EMBO/soda-nel huggingface dataset:
Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons (2020)
Figure 1

Fig 1. Genetic loss of KCC2 promotes apoptosis of embryonic neocortical projection neurons in vivo

A Representative images of E18.5 coronal cortical sections from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmids encoding EGFP, EGFP+Cre (Cre) or EGFP+Cre+KCC2FL (Cre+KCC2FL). DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm.

B Quantification of the number of EGFP+ neurons/ROI from embryos electroporated with constructs in (A). Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01. Data are presented as mean ± S.E.M., n (EGFP) = 8 embryos; n (Cre) = 8 embryos; n (Cre+KCC2FL) = 13 embryos.

C Representative image of cleaved Caspase 3 (Cas-3, upper panel) and TUNEL (lower panel) staining in coronal sections from Kcc2lox/lox cortex at E16.5 electroporated with EGFP or EGFP+Cre are shown. Arrowheads point to neurons expressing Cas-3 (upper panel) and TUNEL (lower panel). DAPI staining (blue) marks cell nuclei. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; Sp, subplate; IZ, intermediate zone. Large scale bar: 50 µm, small scale bar: 20 µm.

D Quantification of the percentage of EGFP+ neurons expressing apoptotic markers at E16.5 from embryos electroporated with EGFP ± Cre. Statistical significance was determined using Mann-Whitney U test (Cas-3); and two-tailed t test (TUNEL), ***P < 0.001. Data are presented as mean ± S.E.M., n (-Cre) = 6 embryos; n (+Cre) = 6 embryos.

E The number of EGFP+Cre neurons as a percentage of neurons expressing EGFP alone. Statistical significance was determined using a two-tailed Student's t test. Data are presented as mean ± S.E.M., n = 6 embryos.
Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons (2020)
Figure 2

Fig 2. Ion transport-independent actions of KCC2 CTD promote the survival of migrating projection neurons

A Top: Representative images of E18.5 coronal cortical brain sections from Kcc2lox/lox embryos electroporated in utero at E14.5 together with plasmid constructs encoding Cre, a fluorescent marker (EGFP or mRFP, both pseudo-colored in green) and one of the following constructs: KCC2ΔNTD, KCC2CTD, KCC2R952H, or cofilinS3A. DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. Bottom: schematic representation of the KCC2 constructs.

B Quantification of the number of transfected neurons/ROI from embryos electroporated with constructs in (A). The mean number of transfected neurons from embryos electroporated with Cre+KCC2FL taken from Fig 1B shown as dotted line. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Cre+KCC2FL. Data are presented as mean ± S.E.M., n (KCC2ΔNTD) = 7 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos; n (Cre+cofilinS3A) = 8 embryos.
Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons (2020)
Figure 3

Fig 3. Cell death mediated by KCC2 deletion differentially affects upper cortical PNs migrating in deep- vs. superficial-layers at E18.5

A Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding EGFP or EGFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. EGFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm.

B Number of EGFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to EGFP. Data are presented as mean ± S.E.M., n (EGFP+Cre) = 8 embryos; n (EGFP) = 8 embryos.

C Representative image of E18.5 coronal brain sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding mRFP or mRFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. mRFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei.

D Number of mRFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to mRFP. Data are presented as mean ± S.E.M., n (mRFP+Cre) = 5 embryos; n (mRFP) = 5 embryos.

E Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos co-electroporated in utero at E14.5 with plasmids encoding a fluorescent marker (green) together and Cre+KCC2FL, Cre+KCC2CTD, or Cre+KCC2R952H. DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm.

F Number of transfected neurons migrating above (II-IV, left) and below (V-VI/IZ-SVZ, right) the upper border of layer V in embryos electroporated with constructs in (E) normalized to respective data from embryos electroporated with Cre. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 to Cre. Data are presented as mean ± S.E.M., n (Cre+KCC2FL) = 13 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos.
Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons (2020)
Figure 4

Fig 4. Constitutive ablation of KCC2 does not alter cortical lamination but increases cell death of deep-layer migrating upper cortical layer PNs at E18.5

A, B, C The layer thickness and neuronal number within each layer was assessed using layer-specific markers in Kcc2-/-and Kcc2+/+ embryos at E18.5. Cux1 was used to label layers II-IV (A), Ctip2 to label layer V (B), and Tbr1 to label layer VI (C). Dashed white lines in the representative images in (A-C) indicate upper and lower layer boundaries. Sp, subplate. Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc (neuronal numbers) and one-way ANOVA with Holm-Sidak's post hoc (layer thickness). Data are presented as mean ± S.E.M., n = 11 embryos for both genotypes. Scale bar = 50 µm.

D Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) in Kcc2-/- and Kcc2+/+ embryos electroporated in utero at E14.5 with a plasmid encoding EGFP (green). DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm.

E Neurons from the brain sections in (D) were quantified and the total number of EGFP+ neurons in the Kcc2-/- sections is presented as a percentage of pooled Kcc2+/+ and Kcc2+/- values. Statistical significance was determined using one-tailed Student's t test, *P < 0.05. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.

F Number of EGFP+ neurons in Kcc2-/- embryos migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective pooled data from Kcc2+/+ and Kcc2+/- embryos. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Kcc2+/+ + Kcc2+/-. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.
Slower growth of E. coli leads to longer survival in carbon starvation (2020)
Figure 3

Figure 3. Normalization of maintenance rate and recycling yield with cell volume of E. coli.

(A-B) Cell size during starvation. Length and width of the cells are measured with phase-contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi-spheres, as described in the graphical synopsis at the top (see also Table EV3 and Methods). Each measurement is an average of 200 cells. (A) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady-state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0.9 h − 1 (see upper color legend). Note that, as shown in Schink et al. (2019), cell widths do not change from steady state growth to starvation, while lengths decrease. Both length and width increase exponentially with growth rate. (B) Starvation volume of the cells described in panel A, computed as explained in the graphical synopsis (see also Methods). In agreement with literature (Schaechter et al., 1958), it increases exponentially with growth rate (black line).

(C-D) Recycling yield and maintenance rate per cell volume. (C) Recycling yields and (D) maintenance rate measured as described in Fig. 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to (see color legend at the top and Table EV3). The normalized yield is constant within the uncertainty. Maintenance rate increases significantly with a slope of (0.99±0.55) h, matching the increase of the death rate, (1.1±0.4) h, shown in Fig 2B. Solid black lines show linear fits on log transformed data, using weighted least square fits.

Data information: Slopes are reported with standard error and take uncertainty in data into account (see Methods). Data shown as mean ± SD. Two replicates per condition.
Slower growth of E. coli leads to longer survival in carbon starvation (2020)
Figure 4

Figure 4. Influence of RpoS knock-out on the death rate dependence. Death rates of ∆rpoS mutants versus the growth rate (grey symbols), compared to wild-type E. coli K-12 (white symbols, data from Fig. 1). Death rate increases with growth rate with a smaller slope (∆rpoS: 0.57±0.13 h) compared to wild-type (WT: 0.88±0.13 h). Black and grey lines show linear weighted least square fits to log-transformed data, which take uncertainty in data points into account (see Methods). Data shown as mean ± SD. Two or three replicates per condition.
Seipin regulates ER-lipid droplet contacts and cargo delivery (2016)
Figure 1

Figure 1. WT-seipin-GFP rescues aberrant LD biogenesis and is enriched at ER-LD contact sites.

(A) A431 WT and SKO cells -/+ GFP-fused WT or A212P-seipin were delipidated, treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (B) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/cell in indicated size category, mean ± SEM, n=57-61 cells/genotype, 2 experiments, p**< 0.005 (unpaired T-test). (C) SKO + WT- or A212P-seipin-GFP cells were stained for calreticulin. Maximum intensity projections of deconvolved confocal z-stacks. (D) SKO + WT- or A212P-seipin-GFP cells were grown in standard growth medium, fixed and stained with anti-GFP antibodies. Widefield and STORM images. (E) SKO + WT-seipin-GFP cells were delipidated and treated with OA for 1h, fixed and stained with LipidTox Red. Single focal plane of a 3D-SIM z-stack. (F) Analysis of (E), bars: % of LDs/cell with the indicated number of WT-seipin-GFP punctae associated, mean ± SEM, n=12 cells (693 LDs), 2 experiments, p**< 0.005 (Tukey multiple comparisons test). (G) SKO + WT- or A212P-seipin-GFP cells were delipidated, incubated with OA for 30 min, and immunolabelled for TEM with anti-GFP antibodies. Black arrowheads: immuno-gold, orange arrowheads: ER-LD junctions. See also Appendix Fig S3. (H) Analysis of (G), bars: relative labeling index in different cellular compartments, n=3456-4512 immuno-gold clusters. Distribution of both WT- and A212P-GFP-seipin immunolabel is non-random (chi square test, **p<0.005), but there is significant enrichment of WT- but not A212P-seipin-GFP at ER-LD contacts relative to the ER (binomial test, **p<0.005). See also Appendix Table S1 and S2. (I) A431 WT cells with seipin tagged with sf-GFP at its chromosomal locus and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 1 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack.
Seipin regulates ER-lipid droplet contacts and cargo delivery (2016)
Figure 2

Figure 2. Seipin is stably associated with LDs and lack of seipin results in loss of LD contacts with the ER.

(A) SKO cells stably expressing WT-seipin-GFP cells were delipidated and then incubated with OA and LipidTox Deep Red for 30-75 min. Airyscan video snapshots, upper panel: an example of 2 seipin-GPF punctae, one stably associated with a LD (blue arrowhead) and another not LD associated (orange arrowhead). Lower panel: an example of a mobile LD, with a single seipin puncta moving together with it (blue arrowhead). (B) Analysis of (A), bars: mean velocity of tracked seipin puncta ± SEM, n=13 ROIs from 13 cells, 90 puncta, 2 experiments, **p<0.005 (unpaired T-test). (C) Analysis of LD mobility in WT and SKO cells. Cells were incubated with OA for 45-75 min, LDs were visualized with LD540 and cells imaged by widefield microscopy. Bars: 1 – [Pearson’s colocalization coefficient between subsequent frames/cell], ± SEM, n= 149-151 cells, 2 experiments, **p<0.005 (Mann Whitney test). (D) Examples of LD trajectories from cells treated and imaged , arrowhead indicates the tracked LD. (E) Analysis of the LD mobility from (C) and (D), individual LDs plotted for volume and mean velocity. (F) WT and SKO cells were transfected with ER marker plasmid (sfGFP-ER-3, green), delipidated overnight and incubated with OA and LipidTox Deep Red (magenta) for 15-75 min. Airyscan video snapshots exemplifying different modes of LD ER-associated movement, note the different time scales. Panel I and II: WT cell, panel III: SKO cell. (G) Analysis of LD ER-associated movement from live airyscan microscopy as in (F). Bars: % of LDs/ROI displaying indicated mode of movement, ± SEM, n= 43-55 ROIs (201-467 LDs) from 4 experiments, **p<0.005 (Mann Whitney test). (H) Analysis of LD velocity. Bars: mean velocity of tracked LDs, ± SEM, n=29-42 LDs/group, 2 experiments, **p<0.005 (Kruskal-Wallis test, followed by Dunn test). (I) WT and SKO cells stably expressing HSP47-APEX were delipidated, treated with OA for 1 h and processed for SB-EM. 3D models of LDs and surrounding ER and mitochondria profiles shown on top of a block face image from SB-EM dataset. Insets are representative cross sections from modeled area highlighting the ER-LD association. Red= LD, Yellow= ER, Green=mitochondria. (J) Analysis of LDs from (I). Black lines indicate median, boxes third quartiles, n= 98-227 LDs. (K) Analysis of LDs displaying no ER contacts from (I). Bars: mean % of LDs in ROIs not in proximity to the ER, ± SEM, n= 11-16 ROIs/genotype, **p<0.005 (unpaired T-test).
Seipin regulates ER-lipid droplet contacts and cargo delivery (2016)
Figure 4

Figure 4. Endogenous ACSL3 accumulates at ER-LD junctions in SKO cells.

(A) WT and SKO cells stably expressing ACSL3-Cherry were transfected with ADRP-GFP for 24 h, incubated with OA for 1 h, fixed and stained with LD540. Single confocal planes of deconvolved confocal z-stacks. (B) Colocalization analysis of (A), from single confocal sections. Bars: correlation coefficient m1 ± SEM, n=34-35 cells/genotype, 2 experiments, **p<0.005 (unpaired T-test). (C) WT and SKO cells were transfected with ADRP-GFP for 24 h, incubated with OA for 2 h and co-plated with delipidated WT or SKO cells stably expressing ACSL3-Cherry. After co-plating, cells were incubated for 2.5 h in normal growth medium, then fused together using PEG. Cells were fixed and imaged 1 h post-fusion. Single confocal planes. (D) Colocalization analysis of (C). Bars: correlation coefficient m1 ± SEM, n=31-39 cells/treatment, 3 experiments, **p<0.005 (Tukey’s test). Similar results were obtained when cells were kept in delipidation medium after co-plating. (E) WT and SKO cells were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, single confocal planes of deconvolved z-stacks. (F) Analysis of (E), insets illustrate assigned categories of ACSL3 LD association. Bars: % of LDs/ROI in the indicated category, mean ± SEM, n= 9-10 peripheral ROIs from 9-10 cells/treatment (616-968 LDs), 3 experiments, **p<0.005 (unpaired T-test). (G) TEM images of anti-ACSL3-labeled WT and SKO cells treated with OA for 1 h. (H) Analysis of (G), n= 66-119 LDs from 8 cells/genotype.
Seipin regulates ER-lipid droplet contacts and cargo delivery (2016)
Figure 6

Figure 6. Older LDs of SKO cells display heterogeneous ER connectivity.

(A) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 20 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack. (B) WT and SKO cells were treated with OA for 20 h, fixed and stained with LD540. Maximum projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (C) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/field of view in indicated size category, mean ± SEM, n=25-30 fields of view (100-120 ROIs of not clustered LD areas from 100-120 cells), 2 experiments, p**< 0.005 (unpaired T-test). See also Appendix Figure S5D. (D) TEM micrographs of WT and SKO cells treated with OA for 20 h. (E) WT and SKO cells were treated as in (B), LDs were visualized with LD540 and cells imaged live with widefield microscopy. Exemplary trajectories of peripheral LDs tracked for 40 s. (F) Analysis of cells treated and imaged as in (E). Bars: 1 – [Pearson’s colocalization coefficient between subsequent frames/cell], ± SEM, n=187-221 cells, 2 experiments, **p<0.005 (Mann Whitney test). (G) WT and SKO cells were treated with OA for 20 h, fixed and stained with anti-ACSL3 antibodies and LD540. Maximum intensity projections of deconvolved confocal z-stacks. Insets show small and large LDs. (H) Analysis of (E). Bars: % of small (diameter <0.5 µm) or large (diameter >0.5 µm) LDs/cell in the indicated category, mean ± SEM, n=10 ROIs from 10 cells/treatment (305-581 LDs), **p<0.005 (unpaired T-test). (I) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 20 h, and LDs were labelled with LipidTox Green prior to imaging. The HPos-Cherry signal was bleached and signal recovery to LDs was monitored. (J) Analysis of (I). Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=51-61 bleached LD areas/genotype, 3 experiments.
Seipin regulates ER-lipid droplet contacts and cargo delivery (2016)
Figure 7

Figure 7. BSCL2 patient fibroblasts display LDs moving independently of the ER, ACSL3 mistargeting and defective fatty acid flux to neutral lipids.

(A) Two control and BSCL2 fibroblast cell lines were delipidated and treated with OA for 1 h, fixed and stained with LD540. Maximum intensity projections of deconvolved confocal z-stacks. (B) Control and BSLC2 fibroblasts were incubated with OA for 75-90 min, LDs were stained with LD540 and cells imaged live with widefield microscopy. Insets show overlay of two subsequent frames (400 ms apart). (C) Analysis of (B). Bars = 1 - [Pearson’s colocalization coefficient between subsequent frames/ROI], ± SEM, n=120-140 ROIs (60-70 cells), 2 experiments, **p<0.005 (Mann Whitney test). (D) Control and BSLC2 fibroblasts were transfected with ER marker plasmid (sfGFP-ER-3) for 24 h and treated with OA for 10-60 min. LipidTox Deep Red or LD540 was used to label LDs and cells were imaged live by airyscan microscopy. Examples of LDs with high mobility are shown, blue arrowheads indicate a LD moving along the ER, orange arrowheads indicate LDs moving independent of the ER. (E) Analysis of ER associated movement of LDs from live airyscan microscopy as in (D). Bars: % of LDs/ROI displaying indicated mode of movement, mean ± SEM, n= 50-42 ROIs (218-374 LDs), 2 experiments, **p<0.005 (Mann Whitney test). (F) Control and BSLC2 fibroblasts were treated with OA for 1 h, fixed and stained with anti-ACSL3 antibodies and LD540. Representative sections of peripheral ER areas, maximum intensity projections of deconvolved confocal z-stacks. (G) Analysis of (F), with insets illustrating assigned categories of ACSL3 LD association. Insets are the same as in Fig 4F. Bars: % of LDs/ROI showing the category indicated, mean ± SEM, n= 11-10 peripheral ROIs from 11-10 cells/group (1200-2019 LDs), 2 experiments, **p<0.005 (unpaired T-test). (H) Delipidated fibroblasts were pulsed for 20 min with 0.1 mM alkyne-OA either directly (1) or after pre-incubation with 0.1 mM OA for 1 h (2), and chased in the presence of 0.1 mM OA for 40 min. Bars: % of alkyne-OA in neutral lipids (triacylglycerol and cholesterol esters), normalized to control cells in (1), mean ± SEM, n=11-12 samples, 2 experiments, *p<0.05 (unpaired T-test). For C,E,G and H the results are pooled of 2 controls and 2 patients.
Neutrophils discriminate live from dead bacteria by integrating signaling via formylated peptide receptors and Toll-like receptors (2022)
Figure 1

Fig. 1. Live, but not killed, bacteria induce high-level Cxcl2 production.

(A-F) TNF-α, Cxcl1 and Cxcl2 (A, C, and E respectively) were measured at 24 h after stimulation of bone-marrow-derived neutrophils (5 x 105 cells) with increasing concentrations of bacterial nucleic acids (1, 5, 10 and 20 µg of DNA + RNA in equal proportions), HK-bacteria (1, 5, 10 or 20 μg/ml) or live bacteria (MOIs of 2, 5, 10 and 20). LPS (1, 10, 100 and 1000 ng/ml) was used as a positive control stimulus. (B, D and F), Effect of FPR antagonist Boc-2 on GBS-induced TNF-α, Cxcl1 and Cxcl2 production, respectively. Bone-marrow-derived neutrophils were exposed to Boc-2 (50 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.
Neutrophils discriminate live from dead bacteria by integrating signaling via formylated peptide receptors and Toll-like receptors (2022)
Figure 3

Fig. 3. FPRs are required for Cxcl2 responses to live streptococci.

(A-F) Cxcl1 and Cxcl2 concentrations in 24-h culture supernatants of neutrophils (A and B), CSF-polarized (C and D) or GM-CSF-polarized (E and F) BMDMs (ΜΦ) from WT or Fpr-deficient mice stimulated with live GBS (MOI of 2, 5, and 10) or HK-GBS (10 µg/ml). LPS (100 ng/ml) was used as a positive control stimulus. Means + SD of data from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT mice, determined by unpaired t-test.