EMBO/soda-nel huggingface dataset:
Extrinsic and intrinsic apoptosis activate Pannexin-1 to drive NLRP3 inflammasome assembly (2019)
Figure 1

Figure 1. Extrinsic apoptosis trigger GSDMD-dependent and caspase-3/7-dependent necrosis.

(A-E) Primary (A, B) or immortalized BMDMs (D, E) were stimulated with recombinant murine TNF (100 ng/ml) in combination with (A, D) SM or (B, E) TAK1i for 6 or 4 h respectively. (C) Time-lapse confocal images (hour:min) of BMDMs stimulated with recombinant murine TNF (100 ng/ml) and SM (250 nM) stained with propidium iodide (red) for 6 h. Black arrowheads indicate membrane ballooning while white arrowheads indicate apoptotic bodies.

Data information: Data are means + SEM of pooled data from (A-B) 5 or (D-E) 8 independent experiments. Statistical analyses for normally distributed data sets were analyzed using the parametric t test whereas non-normally distributed data sets were analyzed using non-parametric Mann-Whitney t tests. Data were considered significant when *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001. (C) Data are representative of 3 independent experiments.
Extrinsic and intrinsic apoptosis activate Pannexin-1 to drive NLRP3 inflammasome assembly (2019)
Figure 2

Figure 2. Extrinsic apoptosis triggers caspase-1/11-independent GSDMD processing and GSDMD/E-independent NLRP3 activation.

(A-B) BMDMs were stimulated with TNF (100 ng/ml) in combination with TAK1i (125 nM) for the indicated time points. (A) LDH release and (B) mixed supernatant and cell extracts were analysed.

(C) Representation of known caspase cleavage site and molecular weight of corresponding cleavage fragment in mouse GSDMD.

(D) BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h in the presence or absence of KCl (50 mM). Where indicated, cells were pre-incubated with MCC950 (10 μM) 20-30 min prior to TNF/TAK1i stimulation.

(E-G) BMDMs were costimulated with TNF (100 ng/ml) and SM (E) (250 nM; 6 h) and mixed supernatant and cell extracts were analysed by immunoblot or (F) LDH release in the cell culture supernatant were quantified at the indicated time points.

Data information: Data are means + SEM of pooled data from (A) 4 or (F) 5 independent experiments. Statistical analyses were performed using a two-way ANOVA. Data were considered significant when *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001. All immunoblots are representative of 3 independent experiments.
Extrinsic and intrinsic apoptosis activate Pannexin-1 to drive NLRP3 inflammasome assembly (2019)
Figure 3

Figure 3. Caspase-3 suppresses caspase-8-dependent GSDMD activation and cell lysis during extrinsic apoptosis.

(A) HEK 293T cells were transfected with doxycycline-inducible DmrB-caspase-8 and the indicated GSDMD constructs. Cells were stimulated with doxycycline (10 μg/ml) for 18 h to induce DmrB-caspase-8 expression and exposed to B/B homodimerizer (12.5 nM) for another 2 h to activate caspase-8. Mixed supernatant and extracts were analysed by immunoblot.

(B-C) Immortalized Gsdmd-/- BDMDs expressing GSDMDWT and GSDMDD88A were (B) costimulated with TNF (100 ng/ml) and SM for 6 h or (C) primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with LCL161 (1 μM) for 24 h and LDH release was quantified.

(D-E) BMDMs were costimulated with TNF (100 ng/ml) and TAK1i for 4 h and (D) mixed supernatant and extracts were analysed by immunoblot or (E) LDH release was quantified at the indicated time points.

Data information: All immunoblots are representative of 3 independent experiments. Data are means +SEM of pooled data from (B-C) 3 or (E) 7 individual experiments. (B-C) Statistical analyses for normally distributed data sets were analyzed using the parametric t test whereas non-normally distributed data sets were analyzed using non-parametric Mann-Whitney t tests. (E) Statistical analyses were performed using a two-way ANOVA. Data were considered significant when *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001.
Extrinsic and intrinsic apoptosis activate Pannexin-1 to drive NLRP3 inflammasome assembly (2019)
Figure 4

Figure 4. RIPK3 promotes caspase-3 activation and Pannexin-1 activity to drive NLRP3 assembly during extrinsic apoptosis.

(A-E) BMDMs were costimulated with TNF (100 ng/ml) and TAK1i (125 nM) for 4 h and (A, B) LDH release was quantified or (C, D, E) mixed supernatant and extracts were analysed by immunoblot. Where indicated, cells were treated with the inhibitors Nec-1s (50 μM), MCC950 (10 μM), GSK'872 (1 μM), probenecid (1 mM) 20-30 min prior to cell stimulation. KCl (50 mM) was added together with TNF and TAK1i. (C) To induce necroptosis, BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and Q-VD-OPh (10 μM) was added at the last 20-30 min of priming and stimulated with SM (500 nM) for 4 h. (E) To activate the NLRP3 inflammasome, BMDM were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 4 h and stimulated with nigericin (10 μM) for 1 h.

(F) BMDMs were primed for 3 h with ultrapure E. coli K12 LPS (100 ng/ml) and stimulated with SM (0.5 μM) for a further 4 h. Probenecid (1 mM) was added 20-30 min prior to cell stimulation and mixed supernatant and extracts were analysed by immunoblot.

(G-H) BMDMs were stimulated with TNF (100 ng/ml) and SM (0.5 μM) for 6 h and (G) mixed supernatant and extracts were analysed by immunoblot or (H) LDH release was quantified.

Data information: All immunoblots are representative of 3 independent experiments. (A, B, H) Data are means +SEM of pooled data from (A, H) 4 or (B) 3 independent experiments. Statistical analyses for normally distributed data sets were analyzed using the parametric t test whereas non-normally distributed data sets were analyzed using non-parametric Mann-Whitney t tests. Data were considered significant when *P<0.05, **P<0.01 or ****P<0.0001.
Extrinsic and intrinsic apoptosis activate Pannexin-1 to drive NLRP3 inflammasome assembly (2019)
Figure 5

Figure 5. Intrinsic apoptosis drives gasdermin-independent cell lysis but promotes NLPR3 assembly through pannexin-1 activity.

(A-B) BMDMs were stimulated with an increasing dose of S63845 in the presence of ABT-737 (0.5 μM) and LDH release was quantified at 6 h.

(C-F) BMDMs were stimulated with ABT-737 (0.5 μM) and S63845 (0.5 μM) and LDH release was quantified (C, E) or mixed supernatant and extracts were analysed by immunoblot at 6 h (D, F).

(G-H) BMDMs were primed with ultrapure E. coli K12 LPS (100 ng/ml) for 3 h and further stimulated with ABT-737 (1 μM) and S63845 (1 μM) for 24 h and mixed supernatant and extracts were analysed by immunoblot (G) and IL-1β in cell-free supernatant were quantified by ELISA (H).

Data information: All immunoblots are representative of 3 independent experiments. Data are means +SEM of pooled data from (A) 4, (B) 5, or (C, E, H) 3 independent experiments.
Expanded View Figure Legends
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 1

Increased levels of GABA inhibit pexophagy and mitophagy, but not other autophagy‐related pathways

Peroxisomes were induced by growing the WT strain expressing Pot1-GFP in oleate medium to mid-log-phase, then transferred to SD-N starvation medium with or without GABA to trigger pexophagy for 6 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.
Mitochondria were induced by growing the WT strain expressing OM45-GFP in YPL medium to mid-log-phase and subsequently transferring cells to either SD-N with or without GABA to trigger mitophagy for 12 h. GFP cleavage was analyzed at the indicated time points by immunoblotting.
Mitophagy was monitored by fluorescence microscopy using a WT strain expressing OM45-GFP grown in YPL medium for 12 h to mid-log-phase in the presence of FM4-64, and transferred to either SD-N medium with or without GABA for 24 h. Bar, 5 µm.
The Cvt pathway was monitored using the WT strain in SD medium with or without GABA, grown to mid-log-phase, after which samples were analyzed for Ape1 maturation.
Ribophagy was monitored by growing the WT strain expressing Rpl25-GFP in SD medium to mid-log-phase and transferring cells to SD-N either with or without GABA for 24 h.
Autophagy was monitored by growing the WT strain expressing GFP-Atg8 in SD medium to mid-log-phase and transferring cells to SD-N either with or without GABA for 6 h.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 2

The GABA‐induced block in pexophagy and mitophagy is overridden by rapamycin

Peroxisomes were induced in oleate medium and pexophagy was monitored as described for Fig .
Pexophagy was monitored by fluorescence microscopy using a WT strain expressing Pot1-GFP grown in oleate medium to mid-log-phase in the presence of FM4-64, and transferred to either SD-N medium with or without GABA or to SD-N with GABA and rapamycin for 6 h. Bar, 5 µm.
Mitochondria were induced in YPL medium and mitophagy was assessed as described for Fig .
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 3

Increasing GABA levels endogenously also inhibits pexophagy and mitophagy, and these defects are suppressed by rapamycin

WT cells expressing OM45-GFP, along with the uga2? strain over-expressing the GAD1 gene and expressing OM45-GFP were grown in YPL medium to mid-log-phase. To monitor mitophagy, strains were transferred to SD-N starvation medium (with or without rapamycin).
WT strain along with the uga2? strain over-expressing the GAD1 gene was grown in oleate medium and pexophagy was monitored as described in Fig , with or without rapamycin. Samples were taken at the indicated time points, and Pot1 degradation was analyzed by immunoblotting (45 kD).
To monitor autophagy, WT cells expressing GFP-Atg8 along with the uga2? strain over-expressing the GAD1 gene and expressing GFP-Atg8 were grown in SD medium and transferred to SD-N.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 4

Increased GABA levels activate Tor in starvation conditions and inhibit pexophagy and mitophagy by acting through Sch9

WT and sch9? strains were grown, as described earlier, for pexophagy or mitophagy assays with or without GABA.

A,B WT cells were cultured under pexophagy (A) or mitophagy (B) conditions with or without GABA and rapamycin. S6 phosphorylation at the indicated time points was analyzed by immunoblotting with a loading control.
C Samples were analyzed for Pot1 degradation by immunoblotting (45 kD).
D GFP production during mitophagy was analyzed by immunoblotting.
E Proposed model for the regulation of pexophagy and mitophagy by GABA. Elevated GABA activates Tor1 in starvation conditions and inhibits pexophagy and mitophagy by activating Sch9.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 5

Increasing GABA concentration further increases Tor activity and inhibits autophagy

WT cells were cultured under autophagy conditions with or without GABA for 6 h.

S6 phosphorylation after 6 h in SD-N was analyzed by immunoblotting with a loading control.
GFP production monitoring autophagy at the indicated time points was analyzed by immunoblotting.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 6

The GABA‐induced block of pexophagy and mitophagy increases reactive oxygen species levels that can be reduced by rapamycin

A,B WT, WT with GABA, WT with GABA and 10 mM GSH and WT with GABA and 200 nM rapamycin were tested for intracellular ROS levels under (A) pexophagy and (B) mitophagy conditions. After 24 h incubation, cells were stained with DHR-123 and propidium iodide for 1 h. Living cells were analyzed for DHR-123 fluorescence by flow cytometry. Data represent mean + s.d. (n = 3). *P < 0.005, **P < 0.01
C,D Yeast cells stained with 5 µM propidium iodide were used to differentiate between living and dead cells under (C) pexophagy or (D) mitophagy conditions. Significant differences between the treatments and strains were determined using an unpaired two-tailed t-test. **P < 0.01.
E Pexophagy assay was monitored by the degradation of Pot1-GFP and analyzed for GFP cleavage by immunoblotting.
F Mitophagy assay was monitored by the degradation of Om45-GFP and analyzed for GFP cleavage by immunoblotting.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 7

Elevated GABA inhibits mitophagy in mammalian cells

A,B Example images of Parkin-expressing HeLa cells analyzed using a tandem fluorochrome protein (mito-RFP-GFP) mitophagy assay under (A) control conditions or (B) displaying mitophagy depicted by the red mitochondrial structures localized to lysosomes. Bar, 10 µm.
C Percentage of cells displaying mitophagy + s.d., **P < 0.01 using an unpaired two-tailed t-test, n > 80.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 8

SSADH‐deficient mice have increased numbers of mitochondria and aberrant antioxidant levels that can be normalized by rapamycin

Electron microscopy images of mitochondria from WT (n = 44) and SSADH-deficient mice (Aldh5a1-/-) (n = 80) were calculated for area size.
Electron microscopy images showing typical sizes of WT and Aldh5a1-/- mice liver mitochondria. Bar, 0.5 µm.
Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1-/- mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.
Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1-/- mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life.
Aldh5a1-/- mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non-disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay.
Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1).
Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1-/- mice treated with vehicle and Aldh5a1-/- mice treated with rapamycin. Bar, 10 µm.

Data information: **P < 0.01, *P < 0.05 using an unpaired two-tailed t-test. Data represent average + s.d.
Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition (2014)
Figure 9

SSADH‐deficient mice have increased levels of S6 phosphorylation compared to WT mice that can be reduced by rapamycin treatment

Aldh5a1-/- mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non-disease controls. After sacrifice, homogenates were used to measure S6 phosphorylation.

Quantification of S6 phosphorylation of liver lysates from WT (n = 5) and Aldh5a1-/- mice treated with vehicle (n = 4) or rapamycin (n = 5) after normalization (WT set to 1).
S6 phosphorylation of liver lysates analyzed by immunoblotting.
Quantification of S6 phosphorylation of brain lysates from WT (n = 2) and Aldh5a1-/- mice treated with vehicle (n = 3) or rapamycin (n = 3) after normalization (WT set to 1).
S6 phosphorylation of brain lysates analyzed by immunoblotting.

Data information: **P < 0.01, *P < 0.05, using an unpaired two-tailed t-test. Data represent average + s.d.
Dynamic properties of noise and Her6 levels are optimized by miR-9, allowing the decoding of the Her6 oscillator (2020)
Figure 1

Figure 1. Her6::Venus protein expression during Zebrafish neural development.

(a) Double fluorescent whole mount in situ hybridization (WM-ISH) to detect her6 (green) and her9 (magenta); coronal view (left panel) and transversal section (right panel), scale bar 20μm; 30-32hpf; annotations denote anterior (A), posterior (P), otic vesicle (ov), dorsal (D) and ventral (V).

(b) Schematic of strategy used to generate the Her6::Venus knock-in; left arm LA, right arm RA.

(c) Representative time series example of Her6::Venus expression during development, in the midbrain and hindbrain. Confocal images represented as 2D Maximum projection; longitudinal view; scale bar 50μm; otic vesicle (ov); also included in Movie EV1. r1: rhombomere 1, r2: rhombomere 2, r3: rhombomere 3, r4: rhombomere 4, r5: rhombomere 5, r6: rhombomere 6.

(d-f) Intensity mean of Her6::Venus per rhombomere area over development grouped by expression level, related to the r1-r6 regions in panel (c) : (d) r1 and r2; (e) r3 and r4; (f) r5 and r6.

(g) Transversal view of r6 in Her6::Venus embryos over time; Her6::Venus protein expression domains: a ventral domain (arrows) and a more dorsal lateral domain (arrowheads); the caax-mRFP was used as membrane marker (magenta); scale bars 20μm; images at 30-40hpf are maximum projection of 4 z-stacks from Movie EV2.

(h) Quantification of Her6::Venus(+) cell number (green) compared to total cell number (black) over development.

(i) Proportional changes in Her6::Venus(+) cell numbers during development; bars indicate median and interquartile range of counts collected from 3 different z-stacks per embryo and one embryo per condition; one-way ANOVA with Bonferroni multiple comparison test with significance p<0.01**.

(j-k) Nuclear abundance and nuclear concentration of Her6::Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy (FCS); bars indicate median and interquartile range of (19hpf: 6 embryos, 50 cells), (29hpf: 6 embryos, 86 cells), (33hpf: 5 embryos, 76 cells), (35hpf: 6 embryos, 59 cells) and (48hpf: 6 embryos, 89 cells); Kruskal-Wallis with Dunn's multiple comparison test, significance p<0.05*, p<0.001***, p<0.0001****.

(l) Quantification of heterogeneity using kurtosis from concentration data in (k); null kurtosis corresponds to a normal distribution.
Dynamic properties of noise and Her6 levels are optimized by miR-9, allowing the decoding of the Her6 oscillator (2020)
Figure 2

Figure 2. Dynamics of Her6::Venus in single neural progenitor cells.

(a) Experimental approach used to image Her6::Venus dynamic expression at single cell resolution.

(b) Schematic representation of genetic auto-repression network of Her6 including miR-9 regulation.

(c) 3D confocal representative images of a single neural progenitor cell (red arrow) tracked over time, data starting from 34hpf; scale bar 10μm, images from Movie EV3.

(d) Single cell time series of Her6::Venus, mKeima-H2B and Her6::Venus signal normalized by mKeima-H2B corresponding to the cell in (c).

(e) Covariance models and parameters used to characterize oscillatory (KOUosc ) and non-oscillatory (KOU ) single cell expression.

(f-h) Quantification of single cell dynamics at different stages in normal development including: (f) Proportion of oscillatory cells at different stages in development. (g) period of oscillators estimated using KOUosc ,(h) noise measured by aperiodic lengthscale (αOU) and (i) local coefficient of variation. Dashed lines indicate median and dotted lines indicate interquartile ranges of 28hpf (14 cells, 1 embryo), 30hpf (14 cells, 1 embryo) and 34hpf (10 cells, 1 embryo); Mann-Whitney two-tailed test, significance p<0.05 *, p<0.0001****.
Dynamic properties of noise and Her6 levels are optimized by miR-9, allowing the decoding of the Her6 oscillator (2020)
Figure 3

Figure 3. A mutation of the miR-9 binding site affects Her6 level over the course of development.

(a) Chromogenic WM-ISH of miR-9 using miR-9 LNA 5'-Dig observed at different stages during development; longitudinal view, anterior to the left.

(b) Transverse section of double fluorescent WM-ISH for her6 (green) and mir-9-4 (magenta) imaged in fixed embryo at 31hpf; scale bar 30μm.

(c) Schematic representing the miR-9 binding site (MBS) of her6::venus mutated by CRISPR-Cas9nls protein; MBSm refers to specific sgRNA to mutate the MBS as opposed to sgRNA that does not produce mutation (control-CTRL).

(d) Confocal imaging of CTRL and MBSm embryo showing Her6::Venus expression in the hindbrain, rhombomeres 3 to 6 (r3-r6) over the course of development; longitudinal view, scale 30μm, otic vesicle (ov). Images are representing 2D maximum projection.

(e) Her6::Venus expression (red arrows) in hindbrain, rhombomere 6 of live CTRL and MBSm embryos at 52hpf; edge of ventricular zone shown in yellow; transversal view; scale 30μm.

(f) Her6::Venus protein abundance in CTRL versus MBSm homozygous embryos observed in hindbrain, r6 between 34hpf and 69hpf; bars indicate median and interquartile range of (34hpf CTRL: 36 cells, 1 embryo), (34hpf MBSm: 35 cells, 1 embryo), (48±2hpf CTRL: 172 cells, 7 embryos), (48±2hpf MBSm: 124 cells, 5 embryos), (56±2hpf CTRL: 69 cells, 4 embryos), (56±2hpf MBSm: 31 cells, 2 embryos), (69hpf CTRL: 12 cells, 1 embryo), (69hpf MBSm: 6 cells, 2 embryos); Kruskal-Wallis with Dunn's multiple comparison test, significance: p<0.05*, p<0.001***, p<0.0001****.

(g) Comparative profiles of Her6 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in (f).
Dynamic properties of noise and Her6 levels are optimized by miR-9, allowing the decoding of the Her6 oscillator (2020)
Figure 4

Figure 4. A mutation of the miR-9 binding site affects Her6 dynamics at single cell level.

(a-b) Representative examples of single cell oscillators observed in CTRL (a) and MBSm (b) embryos imaged from 34hpf onwards; time series represent Her6::Venus expression relative to mKeima-H2B (top panel) and detrended relative signal (bottom panel); parameters reported for log-likelihood ratio (LLR) and period correspond to KOUosc .

(c) Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots; paired t test with two tail significance p<0.01**.

(d) Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells; dots indicate median per experiment per condition; paired t test with two tail not significant.

(e) Analysis of local coefficient of variation (LCOV) as a ratio between MBSm and CTRL; circles indicate median LCOV observed in MBSm cells divided by median LCOV observed in CTRL cells per experiment; bars indicate mean and SEM of LCOV ratios from 4 independent experiments.

(f-g) Representative examples of detrended Her6::Venus relative to mKeima-H2B in non-oscillatory single cells observed in CTRL (f) and MBSm (g) embryos imaged from 34hpf onwards.

(h) Aperiodic KOU covariance model indicating differences in rate of correlation decay between subsequent peaks corresponding to examples in (f-g).

(i) Example of one experiment representing quantification of noise by aperiodic lengthscale in one CTRL versus one MBSm embryo; bars indicate mean with 95% confidence intervals.

(j) Pairwise comparison of aperiodic lengthscale; dots indicate median per experiment from CTRL (15 cells, 5 cells, 13 cells, 7 cells; 4 embryos) and MBSm (14 cells, 5+12 cells, 4+8 cells, 13 cells; 6 embryos); paired t-test with two-tail significance for p<0.05 *.