yeast SG proteins, (2) proteins constituting recently isolated Arabidopsis RBP47-SG proteome (Kosmacz et al. 2019), and (3) novel plant SG components validated in Fig. 3, EV3 and EV4. The full lists of TSN1- and TSN2-interacting proteins, including as yet uncharacterized and potentially novel SG components not belonging to any of the three groups, are provided in Dataset EV1.

B, Co-immunoprecipitation (Co-Ip) of the two TSN isoforms and RH12 in protein extracts prepared from N. benthamiana leaves agro-infiltrated with GFP-TSN1 or GFP-TSN2 and Myc-RH12. Free GFP was used as a negative control. Input and Co-Ip fractions were analyzed by immunoblotting using α-Myc and α-GFP.

C, Localization of RH12 in root cells of 5-d-old Arabidopsis seedlings expressing GFP-RH12 under control of the native promoter. The seedlings were grown under 23°C (NS) or incubated at 39°C for 60 min (HS). Scale bars = 10 μm.