EMBO/soda-nel huggingface dataset:
GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis (2022)
Figure 5

Figure 5. PMA-induced NETosis requires NADPH oxidase activity, but not GSDMD.

(A-H) LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) (A-D), nigericin (100 mins) (E-G), or TcDA (3 hr) (H). Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30) (A), and extracellular release of β-actin and GAPDH (C-F, H). * with Western blots indicates non-specific bands. (B, G) PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).
GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis (2022)
Figure 6

Figure 6. Caspase-1 and GSDMD drive S. Typhimurium-induced neutrophil pyroptosis.

(A-H) Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs taken at different time points were stained with DAPI (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM. (B) Representative confocal micrographs of untreated (Mock), S. Typhimurium-infected or PMA-stimulated wildtype BMNs stained for neutrophil elastase (Red) and SYTOX Green (Green) (Scale bar: 10 μM). (C, D) Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. (E) Cell death was measured by LDH assay 3 h post-infection. (F) IL-1β secretion levels were determined by Luminex assay 1 hr post-infection. (G) Representative confocal micrographs of neutrophils stained with DAPI (dark blue nuclei) and SYTOX Green (light blue) taken at different time points post-infection (scale bar: 20 μM). (H) PMP was assessed by measurement of SYTOX Green incorporation over time.

Data information: In E, F, H, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). S. Typh.: S. Typhimurium.

Expanded View Figure Legends
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes (2011)
figf1

(a) Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP–LC3 and H2B–mCherry. The arrow marks recruitment of LC3 to the entotic vacuole (see Supplementary Movie S1). Scale bar, 15 μm. (b) Images of MCF10A cells expressing GFP–LC3G120A and mCherry–LC3; note no recruitment of GFP–LC3G120A to the entotic vacuole. Scale bar, 15 μm. (c) LC3 is recruited from the host-cell cytosol onto the entotic vacuole membrane. Data points are the mean fluorescence intensity of GFP–LC3 on vacuoles versus cytoplasm for two independent cell-in-cell structures (see Supplementary Movie S2). (d) Correlative video-light–electron microscopy of an MCF10A GFP–LC3 cell-in-cell structure. Cells were imaged by time-lapse microscopy, followed by fixation after LC3 recruitment (see Supplementary Movie S3). Top left, post-fixation images of phase-contrast and GFP-fluorescence signals were taken demonstrating LC3 recruitment. Bottom left and right, electron microscopy (EM) of the same cell shows the entotic vacuole is a single membrane (arrow in right image, which is a higher magnification of the area outlined in the bottom left image). (e) Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of the internalized cell. Data represent mean±s.e.m. from at least three independent experiments;
n, total number of death events;
***P0.0001. (f,g) Time-lapse images of MCF10A cell-in-cell structures expressing either 2×FYVE–mCherry (red) and GFP–LC3 (green; f) or Lamp1–GFP (green) and mCherry–LC3 (red; g). The arrows indicate recruitment to the entotic vacuole (see Supplementary Movies S4 and S5). Times are indicated as h:min:s (a,f) or min (g). Scale bars, 5 μm. (h) Representative images of cell-in-cell structures in which only host cells express Cathepsin-B–mCherry. Left, a live inner cell with no Cathepsin B in the vacuole (arrow); middle, a live internalized cell with Cathepsin B from the host inside of the vacuole (arrowhead); right, a dead internalized cell with Cathepsin B from the host throughout the corpse (asterisk). Scale bar, 15 μm. (i) Five representative timings of GFP–LC3 recruitment to entotic vacuoles (green bar) and death of internalized cells (cross) from time-lapse microscopy. (j) Initiation and duration of 2×FYVE domain (red bar,
n=11), LC3 (green bar,
n=20) and Lamp1 (orange bar,
n=7) recruitment to entotic vacuoles, determined using double-expressing cells shown in
f,g. Error bars represent s.d.
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes (2011)
figf2

(a) Left, model depicting the multiple cell fates of internalized cells; the arrow thickness represents the relative frequency of events. Right, images of MCF10A cell-in-cell structures (1, host cell; 2, internalized cell) that show a cell death (top images) or release (bottom images) event. Scale bar, 15 μm. (b) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition. Data represent the mean from at least three independent experiments;
n, total number of structures analysed;
***P0.007,
**P0.004,
*P0.01. (c,d) Quantification of LC3 recruitment (c), and cell fate (d), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments;
n, total number of structures analysed;
*P0.01,
**P0.004. (e) Left, quantification of type of internalized cell death after autophagy inhibition. Data represent the mean of three independent experiments;
n, total death events analysed;
**P0.03. Right, representative images of non-apoptotic and apoptotic death of an internalized cell; the arrow points to fragmented apoptotic nuclei. Scale bars, 15 μm. (f) Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP–mCherry–LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27;
*P0.03,
**P0.003,
***P0.0004. (g) Quantification of autophagosome and autolysosome area in control MCF10A (n=10) and internalized cell (n=10) cytoplasm imaged by electron microscopy;
**P0.005. (h) Representative electron micrographs of cell-in-cell structures (left) and internalized cell cytoplasm (middle and right) showing autophagosomes (arrow) and autolysosomes (arrowhead).
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes (2011)
figf3

(a) The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells);
*P0.05,
**P0.02. (b) Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments;
**P0.004. (c) Representative images of an MCF10A E7+Bcl2 cell-in-cell structure formed in soft agar. The arrow marks LC3 recruitment around internalized cells. Scale bar, 5 μm. (d) Quantification of MCF10A E7+Bcl2 cell-in-cell structures 48 h after seeding into soft agar (white bars), and colony formation after 2 weeks (grey bars). Data represent mean±s.e.m.from three independent experiments;
*P0.03,
**P0.004,
***P0.001. (e) Representative wells for data in d. The insets show higher magnifications. (f) Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments;
*P0.001. (g) Representative wells for data in f. The insets show higher magnifications. See also Supplementary Movie S6.
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes (2011)
figf4

(a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. (b) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA;
n, number of cells analysed;
***P0.0001, chi-squared test. (c) Apoptotic U937 cells expressing H2B–mCherry phagocytized by J774 macrophages expressing GFP–LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. (d) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells;
***P0.0001, chi-squared test. (e) Correlative light–electron microscopy of an apoptotic phagosome with GFP–LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnification of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus). (f) Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP–LC3 cells;
***P0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3.
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes (2011)
figf5

(a) Representative images of a J774 macrophage expressing GFP–LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. (b) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages;
n, number of macropinosomes analysed;
***P0.0001, chi-squared test. See Supplementary Movie S9. (c) Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP–LC3 incubated with red dextran. Scale bar, 2 μm. (d) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5;
n, number of macropinosomes analysed;
***P0.0001, chi-squared test. (e) J774 GFP–LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10.
Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes (2011)
figf6

(a) A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. (b) Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from a, left arrow). (c) Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. (d) Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes;
bec-1, 9 embryos, 25 phagosomes;
***P0.0001, chi-squared test. (e) Representative DIC images from control- and
bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. (f) Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.;
***P0.0001. (g) Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm.
Dynorphin-Based "Release on Demand" Gene Therapy for Drug-Resistant Temporal Lobe Epilepsy (2019)
Figure 1

Fig. 1: Effect of AAV-pDyn on seizures in mouse and rat models of TLE.

A: Displayed (sc)AAV2-based vector backbones) were packaged in AAV serotype 1 capsids. AAV-ITRs are displayed in grey, ∆ITR refers to the mutated ITR-version of scAAVs. The transgene of AAV-pDyn is a codon-optimized version of the full-length human preprodynorphin cDNA enhanced by a WPRE element. Control vectors carry either a truncated, nonfunctional version of the enhanced GFP gene (AAV-ΔGFP), or its functional counterpart (AAV-eGFP; not displayed). B: Kainic acid mouse model of TLE: Daily EEG recordings obtained from the epileptogenic focus starting from one month after KA injection and spanning the period from 2 days before to 7 days after AAV-pDyn delivery (2x109 gp). C: higher time resolution of the indicated section of the Day +1 in (B), representing a hippocampal paroxysmal discharge (HPD). A generalized seizure is depicted in Fig. EV1 D and E: The characteristic EEG features of this model, secondary generalized seizures (bars) and HPDs (lines) were reduced in number and in duration by AAV-pDyn (red; n = 3 (from day 60); 7 (till day 30) per time-interval), but not by AAV-ΔGFP or after sham treatment (blue; n = 4 (day 90); 5 (day 30 and 60); 6 (before day 30), per time-interval). The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE. Some animals could not be recorded for the entire period due to loss of implant. ** p<0.01; effect of treatment analyzed by two-way ANOVA with Bonferroni correction for both number and time F: Injection of norBNI (20 mg/kg; i.p.) results in a transient reappearance of HPDs immediately and 24 hrs after application. One week after norBNI application (washout) suppression of HPDs was re-established. Data obtained from 4 epileptic animals before (black) and after (red) AAV-pDyn delivery (2x109 gp) are depicted. * p<0.05; ** p<0.01; one-way ANOVA for repeated measures with Friedman post hoc test G: EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self-sustained status epilepticus (SSSE) before (black) and after (red) AAV-pDyn delivery (4x109 gp) are depicted. Spike trains with a frequency of at least 2.5 Hz induced by SSSE were markedly reduced by AAV-pDyn (n=4). * p<0.05 one-way ANOVA for repeated measures with Friedman post hoc test. Data represent mean ± standard error of the mean.
Dynorphin-Based "Release on Demand" Gene Therapy for Drug-Resistant Temporal Lobe Epilepsy (2019)
Figure 2

Fig. 2: Effects of AAV-pDyn on spatial learning and memory

A-I: Spatial learning and memory was tested on the Barnes maze. Quadrant 1 (Q1) contains the target hole (red; A). Unilateral injection of AAV-pDyn (B) or AAV-eGFP (C) into naïve young adult mice (12 weeks age) did not influence the performance as compared to naïve controls (D) when tested 4 weeks after AAV injection. Mice treated two weeks after KA with AAV-pDyn (E,H) performed equally to age-matched naïve controls (D,G) 2 weeks (E) and 5.5 months (H) after treatment. By contrast, animals treated two weeks after KA with AAV-ΔGFP (F,I) gradually lost this ability. Two-way ANOVA revealed significance between AAV-ΔGFP and AAV-pDyn treated groups for interaction 2 weeks (p = 0.0349) and 5.5 months (p = 0.0311) after AAV, respectively, at each time interval and quadrant (p < 0.0001) 2 weeks after AAV. Comparison of AAV-pDyn injected with naïve animals revealed no differences.

J-L: epileptic mice, which were not able to learn the Barnes maze task one month after KA (J, M), AAV-pDyn application restored spatial memory one (K) and 2 months (L) after treatment. Treatment with AAV-ΔGFP did not result in improved memory (N,O). Animal numbers: (B) and (C) n= 9; (D) through (H) n=8; (I) n= 5, note: 3 mice had to be killed due to accelerating seizure activity and resulting weight loss; (J) through (O) n=7; Two-way ANOVA revealed significance for interaction (p = 0.0049) and quadrant (<0.0001) comparing AAV- ΔGFP with AAV-pDyn treated animals at the later time-interval.

Data represent mean ± standard deviation

*** = p<0.001; ** = p<0.01; * = p<0.05 by 1-way ANOVA and Dunnett post hoc test.
Dynorphin-Based "Release on Demand" Gene Therapy for Drug-Resistant Temporal Lobe Epilepsy (2019)
Figure 3

Fig. 3: Distribution and release "on demand" of vector derived dynorphins

A-F: Double-immunofluorescence labeling is depicted for pDyn and NeuN (A-C) or GFAP (D-F) in the ipsilateral dentate gyrus of KA-treated and AAV-pDyn-injected mice. Enlarged view in (F) represents 15 x 30 µm. G: mature Dyn B content (measured by a Dyn B specific EIA) in the dorsal hippocampus of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n = 6) and 6 (filled symbols; n = 3) months after vector treatment. Naïve animals were age matched to the 1.5 months after AAV group. iH stands for ipsilateral hippocampus and cH for contralateral hippocampus.* = p<0.05; ** = p<0.01; paired t-test was used for comparison of ipsi- and contralateral hippocampi. Two-way ANOVA was used to compare Dyn levels between the early and late time interval. H: Mature Dyn B content in the CSF of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n= 6) and 7 (filled symbols; n = 4) months after vector treatment. ** = p<0.01; one-way ANOVA with Dunnett post hoc test

I: The release of fully processed, mature Dyn B under different stimulation conditions was analyzed in microdialysates collected from the hippocampus of pDyn-deficient (KO) animals two weeks after injection of AAV-pDyn. Three baseline samples (BL; 25 min each) were collected. This was followed by 25 min low-frequency stimulation (LF), 25 min baseline, and 25 min high-frequency stimulation (HF; I). J: The microdialysis probe (red cross) was placed in the dentate gyrus, the stimulation electrode (black cross) in the entorhinal cortex. K: Dyn B was quantified by EIA in the dialysate collected during different stimulation intensities. The red line represents the detection limit of the EIA. Data represent mean ± standard error of the mean * = p<0.05; ** = p<0.01; n=4; one-way ANOVA with Tukey post hoc test.
Dynorphin-Based "Release on Demand" Gene Therapy for Drug-Resistant Temporal Lobe Epilepsy (2019)
Figure 4

Fig 4: Effect of Dyn A/B on induced epileptiform activity in human hippocampal slices. A: depicts representative traces under conditions of increased KCl. B: Same as A together with Dyn A/B and C: same as A under co-application of Dyn A/B with 5'-GNTI. D: Burst events and E: interictal spikes were recorded from area CA1. F: Treatment with Dyn A/B markedly reduced the amplitude and number of bursts G: but not of interictal spikes. Each color represents the last 5 minutes of each application for one slice of 4 individual patients. Data sets for each patient represent mean ± SEM for amplitude or absolute number of events. Two-way ANOVA for repeated measurements revealed high significance for interaction (p < 0.0001) and amplitude (p < 0.0001) or inter-event intervals (p < 0.0001), but also for the factor patient (p < 0.0001). Therefore, careful interpretation of the raw data is considered as preferred method. For detailed data see Table EV1.
Dynorphin-Based "Release on Demand" Gene Therapy for Drug-Resistant Temporal Lobe Epilepsy (2019)
Figure 5

Fig. 5: Vector spread and analysis of inflammatory effects of AAV-pDyn

A: In-situ hybridization for AAV-derived pDyn mRNA at 10 weeks post AAV-pDyn delivery in the left dorsal hippocampus is depicted for a single brain at five levels between 1.1 (left) and 2.3 (right) mm from bregma. The probe specifically detects the codon-optimized sequence, therefore the contralateral side represents a control devoid of the respective mRNA.

B-F: At 10 weeks post AAV-pDyn delivery, markers of inflammation were analyzed as follows: Relative optical densities (ROD) were evaluated from film autoradiographs after in-situ hybridization for mRNAs of inflammatory markers (Interleukin-1β (B), Interleukin-6 (C), Interleukin1a receptor agonist (D), Tumor Necrosis Factor-α (E) and Nitric Oxide Synthase-1 (F)) in 3 principal sub-regions of the hippocampus in AAV-pDyn (n=7; red) or AAV-eGFP (n=5; blue) injected animals. GC = granule cell layer; CA = cornu ammonis. Data represent mean ± standard deviation
Mfn2 localization in the ER is necessary for its bioenergetic function and neuritic development (2021)
Figure 1

Figure 1. Defects in mitochondrial bioenergetics in Mfn2 KO cells do not depend on mitochondrial morphology.

(A-D) (A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm. (B) Percentage of cells displaying globular mitochondria (n=310-668 cells analyzed in 3-6 independent experiments). Data are presented as mean ± SEM. (C) Analysis of aspect ratio (AR) calculated as major axis/minor axis and (D) circularity calculated as 4*π*Area/Perimeter2 (n=300 mitochondria in 3 independent experiments). Data are presented as mean ± SEM.

(E) Mitochondrial membrane potential (MMP) was determined by measuring TMRM fluorescence (n= 90 cells analysed in 3 independent experiments). Data are presented as mean ± SEM

(F-H) (F) Oxygen consumption rate (OCR) normalized to the amount of protein in WT, Mfn1 KO and Mfn2 KO fibroblasts. Proton leak was measured after application of oligomycin (1 μM), maximal after CCCP application (10 μM) and oligomycin (n= 3 independent experiments). Data are presented as mean ± SEM. (G) Respiratory control ratio (RCR) was calculated as maximal oxygen consumption rate (OCR)/H+ leak OCR (n= 3 independent experiments). Data are presented as mean ± SEM (H) Spare respiratory capacity was calculated as maximal OCR- basal OCR (n= 3 independent experiments). Data are presented as mean ± SEM.

I) WT MEFs and Mfn1 or Mfn2 KO MEFs were incubated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). Data are presented as mean ± SEM.

Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test.