Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: Internalized ANXA11 fibrils accumulate in lysosomes and trigger lysosomal membrane permeabilization (LMP) in neurons. a Representative confocal microscopy images of SH-SY5Y cells treated with Alexa Fluor 488-labeled ANXA11 preformed fibrils (PFFs-AF488, green) for 24 h. Cytoskeleton was stained with phalloidin (white) and nuclei with DAPI (blue). The images show intracellular localization of ANXA11 aggregates. b Flow cytometry analysis quantifying the uptake of ANXA11 PFFs by SH-SY5Y cells after 24 h treatment. c Confocal images showing the subcellular localization of internalized ANXA11 PFFs-AF647 (red) and the lysosomal marker LAMP1 (green). d Fluorescence intensity profile analysis along the white line drawn in ( c ), demonstrating the colocalization of ANXA11 PFFs with lysosomes. e Transmission electron microscopy images of SH-SY5Y cells treated with ANXA11 PFFs. Arrows indicate electron-dense aggregates within lysosomes and damaged mitochondria with disrupted cristae. f Western blot analysis of cathepsins (CSTA, CTSB, and CTSD) in cytosolic versus whole-cell lysate fractions. The presence of mature cathepsins in the cytosolic fraction upon PFFs treatment indicates lysosomal leakage. GAPDH was used as a loading control. g Representative images of Lysotracker Red staining in SH-SY5Y cells treated with or without ANXA11 PFFs, assessing lysosomal acidity and integrity. h Quantification of Lysotracker Red fluorescence intensity shown in ( g ). i Immunofluorescence images showing the recruitment of galectin-3 (GAL3, purple), a marker of lysosomal rupture, to LAMP1-positive lysosomes (green) containing ANXA11 PFFs-AF647 (red). j Fluorescence intensity profile analysis along the line in ( i ), showing the colocalization of GAL3 with ruptured lysosomes containing ANXA11 PFFs. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( h ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Membrane, Confocal Microscopy, Labeling, Staining, Flow Cytometry, Marker, Fluorescence, Transmission Assay, Electron Microscopy, Western Blot, Control, Immunofluorescence

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: ANXA11 PFFs trigger RB1CC1-dependent lysophagy and autophagic flux. a Western blot analysis of LC3 levels in SH-SY5Y cells treated with or without ANXA11 PFFs. GAPDH served as a loading control. b , c Quantification of the LC3-II/LC3-I ratio ( b ) and LC3-II/GAPDH ratio ( c ) from the blots in ( a ), indicating increased autophagic activity upon PFFs treatment. d Representative confocal images showing the recruitment of LC3 (purple) to LAMP1-positive lysosomes (green) containing ANXA11 PFFs-AF647 (red). Nuclei were stained with DAPI (blue). e Fluorescence intensity profile analysis along the white line in ( d ), confirming the colocalization of ANXA11 PFFs, LAMP1, and LC3. f Transmission electron microscopy images showing double-membrane autophagosomes engulfing electron-dense lysosomal contents (arrows), a hallmark of lysophagy, in cells treated with ANXA11 PFFs. g Western blot confirming the knockdown efficiency of RB1CC1 in SH-SY5Y cells. h Western blot analysis of LC3 turnover in Control KD and RB1CC1 KD cells treated with or without ANXA11 PFFs. i , j Quantification of LC3-II/I ( i ) and LC3-II/GAPDH ( j ) ratios from ( h ), showing that RB1CC1 knockdown prevents the PFF-induced LC3 lipidation. k Immunofluorescence staining of galectin-3 (GAL3, green) in Control KD and RB1CC1 KD cells treated with ANXA11 PFFs-AF647 (red). l Quantification of relative GAL3 fluorescence intensity in ( k ). The increase in GAL3 signal in RB1CC1 KD cells indicates an accumulation of ruptured lysosomes due to impaired clearance. m Analysis of autophagic flux using the mCherry-EGFP-LC3 reporter. Control KD cells treated with PFFs show red puncta (indicating autolysosomes, where GFP is quenched by acidity), whereas RB1CC1 KD cells retain yellow/green fluorescence. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, c ) or one-way ANOVA with Tukey’s post hoc test ( i, j, l ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Western Blot, Control, Activity Assay, Staining, Fluorescence, Transmission Assay, Electron Microscopy, Membrane, Knockdown, Immunofluorescence

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: The FTLD/ALS-linked ANXA11 D40G mutant exhibits enhanced lysosomal disruption and seeding capacity. a Representative immunofluorescence images showing the accumulation of GAL3 (purple) puncta in SH-SY5Y cells treated with WT or D40G mutant ANXA11 PFFs-AF647 (red). Lysosomes were labeled with LAMP1 (green) and nuclei with DAPI (blue). b Quantification of relative GAL3 fluorescence intensity from ( a ). The results indicate that D40G fibrils induce more severe lysosomal membrane rupture compared to WT fibrils. c Representative confocal images displaying the recruitment of LC3 (purple) to LAMP1-positive lysosomes (green) containing internalized WT or D40G ANXA11 PFFs (red). d Quantification of relative LC3 fluorescence intensity from ( c ), showing enhanced autophagic response to D40G-induced damage. e Representative images of LysoTracker Red staining in cells treated with WT or D40G ANXA11 PFFs to assess lysosomal integrity. f Quantification of LysoTracker Red fluorescence intensity from ( e ). The D40G mutant caused a more pronounced reduction in lysosomal acidity. g Representative images of HEK293T cells expressing ANXA11-GFP treated with WT or D40G ANXA11 seeds, showing induced intracellular aggregation. h Quantification of the percentage of cells containing GFP-positive inclusions from ( g ). Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, d ) or one-way ANOVA with Tukey’s post hoc test ( f, h ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Mutagenesis, Disruption, Immunofluorescence, Labeling, Fluorescence, Membrane, Staining, Expressing

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: Lysosomal damage by ANXA11 fibrils activates the p38/MK2/HSP27 signaling axis to promote lysophagy. a Western blot analysis of phosphorylated HSP27 (p-HSP27) and total HSP27 expression in SH-SY5Y cells treated with or without ANXA11 PFFs. GAPDH was used as a loading control. b , c Quantification of p-HSP27 levels normalized to total HSP27 ( b ) and GAPDH ( c ) from ( a ). d Representative immunofluorescence images showing the formation of HSP27 puncta (purple) and their colocalization with ANXA11 PFFs-AF647 (red). Phalloidin (green) stains F-actin and DAPI (blue) stains nuclei. Arrows indicate colocalization sites. e Western blot analysis of phosphorylated p38 MAPK (p-p38, T180/Y182) and total p38 MAPK levels. f , g Quantification of p-p38 levels normalized to total p38 ( f ) and GAPDH ( g ) from ( e ). h Western blot analysis of p-HSP27 and p-p38 MAPK levels in cells treated with ANXA11 PFFs in the presence or absence of the p38 MAPK inhibitor SB203580. i – l Quantification of relative protein levels from ( h ), showing that p38 inhibition effectively blocks the phosphorylation of HSP27. m Western blot analysis of phosphorylated MK2 (p-MK2, Thr334) and total MK2 levels. n , o Quantification of p-MK2 levels normalized to total MK2 ( n ) and GAPDH ( o ) from ( m ). p Western blot analysis of the p38/MK2/HSP27 pathway in cells treated with ANXA11 PFFs in the presence or absence of the MK2 inhibitor PF-3644022. q–v Quantification of phosphorylation levels from ( p ). Inhibition of MK2 significantly reduces the phosphorylation of HSP27 and upstream signaling components. w Representative immunofluorescence images of galectin-3 (GAL3, green) in cells treated with ANXA11 PFFs-AF647 (red) in the presence or absence of the p38 inhibitor PF-3644022. The increase in GAL3 puncta upon inhibition indicates exacerbated lysosomal damage due to impaired protective signaling. Data are presented as mean ± SEM. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, c, f, g, i, j, k, l, n, o, q, r, s, t, u, v ). Exact P -values are indicated in the corresponding graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Inhibition, Phospho-proteomics

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: Lysosomal rupture and autophagy deficiency synergistically promote the cytoplasmic seeding and neuronal propagation of ANXA11 aggregates. a Representative confocal microscopy images of SH-SY5Y cells stably expressing ANXA11-GFP. Cells were treated with ANXA11 PFFs (5 µg/mL) for 24 h, followed by exposure to the lysosomotropic agent LLOMe (1 mM) to induce lysosomal membrane permeabilization. Nuclei were counterstained with DAPI (blue). Note the marked increase in intracellular ANXA11-GFP inclusions in the combined treatment group. b Quantification of the percentage of cells containing ANXA11-GFP positive inclusions from the experiment in ( a ). c , d Dot blot analysis of cell lysates fractionated into Triton X-100 soluble and insoluble fractions. Membranes were probed with the conformation-dependent antibodies OC (targeting amyloid fibrils) ( c ) and A11 (targeting prefibrillar oligomers) ( d ). e , f Densitometric quantification of the relative signal intensity of OC ( e ) and A11 ( f ) in the insoluble fractions, normalized to controls. g Schematic illustration of the neuron-to-neuron transmission assay. Donor iPSC-derived neurons were loaded with AF647-labeled ANXA11 PFFs, washed to remove extracellular fibrils, and the conditioned medium/lysate was transferred to recipient neurons. Recipient cells were subsequently treated with LLOMe or BafA1 to assess the impact of lysosomal integrity and autophagic flux on aggregate propagation. h Representative confocal immunofluorescence images of recipient iPSC-derived neurons stained for the neuronal marker MAP2 (green). Internalized intracellular ANXA11 PFFs-AF647 are shown in red. The confocal imaging ensures that the red fluorescent signals represent genuine intracellular aggregates rather than extracellular membrane-bound particles. i Quantification of the percentage of recipient neurons containing ANXA11 PFFs-positive aggregates. The data demonstrate that both lysosomal rupture (LLOMe) and autophagy inhibition (BafA1) significantly enhance the accumulation of pathological seeds in recipient neurons. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( b, e, f, i ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Confocal Microscopy, Stable Transfection, Expressing, Membrane, Dot Blot, Transmission Assay, Derivative Assay, Labeling, Immunofluorescence, Staining, Marker, Imaging, Inhibition

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: ANXA11 fibril-induced lysosomal rupture triggers a cathepsin B-dependent cascade of mitochondrial dysfunction, oxidative stress, and apoptosis. a Schematic illustration of the proposed cytotoxic mechanism: lysosomal membrane permeabilization (LMP) induced by ANXA11 fibrils leads to the cytosolic release of cathepsin B, which subsequently triggers mitochondrial dysfunction, ROS accumulation, and Caspase-3-mediated apoptosis. b Representative transmission electron microscopy images of SH-SY5Y cells. Control cells exhibit healthy mitochondria with intact cristae, whereas ANXA11 PFFs-treated cells display swollen mitochondria with disrupted cristae (black arrows). c Representative confocal images showing mitochondrial morphology. Cells were stained for the mitochondrial outer membrane marker TOM20 (green), F-actin (Phalloidin, white), and nuclei (DAPI, blue). ANXA11 PFFs (red) induce mitochondrial fragmentation and network disruption. d Flow cytometric analysis of intracellular reactive oxygen species (ROS) levels using the DCFH-DA probe. Treatment with the specific Cathepsin B inhibitor CA-074Me attenuates the ROS surge induced by ANXA11 PFFs. e Representative fluorescence images of ROS generation in SH-SY5Y cells and iPSC-derived neurons. The green signal represents the fluorescence emitted by the oxidized DCF probe, visually indicating the levels of intracellular ROS. These images confirm the induction of oxidative stress upon PFFs exposure and its mitigation by lysosomal protection. f Assessment of mitochondrial membrane potential using JC-1 staining. In healthy cells, JC-1 forms red aggregates; in depolarized mitochondria, it exists as green monomers. ANXA11 PFFs induce a shift toward green fluorescence (depolarization), which is prevented by CA-074Me. g Quantification of the JC-1 aggregate-to-monomer fluorescence ratio from ( f ). h Western blot analysis of apoptotic signaling. ANXA11 PFFs increase levels of cleaved Caspase-3 (Asp175). This activation is partially blocked by inhibiting cathepsin B (CA-074Me) or autophagic flux (Bafilomycin A1). GAPDH served as a loading control. i, j Densitometric quantification of total Caspase-3 ( i ) and cleaved Caspase-3 ( j ) levels normalized to GAPDH. k Flow cytometric quantification of apoptosis using Annexin V-PE/Propidium Iodide (PI) double staining. The proportion of early (Q3) and late (Q2) apoptotic cells was significantly increased by ANXA11 PFFs but rescued by CA-074Me treatment. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( g , i , j ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Control, Staining, Marker, Disruption, Fluorescence, Derivative Assay, Western Blot, Activation Assay, Double Staining

Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: The FTLD/ALS-linked D40G mutation exacerbates lysosomal rupture and drives a hyperactive lysophagic response in human cerebral organoids. a Schematic timeline illustrating the generation of human iPSC-derived cerebral organoids and the experimental window for ANXA11 PFFs treatment (Day 40 +). b Multiplex immunofluorescence characterization of Day 40 organoids. The tissue exhibits a complex cytoarchitecture containing PAX6 + neural progenitors (red), TUJ1 + early neurons (purple), MAP2 + mature neurons (green), TBR1 + deep-layer cortical neurons (orange), and NeuN + neuronal nuclei (white/cyan). Nuclei are counterstained with DAPI (blue). c Representative confocal images showing the internalization of AF647-labeled ANXA11 PFFs (red) into the organoid parenchyma and their specific colocalization with LAMP1 + lysosomes (green). d Assessment of lysosomal membrane permeabilization (LMP). Organoids were treated with WT or D40G ANXA11 PFFs. Note the widespread accumulation of galectin-3 (GAL3, purple) puncta on lysosomes in the D40G group, indicating severe membrane rupture. e , f Fluorescence intensity profile analyses along the white lines in ( d ), demonstrating the recruitment of GAL3 to lysosomes containing WT ( e ) and D40G ( f ) fibrils. The D40G profile shows a higher degree of signal overlap. g Evaluation of the lysophagic response. Immunostaining revealed the recruitment of the autophagy marker LC3 (purple) to LAMP1 + lysosomes (green) containing ANXA11 aggregates (red). h , i Fluorescence intensity profile analyses along the white lines in ( g ) , confirming the colocalization of LC3 with ruptured lysosomes in WT ( h ) and D40G ( i ) treated organoids. j Western blot analysis of the p38/MK2/HSP27 signaling axis in organoid lysates following treatment with WT or D40G ANXA11 PFFs. GAPDH served as a loading control. k – p Densitometric quantification of the phosphorylation levels of HSP27 ( k , l ), p38 MAPK ( m , n ), and MK2 ( o , p ). The data indicate that the D40G mutant induces a significantly more robust activation of this protective stress response pathway compared to WT fibrils. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( k , l , m , n , o , p ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Mutagenesis, Derivative Assay, Multiplex Assay, Immunofluorescence, Labeling, Membrane, Fluorescence, Immunostaining, Marker, Western Blot, Control, Phospho-proteomics, Activation Assay

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: Nuclear cathepsin translocation occurs already from 8 hpi, is independent of cathepsin activity and does not result in increased Histone 3 cleavage. a) Time course of nuclear cathepsin activity. Nuclei and S Tm were enriched by sequential 50 x g and 8,000 x g centrifugation steps, respectively. RAW264.7 cells were infected with wildtype S Tm 14028s and treated with DCG04-Bodipy-FLike (5μM) for 4 hours prior to harvesting. Samples were separated by SDS-PAGE and visualised using a fluorescent scanner (Ex 405 nm/Em 520 nm), followed by immunoblotting for the soluble cytoplasmic protein GAPDH, the bacterial protein RpoD and Coomassie staining for histones as a loading control for nuclear extracts. L = lysatome, N = nucleome, S Tm = S Tm enriched fraction. Experiment was performed once. b) RAW264.7 cells were infected with wildtype S Tm at MOI 100:1 and harvested at 20 hpi. Whole cell lysates were immunoblotted with the CtsL specific cleavage product of H3 (H3.cs1) 16 and histone 3 (H3) as loading control. The experiment was performed in biological duplicate per condition and were analysed in adjacent lanes. c) Nuclear extracts from RAW264.7 cells either mock infected, infected with wildtype S Tm or heat killed wildtype ( S Tm-HK) for 20 hours. CA-074-Me was present throughout the infection experiment at the indicated concentrations. Nuclear extracts were analysed by immunoblot for CtsB (1:2,000), Lamin A (1:5,000) and histones by Coommassie stain (see for second replicate data).

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Translocation Assay, Activity Assay, Centrifugation, Infection, SDS Page, Western Blot, Staining

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: a ) Nuclei were extracted from RAW264.7 cells treated with DCG04-Boclicky-TAMRA (5 μM) for 2 hours prior to harvest at 20 hpi with wildtype (Wt), a SPI-2 mutant (Δ ssaV ) or heat killed wildtype bacteria (Wt HK) (MOI = 100:1). Formaldehyde fixed nuclei were subsequently counterstained with Hoechst 33342 and analysed by flow cytometry. Cells in G1, S or G2 phase of the cell cycle are separated by Hoechst 33342 staining on the x-axis. Cathepsin activity (DCG04-Boclicky-TAMRA) is indicated by the % of total DCG04-Boclicky-TAMRA positive nuclei in either sub-G1 or G1/S/G2 or combined total of sub-G1 and G1/2/G2. Data combining all biological replicates from two independent experiments can be seen in Extended Data Fig. 4. b ) Pyroptotic cell death was assessed by quantifying LDH release into culture supernatants of RAW264.7 cells infected with wildtype, SPI-2 (Δ ssaV ) and the effector deletion (Δ sifA) mutants in the presence of 25 μM CA-074-Me or DMSO solvent control at 19 hpi. The Δ sifA mutant has a strong replication defect and readily escapes into the cytoplasm, hyperactivating Caspase-11 dependent cell death . (n) denotes biologically independent samples combined from three independent experiments (batches); each batch contained a minimum of 4 biological replicate wells per condition. Data represents the % LDH release per condition relative maximum LDH release (see Methods). Box plots are depicted as in Fig. 3C. An unpaired t-test (two-sided) was used to calculate p . c ) BMDMs were infected with wild-type S Tm (MOI 100:1), followed by incubation in the presence of cathepsin inhibitor CA-074-Me at the indicated concentrations. At the indicated hpi’s, cell death was measured as the % of LDH released into culture supernatants. Data points represent the mean and error bars indicate the 95% CI. (n) denotes biologically independent samples. Time points 0, 10 and 14 hours are derived from three biological replicates per condition (n=3 per condition) from a single batch, whereas the 18-hour time point contains combined data from 3 or more independent experiments (batches), each batch containing 3-4 biological replicates per condition (DMSO n=30; CA-074-Me (12.5 μM) n=28; CA-074-Me (25 μM) n=13). d ) related to a ), wildtype, caspase-11/1 -/-, caspase-11 -/-, NLRP3 -/-, NLRC4 -/- BMDMs were infected with wildtype S Tm (MOI 100:1), followed by incubation in the presence of cathepsin inhibitor CA-074-Me (12.5 μM) for 18 hpi. The % LDH released into culture supernatants was measured 18 hours post-infection. n denotes biologically independent samples combined from >3 (wildtype) and 3 (mutant genotypes) independent experiments (batches), each batch containing 2-4 biological replicates per condition. A two-sided unpaired t-test was used to calculate p . e ) RAW264.7 cell were transfected with LPS with Fugene (Promega) for 20 hours. Pyroptotic cell death was assessed by quantifying LDH release into culture supernatants in the presence of 25 μM CA-074-Me relative to a DMSO solvent control. (n) denotes biologically independent samples combined from three independent experiments (batches), each batch consisting of a minimum of 3 biological replicate wells per condition. Boxplots are depicted as in Fig. 3C. A two-sided unpaired t-test was used to calculate p .

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Mutagenesis, Flow Cytometry, Staining, Activity Assay, Infection, Incubation, Derivative Assay, Transfection

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: S Tm growth is unaffected by the cathepsin inhibitor CA-074-Me in batch culture conditions. S Tm 14028s growth was measured in the presence of the selective cathepsin inhibitor CA-074-Me in LB 6, 12.5 and 25 μM and DMSO solvent controls. Drug concentrations used are indicated. Experiment was performed in biological triplicate.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques:

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: CA-074-Me inhibits cathepsins and reduces expression of proinflammatory proteins. a) 2D-TPP of RAW264.7 cells infected with wildtype S Tm 14028s for 20 hours in the presence of increasing concentrations of CA-074-Me (1-100 μM). To assess CA-074-Me specificity, we applied 2-dimensional Thermal Proteome Profiling (2D-TPP). 2D-TPP enables proteome assessment of protein ligand binding based on the principle that ligand-bound proteins are more thermally stable than proteins not bound to a ligand 20,21 , and in parallel provides information of proteome-wide protein abundance. We subjected RAW264.7 cells infected with wildtype S Tm to increasing doses of CA-074-Me and compared proteome-wide thermostabilisation relative to solvent controlled cells (see methods). Increased (blue) or decreased fold changes are calculated by normalising the abundance of each protein relative to the abundance in the DMSO vehicle per temperature. Protein stabilization (red framed) is demonstrated by increasing (blue) fold changes with respect to increasing temperature (top to bottom) as well as with increasing drug concentrations (left to right). Changes in protein expression (green framed) can be detected if abundance changes are already evident from low temperatures i.e. before proteins melt. All cathepsins detected by 2D-TPP are displayed. CtsA and CtsD are serine and aspartic-acid proteases, respectively, and are therefore not targeted by CA-074-Me and serve as negative controls. Contrary to evidence demonstrating CA-074-Me targets CtsL 35 , we failed to detect its stabilisation in our experiment. Cathepsins are arranged in the top row, followed by caspases and other cellular proteases in descending order. Note Caspase-1 stability was not affected by CA-074-Me, in line with previous reports 22 . Additionally, CA-074-Me reduced IL-1b expression as previously shown 35,36 . 2D-TPP data can be found in . b) Caspase-11-like proteolysis activity was assessed using purified active Caspase-11 and measuring cleavage of the fluorescent substrate AcLEHD-afc as previously described 37 . Caspase-11-like proteolytic activity was assessed in the presence of indicated concentrations of CA-074-Me and the Caspase-11 inhibitor Z-LEHD-FMK as a positive control. Activity is expressed as the % of AcLEHD-afc cleavage relative to the DMSO solvent control. Data is combined from n = 3 biologically independent experiments; error bars depict standard deviation and the center value denotes the mean. c) 2D-TPP profiles of inflammation related proteins reduced in abundance upon CA-074-Me treatment. Data presented as in (a).

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Expressing, Infection, Ligand Binding Assay, Activity Assay, Purification, Positive Control, Standard Deviation

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: Rapid cell death induced by SPI-1 ON S Tm is independent of cathepsin activity. Wildtype and Caspase-1/11 -/- BMDMs were infected with wildtype S Tm and a SPI-1 mutant (Δ prgK ), late-exponential growing S Tm (SPI-1 ON) in the presence of CA-074-Me at the indicated concentrations. LDH release was measured 1.5 hpi. Boxplots are depicted as in . n denotes the number of biologically independent samples.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Activity Assay, Infection, Mutagenesis

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: CA-074-Me does not affect Caspase-11 expression or processing. Wildtype BMDMs were infected with wildtype S Tm for 20 hours in the presence of CA-074-Me (25 μM) or DMSO control. Lysates were probed for Caspase-11 by immunoblot and using GAPDH as a loading control. Experiment was performed once.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Expressing, Infection, Western Blot

Journal: Nature microbiology

Article Title: Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

doi: 10.1038/s41564-020-0736-7

Figure Lengend Snippet: a ) Two-dimensional thermal proteome profiling (2D-TPP) of RAW264.7 cells infected with wildtype S Tm for 20 hours in the presence of increasing concentrations of CA-074-Me post S Tm uptake. Heatmap of Gasdermin D shows decreased abundance (evident from decrease in protein abundance at lower temperatures, before protein melts) with increasing CA-074-Me concentrations. Key is presented to the left of the heatmap; for each protein and temperature, the signal intensity was normalized to the DMSO vehicle control. 2D-TPP data can be found in Supplementary Table 6 and Extended Data Fig. 6. b ) BMDMs were infected with wildtype Salmonella 14028s (20 hpi) and cell lysates analysed by immunoblot for Gasdermin-D and GAPDH as a loading control. A replicate blot from an independent experiment is located in the Supplementary Information (SI). c ) BMDMs infected with wildtype S Tm (MOI = 100:1) were treated with CA-074-Me (12.5 or 25 μM) or DMSO control at 1 or 9 hpi. Box plots are depicted as in Fig. 3C. A two-sided unpaired t-test was used to calculate p . n denotes the combined data from 3 independent experiments (batch), each batch containing 3-4 biological replicates per condition. d ) Schematic depicting Stefin B constructs used to generate iMAC cell lines targeting Stefin B to the nucleus (Nuc) with a 3x NLS sequence or expressed in the cytoplasm (Cyto). Stefin B potently inhibits several cathepsins (e.g. CtsL, CtsS, CtsH and to a lesser extent CtsB) and has been previously used to block cathepsin activity and overcome cathepsin redundancy . Retrovirally transduced iMACs were FACS sorted for low (Lo) and high (Hi) expression of the Stefin B fusion proteins, which was then verified by immunoblot (Extended Data Fig. 10a) as well as localisation of the Stefin B fusion protein by microscopy (Extended Data Fig. 10b). We tested the sensitivity of Stefin B expressing cells to conditions known to activate the canonical inflammasome (i.e. nigericin + LPS) and the lysosome destablising agent, LLoMe, which triggers a distinct form of cell death. In line with our previous observations that cathepsin activity is not required for canonical inflammasome activation, cells with nuclear-expressed Stefin B were refractory to nigericin+LPS treatment, but also to LLoMe induced cell death (Extended Data Fig. 10c, d). e ) iMACs expressing Stefin B targeted to the nucleus, or expressed in the cytoplasm, were infected with wildtype S Tm (MOI = 100:1; 19 hpi). LDH release was quantified as in Fig 5 b . Sample labels are as described in d . Box plots are depicted as in Fig 3 c . A one sided unpaired t-test was used to calculate p . n denotes the combined data from 4 independent experiments (batches), each batch containing >8 biological replicates per condition.

Article Snippet: In brief, 10 U/100 μL of recombinant Caspase-11 (mouse, Enzo life sciences; BML-SE155-5000) in caspase activity buffer (200 mM NaCl, 50 mM HEPES pH 8.0, 50 mM KCl, 10 mM DTT) supplemented with 100 μMAcLEHD-afc (Santa Cruz; sc-311277) was incubated in the presence of CA-074-Me, E64d (Sigma; E8640) and the Caspase-9/11 inhibitor Z-LEHD-FMK (Abcam; ab142026) or a DMSO solvent control as the indicated concentrations on the figure legend.

Techniques: Infection, Western Blot, Construct, Sequencing, Blocking Assay, Activity Assay, Expressing, Microscopy, Activation Assay