EMBO/soda-nel huggingface dataset:
An immediate-late gene expression module decodes ERK signal duration (2017)
Figure 3

Figure 3 - Model fitting and classification of primary response genes.

A: HEK293∆RAF1:ER cells were treated with 4OHT for constitutive induction of ERK signalling. Phosphorylation levels of ERK2 were measured with bead-based ELISA (Bio-Plex). RNA time course expression data was measured using microarrays. This data was the basis to train gene-wise model parameters.

B: pERK2 log2 fold change upon sustained activation (left) and deduced input function (right) used for model fitting. Average pERK2 log2 fold change upon 4OHT treatment equals 100% signalling amplitude.

C: Measured gene expression kinetics and the resulting maximum likelihood fit of the gene expression model of all significantly induced primary response genes (FDR = 1%). Gene expression is shown as percentage of response amplitude. Mean error is calculated as the mean of absolute residuals serving as a goodness of fit measure.

D: Classification of primary response genes. For each gene, response time r is calculated as the sum of deduced transcriptional delay ∆t and mRNA half-life t1/2 and used for ranking. In general, immediate-early genes (IEGs) have short response times, whereas both immediate late genes (ILGs) and delayed early genes (DEGs) have longer response times. For ILGs, long response times are due to long mRNA half-lives. For DEGs, long response times are mainly due to long transcriptional delays (cf. boxplots).

E: Response times for IEGs, DEGs and ILGs. Genes on the same trajectory have the same response time, but response times are composed differently. For IEGs and ILGs, response times are solely determined by mRNA half-life, whereas DEGs have response times that are mixtures of half-life and transcriptional delay.
An immediate-late gene expression module decodes ERK signal duration (2017)
Figure 4

Figure 4 - Semi-quantitative prediction of mRNA log2 fold changes upon different signalling scenarios.

A: Gene expression upon different stimulations was predicted based on fitted model parameters and measured pERK2 levels. Predictions were verified with gene expression time-course data.

B: Signalling input conditions (left side shows deduced input function, right side shows pERK2 measurements): Sustained ERK signalling (4OHT), two-hour pulse ERK signalling (4OHT+U0126), growth factor signalling (EGF: Epidermal growth factor, FGF: Fibroblast growth factor). Deduced input functions: 100% signalling amplitude corresponds to mean induction in training condition (4OHT). Growth factor-induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in test condition.

C: Predictions are verified with actual gene expression data. Heat maps show log2 fold changes of induced mRNAs. P: Model prediction. D: Gene expression data. E: Mean error = mean of absolute residuals.
An immediate-late gene expression module decodes ERK signal duration (2017)
Figure 6

Figure 6 - Immediate late genes (ILGs) translate signal duration into response amplitude.

A: Upper panel: pERK2 log2 fold changes upon different input scenarios (sustained: 4OHT; two-hour pulse: 4OHT+U0126). Lower panel: Response amplitude across temporal clusters and signal durations. Bold lines show median cluster amplitude at each time point.

B: Capacity to decode signal duration. ILGs translate ERK signal duration into response amplitude. IEGs are only partially able to do so, as many of these genes are still strongly induced upon shortened signal durations.

C: qPCR validation to test different ERK signal durations. HEK293∆RAF1:ER cells were treated with 4OHT and U0126 for different periods of time to generate signal duration scenarios of 0.5 to 8 hours (cf. Fig EV1 C). mRNAs of IEGs EGR1 and FOS relay signal duration to response duration, whereas ILGs CLU and FOSL1 decode signal duration to response amplitude (qPCR data for all 17 validated mRNAs is shown in Fig EV6 A).

D: Relation between signal duration and response amplitude for IEGs and ILGs derived from qPCR validation data. Median amplitude is based on six qPCR-validated ILGs and six qPCR-validated IEGs.

E: Quantification of western blots to present protein log2 fold changes of sample genes upon sustained ERK signalling (4OHT-induced) and transient ERK signalling (EGF-induced) in HEK293∆RAF1:ER cells.

F: Conservation of signal duration decoding to response amplitude in two prominent model systems for ERK signal duration: NGF and HRG cause more sustained ERK signalling compared to EGF treatment in PC12 and MCF7 cells respectively. Decoding of signal duration to response amplitude is clearly governed by ILGs.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 1

Figure 1. Nuclear lamin B is a substantial component of nuclear envelope that is involved in NET formation

(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm.

(B) Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h, and stained for lamin B and DNA as described in panel B. The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 µm.

(C) Schematic cross-section of a cell and portion of the nucleus and the nuclear envelope, as well as the ones with corresponding overexpression of lamin B. The two lipid bilayers of the nuclear envelope are the inner and outer nuclear membranes (INM and ONM, respectively). The meshwork, nuclear lamin B is anchored to INM through lamin B receptor (LBR).

(D) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control.

(E) Summary analysis of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, then fixed by 2% PFA and stained by Sytox Green, following by detection with fluorescent microplate reader.

(F) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.

(G,H) Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red and cell impermeable Sytox Green, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (G) Scale bars, 40 μm.

Data information: Panels A,D,E,F,H were summary analysis of NET formation (A,E), or summary analyses of immunoblots (D), DNA% release index (F) that were calculated based on the arbitrary fluorescent readout unit (A,E), immunoblot arbitrary unit (D). NET-DNA release index calculated based on fluorescent readout (F), or % cells with NET formation by image analysis (H). Data in A,D,E,F,H represent mean ± SD (n=3-5 biological replicates). P*<0.05, P**<0.01, P***<0.001 between groups as indicated. Comparisons amongst three or more groups were performed using ANOVA, followed by Student-Newman-Keuls test. Comparison between two groups was analyzed by student t test.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 2

Figure 2. Nuclear lamin B disassembly, but not proteolytic cleavage, is responsible for nuclear envelope rupture in neutrophils with NET formation

(A,B) Representative and summary of immunoblots of full-length lanes of lamin B in human dPMNs that were treated with PMA for 0, 0.5, 1, 2, 3 h during NET formation, or apoptotic dPMNs that were induced by PMA for longer term (12 h) treatment (A).

(C,D) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0, 0.5, 1, 2, 3 h during NET formation.

(E) Representative confocal microscopy image of a group of pPMNs with NET formation (enclosed by a light blue trapezoid) and their extracellular NETs (indicated by a purple rectangle square) released by these cells, as well as the immunoblot analysis of un-cleaved lamin B and cleaved histone H3 with the whole cell lysates, as well as immunoblot analysis of un-cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment.

(F) Representative immunoblots with full-length lanes for lamin B analysis, and pro-caspase-3 and its activated form caspase-3 in human dPMNs with NET formation that were treated with PMA for 0, 0.5, 1, 2, 3 h.

(G) Representative immunoblots display full-length lamin B (69 kDa) and their cleaved fragments (45 and 25 kDa), and pro-caspase-3 and its activated form caspase-3 in the apoptotic human dPMNs that were treated with PMA for 12, 24h.

Data information: The anti-human lamin B was used in all immunoblots (A-G) and the confocal image (E), and the latter was further detected by FITC-labeled 2nd Ab, scale bar, 20 μm. Anti-human caspase-3 was used (F,G), and β-actin served as loading control (A-D,F,G). Data represent mean ± SD (n=3 biological replicates) for B and D. Comparisons amongst three or more groups were performed using ANOVA, followed by Student-Newman-Keuls test.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 3

Figure 3. Nuclear translocation and phosphorylation of PKCα is accompanied by nuclear envelope rupture during NET formation

(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining.

(B) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody.

(C,D) Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) or PAF (D) for 0, 0.5, 1, 2, 3 h.

Data information: The summary analyses of panel B were calculated based on the circumferences/perimeters of the cell nuclei of 7-10 cells from different time points from 3-6 independent experiments. Data in B represent mean ± SD (n=3-6 biological replicates). P*<0.05 between groups as indicated. Comparisons amongst three or more groups were performed using ANOVA, followed by Student-Newman-Keuls test.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 4

Figure 4. Accumulation of phosphorylated PKCα in nucleus results in nuclear lamin B phosphorylation, disassembly, and the consequent nuclear envelope rupture during NET formation

(A) Representative immunoblots of p-PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h.

(B) Representative sectional images of nuclei of two neutrophils treated by PMA, stained for lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), p-PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody), and nuclear DNA (DAPI). The accumulation of p-PKCα (red arrow pointed) at the site of ruptured/interrupted (green arrow) nuclear lamin B, and swollen/de-condensed DNA (blue arrow) were indicated (b1). A neutrophil (white empty arrow) with NET formation that contained the mixture of DNA with p-PKCα and lamin B (white arrow) was also demonstrated (b2). Scale bars, 10 µm.

(C) Immunoblot images of lamin B, then stripped and probed for p-PKCα, of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA.

(D,E) Representative and summary immunoblot (IB) detection of phospho-lamin B and total lamin B with the lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were treated either by PMA (D,E) for 0 or 3 h. Data represent mean ± SD (n=3-5 biological replicates) for E. P*<0.05 between groups as indicated. Comparison between two groups was analyzed by student t test.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 5

Figure 5. Inhibition of lamin B phosphorylation, or mutation of phosphorylation sites in lamin B, attenuates nuclear envelope rupture and NET release

(A) Summary and representative immunoblots of total PKCα and p-PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated without or with PMA (A) for 3 h.

(B) Immunoblot (IB) detection of the phospho-lamin B and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated by PMA (B) for 3 h.

(C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C).

(D) Schematic representation of lamin B domain structure and several mitotic serine phosphorylation sites (indicated by blue font) that exhibit PKC consensus motifs ([30]) and overview of lamin B reporters containing serine (S) to alanine (A) mutations at PKC consensus phosphorylation sites.

(E) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B (WT control), or mutants with single or multiple defined point mutations at PKCα-consensus-phosphorylation sites (S395A, S405A, S408A) of lamin B, and treated for 3 h by 10 μM PAF in medium containing cell-permeable dye SYTO Red (500 nM) for the total cell count, while the cells with NET formation were stained by cell impermeable dye SYTOX Blue (5 μM). Then the images were taken with Olympus confocal microscopy, followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation.

Data information: Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit (A), or percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ (E), from 3-5 independent experiments. Data represent mean ± SD (n=3-5 biological replicates). P*<0.05, P**<0.01, P***<0.001, between groups as indicated. Comparisons amongst three or more groups were performed using ANOVA, followed by Student-Newman-Keuls test.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 6

Figure 6. Genetic deficiency of PKCα attenuates lamin B phosphorylation and NET formation in primary neutrophils from PKCα knockout mice

(A) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα-/-, PKCα+/-, and their littermate WT (PKCα+/+) mice. Vinculin served as loading control.

(B) Representative immunoblot (IB) detection of the phospho-lamin B (Ser) and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from mouse peritoneal mPMNs from WT vs PKCα-/- mice, stimulated without or with PAF for 3 h.

(C) Summary analyses of NET formation in peritoneal mPMNs, from WT, PKCα+/-, or PKCα-/- mice, which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2% PFA fixation and fluorometric microplate analysis.

(D) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα-/- mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.

(E, F) (E) Representative and (F) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα-/- mice stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (E) Scale bars, 50 μm.

Data information: Panels A,C,D,F were summary analyses that were calculated based on the arbitrary unit (A) or arbitrary fluorescent readout (C), endpoint analysis of NET-DNA release index (D), or % cells with NET formation by image analysis using ImageJ (F) from 3-6 independent experiments as compared to their WT or untreated controls. Data represent mean ± SD (n=3-6 biological replicates), P*<0.05, P**<0.01 between groups as indicated. Comparisons amongst three or more groups were performed using ANOVA, followed by Student-Newman-Keuls test.
Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly (2020)
Figure 7

Figure 7. Strengthening nuclear envelope integrity by lamin B overexpression decreases NET release in vivo and alleviates NET-associated proinflammatory cytokine accumulation in UVB irradiated skin of the lamin B transgenic mice

(A-C) (A,B) representative (C) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.

(D,E) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL-17A (D) or TNFα (E), in the skin tissue of WT vs Lmnb1TG mice without (sham) or with UVB exposure, by co-staining of Ly6B (stained as described in panels A-C) with IL-17A (D) or TNFα (E) using FITC-conjugated rat anti-mouse IL-17A, or FITC conjugated rat anti-mouse TNFα Abs.

Data information: DNA was stained by DAPI. Panels C,D,E are summary analyses that were calculated based on the arbitrary fluorescent unit (C,D,E) from 5-6 independent experiments of different groups as indicated. Data represent mean ± SD (n=5-6 biological replicates). P**<0.01 between groups are indicated. Comparisons amongst three or more groups were performed using ANOVA, followed by Student-Newman-Keuls test.

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A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication (2021)
Figure 1

Figure 1. BspF is required for Brucella replication within rBCVs.

(A) rBCV biogenesis in BMMs infected with either wild type (2308), VirB-deficient (ΔvirB11), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as the percentage of LAMP1-positive BCVs over time. Data are means ± SD of n=3 independent experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to control (2308).

(B) Brucella replication in BMMs infected with either wild type (2308), VirB-deficient (ΔvirB11), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) compared to control (2308).

(C) Brucella replication in BMMs expressing either GFP or GFP-BspF and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to controls.