EMBO/soda-nel huggingface dataset:
USP7 and USP47 deubiquitinases regulate the NLRP3 inflammasome activation. (2018)
Figure 2

Figure 2. Transcriptional inflammasome priming is not required for USP7 and USP47 mediated inflammasome activation.

Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) control, ± S.D. n = 4. *** = P < 0.001, using a one-way ANOVA analysis comparing the mean of each column to the LPS treated sample.

Western blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.

IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors. ** = P < 0.01 using a one-way ANOVA.

Western blots of supernatants and cell lysates from MDMs as treated in C. Bands in the figure represent: Pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa) and β-actin as a loading control. Blots are representative of at least 3 independent blood donors.

LDH-release from MDMs as treated in C. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 9 independent blood donors. ** = P < 0.01 using a one-way ANOVA.
USP7 and USP47 deubiquitinases regulate the NLRP3 inflammasome activation. (2018)
Figure 3

Figure 3. USP7 and USP47 inhibition impairs ASC speck formation and oligomerisation.

Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in insoluble fractions from THP1 cell lysates. PMA differentiated THP1 cells were LPS-primed (1 μg/mL, 4 hrs.) and pre-incubated for 15 min with either 0.1% DMSO or P22077 (2.5 µM). followed by treatment with nigericin (10 μM, 45 mins.). The 'input' represents the abundance of ASC in cell lysates prior to isolation of the insoluble fraction. Blots show membranes that were probed with an anti-ASC antibody. Data is representative of at least three independent experiments.

Western blots showing ASC-oligomerisation after DSS-mediated crosslinking in supernatants from THP1s treated as in A. Blots show membranes that were probed with an anti-ASC antibody. The input represents non-crosslinked supernatant. Data is representative of at least three independent experiments.

Images from an immunofluorescence experiment showing ASC-speck formation in THP1 cells treated as in A. All images show cells that were stained with an anti-ASC antibody. Images are representative of 3 independent experiments. Scale bar 20µm.

Quantification of ASC-speck formation in images depicted in C. The number of ASC specks was quantified and expressed as the percentage of cells containing specks. Bars represent the mean ± S.D. n=3 independent biological replicates * = P < 0.05 using a t-test analysis.
USP7 and USP47 deubiquitinases regulate the NLRP3 inflammasome activation. (2018)
Figure 4

Figure 4. USP7 and USP47 inhibition regulates post-translational NLRP3 modifications.

NLRP3 oligomerisation analysed by native PAGE. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 min.). Digitonin-solubilised cell lysates were analysed by native-PAGE or SDS-PAGE and NLRP3 western-blotting. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inflammasome inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120 kDa). β-actin is shown as a loading control. Blots are representative of 4 independent murine donors.

NLRP3 ubiquitination analysed by western blot. LPS-primed (1 μg/mL, 4 hrs.) murine BMDMs pre-incubated with either 0.1% DMSO or P22077 (at the indicated concentrations) for 15 mins. before treatment with ATP (5 mM, 30 mins.). Endogenous NLRP3 was immunoprecipitated (IP) with anti-NLRP3 and probed with either anti-ubiquitin (Poly-Ub), anti-K63-ubiquitin (Poly-K63-Ub) or anti-NLRP3. As a negative control, cell lysate made from LPS-primed and 10 μM P22077 treated cells was immunoprecipitated with a nonspecific IgG. Total levels of NLRP3 in cell lysates (input) were also probed. Western blots of supernatants and cell lysates for IL-1β are also shown as a control for P22077 inhibition. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa) and NLRP3 (120kDa). β-actin is shown as a loading control. Blots are representative of 2 independent murine donors for 2.5µM P22077 and 4 independent murine donors for 10 µM.
USP7 and USP47 deubiquitinases regulate the NLRP3 inflammasome activation. (2018)
Figure 5

Figure 5. USP7 and USP47 are regulated post-transcriptionally in response to danger signals.

Western blots showing activity changes in both USP7 and USP47 upon treatment with nigericin. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then treated with nigericin (10 μM) for differing lengths of time, as indicated. Relative, activity-based labelling of USP7 and USP47 was analysed by western blot after incubation with ABPs. Higher panels show lysates incubated with ABPs (labelled), with the active DUB bound to ABP indicated by the suffix '-VME'. Lower panels, without probe labelling, show total USP7 or USP47 levels. (Unlabelled). Western blots are representative of 3 independent MDM donors.

Western blots showing activity changes in both USP7 and USP47 upon treatment with CPPD. MDMs were either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and then pre-incubated with either 0.1% DMSO or P22077 (2.5 μM) prior to incubation with CPPD crystals (250 µg/mL, 3 hrs.). Blots are labelled as in B and are representative of 3 independent MDM donors.

Western blots showing activity changes in both USP7 and USP47 in LPS-primed (1 μg/mL, 4 hrs.) MDMs pre-incubated with different P22077 concentrations, as indicated, prior to activation by nigericin treatment (10 μM; 45 mins.). Blots are labelled as in B and are representative of 3 independent MDM donors.

Western blots showing activity changes in both USP7 and USP47 upon activation in high extracellular potassium (High K+) conditions. MDMs were LPS-primed (1 μg/mL, 4 hrs.) and then changed to media that either had normal or high concentrations of K+, and subsequently treated with nigericin (10 μM; 45 mins.). Blots are labelled as in B and are representative of at least 2 independent MDM donors. Pro-IL-1β (31 kDa) and mature IL-1β (mIL-1β, 17 kDa) in the supernatants of these cells are shown as a control. Non-specific bands are highlighted with an asterisk.
USP7 and USP47 deubiquitinases regulate the NLRP3 inflammasome activation. (2018)
Figure 6

Figure 6. Inducible CRISPR/Cas9 knockout for USP7 and USP47 confirms their role in NLRP3 inflammasome activation.

Western blots of supernatants and cell lysates from THP-1 cells engineered by CRISPR/Cas9 to induce USP7 and/or USP47 KOs. Deficiency of both USP7 and USP47 was induced by doxycycline (Doxy) treatment (1 µg/mL; 3 days), as indicated. All cells were differentiated with PMA and either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and treated with nigericin (10 μM, 45 mins.), as indicated. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-caspase-1 (pro-Casp-1; 45 kDa); mature caspase-1 (mCasp-1; 20 kDa); USP7 and USP47. β-actin is shown as a loading control. Blots are representative of at least 3 independent experiments.

IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. n = 4 independent biological replicates, plotted as open circles on each bar. ** = P < 0.01 and *** = P < 0.001 using a one-way ANOVA.

LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D. n = 4 independent biological replicates, plotted as open circles on each bar. *** = P < 0.001 using a one-way ANOVA.

Western blots of supernatants and cell lysates from THP-1 cells engineered by CRISPR/Cas9 to induce USP7 and/or USP47 KOs. Deficiency of both USP7 and USP47 was induced by doxycycline (Doxy) treatment (1 µg/mL; 3 days), as indicated. All cells were differentiated with PMA and either unprimed or LPS-primed (1 μg/mL, 4 hrs.) and treated with nigericin (10 μM, 45 mins.), as indicated. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-caspase-1 (pro-Casp-1; 45 kDa); mature caspase-1 (mCasp-1; 20 kDa); USP7 and USP47. β-actin is shown as a loading control. Blots are representative of at least 3 independent experiments.

IL-1β release measured from doxycycline treated (1 µg/mL; 3 days) PMA-differentiated and LPS-primed (1 μg/mL, 4 hrs.), nigericin activated (10 μM, 45 mins.) THP-1 cells, which contain the indicated sgRNA. Bars represent the mean percentage of IL-1β release relative to their respective non-doxycycline treated control cells, ± S.D. n = 4 independent biological replicates, plotted as open circles on each bar. ** = P < 0.01 and *** = P < 0.001 using a one-way ANOVA.

LDH-release measured from cells as treated in B. Bars represent the mean percentage of LDH release relative to their respective non-doxycycline treated control cells, ± S.D. n = 4 independent biological replicates, plotted as open circles on each bar. *** = P < 0.001 using a one-way ANOVA.
Cardiolipin remodeling enables protein crowding in the inner mitochondrial membrane (2021)
Figure 1

Figure 1. Membrane proteins alter CL remodeling in vitro.

A: Myelin basic protein (MBP) or albumin (Alb) were added to liposomes that contained purified tafazzin (TAZ). Acyl transfer reactions were measured by mass spectrometry.

B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb.

C: CL formation was measured as in B with different amounts of MBP or Alb.

D: PC16:0/18:3 formation was measured in the presence of 85 nmol LPC16:0, 15 nmol PC18:3/18:3, 4 µg TAZ, and MBP.

E, F: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:3)2, 4 µg TAZ, and 0.2 mg MBP (2 independent measurements per time point).

G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR).

H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR.

I: Transacylations were measured in mixtures containing 90 nmol CL(18:1)4, 90 nmol CL(18:2)4, 10nmol LPG18:1, 10 nmol LPE18:1, 8 µg TAZ, 20 mM Ca2+, and different amounts of BR (2 independent measurements per BR/Lipid ratio).

J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.

Data information: The graphs show replicas of independent experiments, their mean values, and standard deviations. Means were compared by Student's t-test.
Cardiolipin remodeling enables protein crowding in the inner mitochondrial membrane (2021)
Figure 3

Figure 3. TAZKO reduces the concentration of proteins in the IM of mouse heart mitochondria.

Hearts were harvested from wild-type (WT) or TAZKO mice at the embryonic age of 14.5 days and at the adult age of 5 months.

A: Mitochondrial membranes were traced in electron micrographs to determine the IM/OM surface area ratio. Tracings of the IM are shown in yellow boxes.

B: Cross sectional areas of mitochondria and IM/OM surface area ratios were determined in randomly collected electron micrographs. Multiple micrographs were analyzed per biological replica.

C: Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence-labeled secondary antibodies. The abundances of IM proteins (NDUFB6, UQCR2, ATP5A1) are expressed relative to the abundance of the OM protein TOM70.

D-F: Relative abundances of mitochondrial proteins were measured in adult hearts by label-free quantitative proteomics. C I-V, complex I-V; CAC, citric acid cycle enzymes; MICOS, contact site complex; mt TA, mitochondrial transaminases; NS, not significant; PDH pyruvate dehydrogenase; VDAC, voltage-dependent anion channel.

Data information: The graphs show mean values and standard deviations of 3 biological replicas. Means were compared by Student's t-test. Data in F represent 77 OXPHOS subunits measured in 3 biological replicas.
Cardiolipin remodeling enables protein crowding in the inner mitochondrial membrane (2021)
Figure 4

Figure 4. CL deficiency causes reduced protein concentration and localized defects in the IM of Drosophila flight muscle mitochondria.

Indirect flight muscles were harvested from adult flies with mutations in the tafazzin gene (∆TAZ) or the CL synthase gene (∆CLS) or from wild type (WT).

A-E: Tissues were analyzed by quantitative electron microscopy. Multiple micrographs were analyzed per biological replica.

F, G: Tissues were analyzed by label-free quantitative proteomics. C I-V, complex I-V; CAC, citric acid cycle enzymes; MICOS, contact site complex; mt TA, mitochondrial transaminases; NS, not significant; PDH pyruvate dehydrogenase; VDAC, voltage-dependent anion channel.

Data information: The graphs show mean values and standard deviations of 3 biological replicas. Mutants were compared to the wild-type by Student's t-test. In B, each data point is the mean value of 40-70 mitochondrial cross sections. In C, data were collected from 10 electron micrographs per strain. In G, data represent 35 OXPHOS subunits measured in 3 biological replicas.
A common molecular logic determines embryonic stem cell self-renewal and reprogramming (2018)
Figure 1

Figure 1: Network models consistent with naïve state maintenance predict the effect of TF forced expression in resetting from primed pluripotency. See also Appendix Fig S1, S2.

(A) Flow-chart describing the methodology. Network components were identified based on functional studies from the literature, and possible interactions between components defined based on pairwise gene expression correlation. A set of experimental results served as constraints. The software RE:IN synthesises all possible interaction networks consistent with the constraints, which is termed the cABN. The cABN is used to formulate predictions to be tested experimentally. Importantly, predictions do not overlap with imposed constraints. If predictions are falsified, the cABN can be further refined by incorporating new experimental results as constraints. The refined cABN is used to generate further predictions.

(B) cABN derived from a Pearson coefficient threshold of 0.832, consistent with constraints previously defined for ESC self-renewal (Dunn et al. 2014). Solid arrow, required interaction; dashed arrow, possible interaction; black arrow, activation; red arrow, inhibition. There is no regulation hierarchy associated with component positioning.

(C) Illustration of EpiSC resetting constraints. See Appendix Fig S1F.

(D) Predicted number of regulation steps required for all models to stabilise in the naïve state under forced expression of a single network component. The red dashed line indicates the number of steps required under empty vector control.

(E) Fold increase of Oct4-GFP+ colony number over control under forced expression of individual factors. n ≥ 5, where each dot indicates an independent experiment. Box-plots show median, 1st and 3rd quartile values. One-sample Wilcoxon test p-values are as indicated, with p values < 0.05 shown in red.

(F) cABN derived from a Pearson coefficient threshold of 0.782.

(G) Predictions from the 0.782 cABN. Light green regions indicate where some, but not all, concrete networks allow stable conversion to the naïve state. Sall4 is indicated in green, as this was imposed as a constraint and therefore is not a model prediction.
A common molecular logic determines embryonic stem cell self-renewal and reprogramming (2018)
Figure 2

Figure 2: Models predict the sequence of gene activation during resetting to naïve pluripotency.

(A) Model predictions of the number of regulation steps required for permanent activation of each network component. Light blue regions indicate where only some concrete networks predict that the given gene has permanently activated.

(B) Heatmap of average gene expression normalised to β-actin over an EpiSC resetting time course in 2i+LIF. Each row is coloured according to the unique minimum and maximum for that gene. The values shown are average expression of four independent experiments.

(C) Gene expression for Stat3, Klf2, Esrrb and Tfcp2l1 during EpiSC resetting relative to established mouse ESCs. β-actin serves as an internal control. Mean +/- SEM, n=4 independent experiments. * = p<0.05 Student's t-test.

(D) Left: Local network topology for Tfcp2l1 and Esrrb. Right: Summary of regulation conditions required by Tfcp2l1 and Esrrb in the 0.782 cABN.
A common molecular logic determines embryonic stem cell self-renewal and reprogramming (2018)
Figure 3

Figure 3: Combinations of potent factors enhance resetting by accelerating network activation. See also Appendix Fig S4.

(A) Left: Comparison of the number of steps required for all concrete networks to stabilise in the naïve state under single and dual factor expression. Right: Experimental results showing the fold increase in colony number over empty vector control under single and dual factor expression. Y = Yes, N = No, * = incorrect prediction.

(B) Predictions and experimental validation of examples of synergistic and non-additive factor combinations. Experimental measurement of fold increase over empty vector control of Oct4-GFP+ colony numbers. Bars indicate the mean of 2 independent experiments, shown as dots.

(C) Scheme for DOX inducible constructs used for dual factor expression.

(D) Experimental setup for the functional characterisation of Esrrb-T2A-Klf4 or Esrrb-T2A-Tbx3 forced expression in EpiSC resetting.

(E) Representative confocal images (top) and quantification (bottom) of Oct4-GFP reporter mean intensity. The indicated cell lines were treated with DOX for the first 2 days and imaged at Day 6. Mean +/- SD of 9 technical replicates. One representative experiment of two is shown. Scale bars=300µm.

(F) Representative alkaline phosphatase (AP) staining images (left) and quantification (right) of AP+ colonies after clonal replating, as described in panel. Mean +/- SEM, n=3 independent experiments.
A common molecular logic determines embryonic stem cell self-renewal and reprogramming (2018)
Figure 4

Figure 4: Co-expression of factors activated late in EpiSC resetting increases pluripotency marker expression and significantly reduces the resetting time scale. See also Appendix Fig S3, S5, Table EV1.

(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), captured at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments.

(B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels.

(C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments.

(D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green).

(E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell.
A common molecular logic determines embryonic stem cell self-renewal and reprogramming (2018)
Figure 6

Figure 6: Klf2 and Stat3 are required for EpiSC resetting, but not for naïve state maintenance. See also Appendix Fig S6, S7, Tables S2-4.

(A) 0.782 cABN predictions of essential or dispensable factors for EpiSC resetting, compared with the experiment results shown in panel (B).

(B) siRNA knockdown effects measured by Oct4-GFP+ colony formation at Day 6 of resetting. n=4 independent experiments. Box-plots indicate 1st, 3rd quartile and median. * = p<0.05 Student's t-test; n.s. = not significant.

(C) Left, resetting capacity of Klf2 and Klf4 KO EpiSCs measured by Oct4-GFP+ colony formation at Day 6 of resetting. Data points represent two independent experiments. Right, representative fluorescent and bright field images of wild type and Klf2 KO EpiSCs at Day 6 of resetting in 2i+LIF. Scale bars=100µm.

(D) Expression of naïve pluripotency, transition and somatic lineage markers in wild type and Klf2 KO EpiSCs during a resetting time course in 2i+LIF. Expression is normalised to wild type EpiSCs in A/F, and β-actin was used as internal control. The values shown correspond to the average expression of three technical replicates from one representative experiment out of two.

(E) Rescue of Klf2 KO EpiSC resetting by forced expression of individual network components measured by Oct4-GFP+ colony formation at Day 6 of resetting in 2i+LIF. Data points represent two independent experiments.

(F) Comparison between the effect of single factor knockdowns on ESC maintenance (n=4 independent experiments) and EpiSC resetting using experimental results.

(G) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation after Stat3 siRNA in wild type EpiSCs (left), or Klf4 KO EpiSCs transfected with Tfcp2l1 and Gbx2 siRNAs. n=3 independent experiments in wild type cells (box plots indicate min, median, max), n=2 for Klf4-/- cells (box plots indicate min, mean, max).

(H) EpiSC resetting in 2i+LIF measured by Oct4-GFP+ colony formation of Stat3 knockdown EpiSCs transiently transfected with Tfcp2l1, Gbx2 and Klf4. n=4 independent experiments: Student's t-test, p-value indicated on plot. Box plots indicate min, median, max. See also Appendix Fig S7C.

(I) The 0.717 cABN, used to illustrate the kinetics of EpiSC resetting as determined experimentally. Left: Genes coloured according to the order of activation during resetting in 2i+LIF. Right: Genes coloured according to their potency in enhancing the efficiency of resetting. TFs with a green border are the common factors required for ESC self-renewal and EpiSC resetting. See also Appendix Fig S6A.